The role of the gut in reverse cholesterol transport – Focus on the enterocyte

Progress in Lipid Research 52 (2013) 317–328

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Progress in Lipid Research

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Review

The role of the gut in reverse cholesterol transport – Focus on the enterocyte

Miriam Lee-Rueckert a, Francisco Blanco-Vaca b,c, Petri T. Kovanen a,⇑, Joan Carles Escola-Gil b

a Wihuri Research Institute, Helsinki, Finlandb IIB Sant Pau-CIBER de Diabetes y Enfermedades Metabolicas Asociadas, Spainc Departament de Bioquímica i Biologia Molecular, Universitat Autonoma de Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 January 2013Received in revised form 1 March 2013Accepted 10 April 2013Available online 20 April 2013

Keywords:AtherosclerosisCholesterol absorptionEnterocyteIntestinal cholesterol transportersMacrophageReverse cholesterol transport

0163-7827/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.plipres.2013.04.003

⇑ Corresponding author. Address: Wihuri ResearchE-mail address: [email protected] (P.T. Kovanen

In the arterial intima, macrophages become cholesterol-enriched foam cells and atherosclerotic lesionsare generated. This atherogenic process can be attenuated, prevented, or even reversed by HDL particlescapable of initiating a multistep pathway known as the macrophage-specific reverse cholesterol trans-port. The macrophage-derived cholesterol released to HDL is taken up by the liver, secreted into the bile,and ultimately excreted in the feces. Importantly, the absorptive epithelial cells lining the lumen of thesmall intestine, the enterocytes, express several membrane-associated proteins which mediate the influxof luminal cholesterol and its subsequent efflux at their apical and basolateral sides. Moreover, genera-tion of intestinal HDL and systemic effects of the gut microbiota recently revealed a direct link betweenthe gut and the cholesterol cargo of peripheral macrophages. This review summarizes experimental evi-dence establishing that the reverse cholesterol transport pathway which initiates in macrophages is sus-ceptible to modulation in the small intestine. We also describe four paths which govern cholesterolpassage across the enterocyte and define a role for the gut in the regulation of reverse cholesterol trans-port. Understanding the concerted function of these paths may be useful when designing therapeuticstrategies aimed at removing cholesterol from the foam cells which occupy atherosclerotic lesions.

� 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3182. Inhibition of cholesterol absorption in the gut also hinders the reabsorption of macrophage-derived cholesterol . . . . . . . . . . . . . . . . . . . . . . . 320

2.1. Niemann–Pick C1-like 1 (NPC1L1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3212.2. Effects of transcription factors on intestinal NPC1L1 expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3212.3. NPC1L1, m-RCT, and atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

3. Stimulation of cholesterol secretion from the enterocyte to the intestinal lumen also favors fecal excretion of macrophage-derived cholesterol 322
3.1. ABCG5/G8 heterodimer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3223.2. Effects of transcription factors on intestinal ABCG5/G8 expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
4. Biogenesis of HDL particles and their export from the enterocyte to blood stimulate initiation of macrophage-RCT . . . . . . . . . . . . . . . . . . . . . 3235. Direct transfer of cholesterol from the blood to the enterocyte and the intestinal lumen may contribute to macrophage-RCT . . . . . . . . . . . . . 3236. Nutritional and physiological conditions may affect macrophage-RCT in the intestinal compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

6.1. Dietary components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3246.2. Gut microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3246.3. Psychological stress and diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

7. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

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318 M. Lee-Rueckert et al. / Progress in Lipid Research 52 (2013) 317–328

1. Introduction

Atherosclerosis initiates with the accumulation of excess choles-terol into cytoplasmic lipid droplets of macrophages in the arterialintima. The physiological pathway to transfer cellular cholesterolfrom the multitude of peripheral body compartments to the liverand to the intestine for its fecal excretion is designated the reversecholesterol transport (RCT) [1]. Since the introduction by Glomsetof a reverse route to transfer peripheral cholesterol to liver [2],RCT has gained substantially in scientific interest and in the recentyears the concept has evolved by focusing on the events of smallmagnitude, but of potentially high cardioprotective impact, whichtake place in the arterial intima [3]. The key classic steps of the bil-iary RCT pathway are the efflux of cholesterol from peripheral cellsto extracellular cholesterol acceptors, followed by cholesteroltransfer from interstitial fluids to plasma and liver, and the excre-tion of hepatic cholesterol via bile into the intestine, and ultimatelyto feces. Moreover, an alternative non-biliary pathway may also

Non-regulated cholesterol

influx (accumulation)

Macrophfoam c

Regulated cholesterol

influx (no accumulation)

tissue1

Fe

Peripheratissues

LDLLDL-R

tissue2

tissue3, etc

LDL

LDL

mod-LDL

LDL CE

Cholesterol reabsorption

Scavenger receptors

Fluid-phasepinocytosis

LDL-R

LDL-R

Fig. 1. Macrophage-specific reverse cholesterol transport (m-RCT) versus total body RCT.cytoplasmic lipid droplets via non-regulated uptake of modified-LDL by scavenger recepsuch cholesterol-laden macrophage foam cells builds up formation of atherosclerotic lesiEfficient removal of cholesterol from macrophage foam cells is mediated by the ABC transthe foam cells and initiates m-RCT, so attenuating the atherosclerotic burden. In contrreceptor-regulated pathway, which, in concert with a basal level of cholesterol effluxmacrophage peripheral cells quantitatively represents the major contribution to the net mRCT that originates in the intimal foam cells (red arrows) is directly relevant for atherostransferred in multiple subsequent steps of the reverse pathway to the intestine. On avereabsorbed, and the remainder will be ultimately excreted from the body as fecal cholestsmall intestine play an important role in determining the efficiency of cholesterol absorphave demonstrated that modulation of the intestinal signaling function in the multi-dirspecific itinerary of cholesterol from peripheral macrophages into feces (see Fig. 2). ABC, ALDL-R, LDL receptor; mod-LDL, modified LDL.

contribute to RCT by delivering cholesterol directly from blood togut (reviewed in [4]), as will be discussed in Section 5.

One physiological example of the major role of the gut in lipidmetabolism is provided by the activity of the intestinal microbiota,which is known to provide an internal environmental signalingsystem. Recent studies have found that, by producing certainmetabolites, this complex microbial system may exert either pro-or antiatherosclerotic effects (recently summarized in [5]). Orallygiven antibiotics can change the microflora enterotype and so alterthe intestinal conversion of cholesterol to coprostanol and, moreimportantly, they also can influence processes involved in thedevelopment of atherosclerosis, as will be discussed in Section 6.2.

Efflux of cellular cholesterol to HDL initiates RCT in all tissues.Thus, measuring the magnitude of the entire centripetal sterol fluxdoes not provide quantitative information about the tiny fractionthat originates in the cholesterol-loaded macrophage foam cells lo-cated in the intima, which is considered the only RCT componentdirectly involved in atherosclerosis [6]. Importantly, cholesterol

HDL

HDL

HDL

HDL

ageell

m-RCT

final step of RCT in the gut

ABCA1ABCG1

FC

FC

FC

FC

FC

ces

l

Total RCT

Cholesterol efflux

In the LDL-rich interstitial fluid of the arterial intima, macrophages accumulate CE intors or via fluid-phase pinocytosis of native LDL particles. Abundant generation of

ons. After CE hydrolysis, the resultant FC is available for various efflux mechanisms.porter-mediated efflux of FC to HDL particles, which reduces the cholesterol cargo ofast, non-macrophage peripheral cells take-up LDL-derived cholesterol via the LDL, maintains the intracellular cholesterol balance. Although the efflux from non-ass of cholesterol that flows through the total body RCT, only the minor fraction of

clerosis. Both macrophage- and non-macrophage-derived cholesterol molecules arerage, 50% of the cholesterol from any source present in the intestinal lumen may beerol to complete the RCT pathway. The final regulatory steps of RCT that occur in thetion and thus can influence the rate of the total body RCT. Moreover, recent studies

ectional traffic of cholesterol across the enterocyte is able to modify the rate of theTP-binding cassette transporter (A1, G1); CE, cholesteryl esters; FC, free cholesterol;

M. Lee-Rueckert et al. / Progress in Lipid Research 52 (2013) 317–328 319

contained in macrophage lipid droplets is exposed to specific cellu-lar regulatory pathways that are not present in other peripheralcells and may not be responsive to conditions that influence thetransport of peripheral cholesterol to liver [6]. Macrophages andadipocytes, in which different cholesterol transporters are involvedin the cholesterol efflux mechanisms, well exemplify this dichot-omy [7,8]. Reduction of the cholesterol pool in the arterial macro-phages can prevent or induce regression of atherosclerosis. In thisfunction of HDL, the dynamics of HDL-cholesterol flux seems tocorrelate better with the disease than the static measurement ofplasma HDL-cholesterol levels [9,10]. Interestingly, an acute inter-vention to promote RCT in familial hypercholesterolemia (FH)patients with massive atherosclerotic lesions by infusing lipo-somes containing proapoA-I (precursor of apoA-I, the main HDLapolipoprotein) increased the fecal cholesterol mass by about40% [11]. However, this study did not include individuals devoidof atherosclerotic lesions, and therefore did not allow estimationof the potential contribution to the net cholesterol excretion fromthe body (i.e. total RCT) of the expanded cholesterol pool containedin the foam cells of atherosclerotic lesions and xanthomas of theFH patients. Importantly, apoA-I/HDL-based infusion therapieshave led to regression of coronary atherosclerotic lesions [12] butin these studies RCT has not been measured. Taken together, thefragmentary pieces of evidence available today favor the hypothe-sis that functional apoA-I is able to stimulate initiation of RCT frommacrophages in vivo in humans.

An illustration of the specific and minor contribution of the RCTfraction derived from macrophages to the total body RCT is shownin Fig. 1. To overcome this quantitative problem, Rader andcoworkers developed an assay in which cultured macrophagesare first radiolabeled with cholesterol in vitro, and then injectedintraperitoneally into mice, after which it is possible to measurethe tracer in the plasma, liver, bile, and ultimately the feces withina period of 48 h [13]. This macrophage-specific RCT (m-RCT) assaynicely measures the ultimate fecal elimination of the tiny but crit-ical cargo of cholesterol in macrophages, which, once released fromthese cells can be routed into the much larger total body RCT. Nota-

Table 1Effect on the in vivo m-RCT of experimental conditions affecting at least one of the intesti

Intestinal path affected Experimental treatments or pathophysiological conditions

Path 1 EzetimibePPARd agonistRestraint stressCorticosteroneFish oil

Paths 1–3 Systemic LXR agonistsIntestinal-specific LXRa transgene expressionIntestinal-specific LXR agonist

Path 4 Liver-specific human NPC1L1 transgene expressionSurgical diversion of bile

ABCB4-targeted inactivationUndetermined FXR agonist

Plant sterolsHigh-fat and high-fructose dietType I-induced diabetes in alloxan-treated miceSitagliptin

The direction of an arrow (up or down) denotes that the measured parameter increased o‘‘=’’, and when it was not determined by ‘‘N.D.’’.* In Ref. [102], biliary cholesterol excretion was completely abolished. It was hypothesize

a FXR agonist and alloxan downregulated and upregulated, respectively, liver cytochintestinal cholesterol absorption. Intestinal cholesterol transporters expression was not

b Several reports using ABCA1 and ABCG5/G8-deficient mice have demonstrated thatpendent of these ABC transporters. It remains to be elucidated whether dietary plant sterimpairment of cholesterol absorption, m-RCT was found to decrease (Fig. 3, unpublishe

c The discrepancy between increased intestinal cholesterol absorption despite a low jestimulation of TICE in high fat-fed hamsters, in which hepatic ACAT2 expression also de

bly, variation in the total body RCT rate may not necessarily reflectvariation of the m-RCT rate. Extensive applications of the macro-phage-to-feces assay have demonstrated during the last 10 yearsthat the pathway involves multiple regulatory events at each ofits individual steps [14,15]. In a recent short-term (3 h) applicationof this in vivo assay, functional (intact), but not dysfunctional (pro-teolyzed), apoA-I increased the transfer of macrophage-derivedcholesterol into the intestinal contents [16]. Given the practicalimpossibility of directly measuring m-RCT in humans, m-RCT eval-uation in rodents constitutes so far the only surrogate to predictthe effect of various physiological or pharmacological conditionson its rate in vivo. Importantly, the magnitude of m-RCT has beenproven to correlate closely to atherosclerotic burden in mice underdifferent pathophysiological conditions [15,17]. One technical lim-itation of the m-RCT assay is that it only quantifies the flux of radi-olabeled cholesterol tracer, i.e. a measure that can be influenced byisotopic dilution in various endogenous pools, the sizes of whichmay vary depending on the experimental setting. More recently,measuring cholesterol mass changes in vivo in macrophages en-trapped in semipermeable hollow fibers and surgically implantedinto the peritoneum of recipient mice has been found to be feasible[18], but further applications of this modified m-RCT assay are stillawaited. Because mice, in contrast to humans, do not express cho-lesteryl ester transfer protein (CETP), cholesterol contained in HDLis delivered directly to liver (mostly scavenger receptor class B typeI (SR-BI)-dependently), instead of being transferred first to apoB-containing lipoproteins and then taken by the hepatic LDL recep-tors. To overcome this shortcoming, a number of the m-RCT studieshave been also performed in the CETP-transgenic (Tg) mice and innaturally CETP-expressing rodents, such as the hamster [17]. A re-cent review discussed more extensively the potential caveats in theinterpretation of m-RCT results [17].

Whereas the significance of HDL particle quality in promotingcholesterol flux from macrophages to feces is now well recognized,the role of the small intestine in the regulation of the rate of the m-RCT pathway is less appreciated. The gut acts as a gate for�1500 mg of cholesterol entering the intestinal lumen per day,

nal cholesterol paths described in Fig. 2.

Intestinal targets Cholesterol absorption m-RCT References

NPC1L1 ABC (A1,G5,G8)

; N.D. ; " [46,48,49]; = ; " [46,49]; = ; " [46]; " N.D. " [46]; = N.D. " [119]; " ; " [50–52]; " ; " [42]= " N.D. " [76]N.D. N.D. = = [102]*

N.D. N.D. N.D. =(102); (103)

[102,103*]

N.D. = N.D. ; [103*]N.D. N.D ; " [53a]= = ; ; [106b]; = " " [127c]N.D. N.D. " ; [128a]N.D. N.D. ; " [129]

r decreased, respectively. When the parameter remained unchanged it is denoted by

d that TICE maintains m-RCT. However, this effect was not reproduced in Ref. [103].rome P450 (Cyp) 7a1 and 8b1 expression and regulated liver bile acid levels anddetermined.the plant sterols-mediated inhibition of intestinal cholesterol absorption is inde-

ols disrupts cholesterol homeostasis by affecting other regulatory pathways. Despited results).junal NPC1L1 expression, associated with a high mass of fecal cholesterol suggestedcreased.


and it is actively involved in cholesterol transport mechanisms. In-deed, via a complex network of cholesterol transporters the smallintestine determines the net balance between absorbed and ex-creted cholesterol and so can effectively influence RCT. Since theenterocyte cannot distinguish the source of cholesterol present inthe intestinal lumen, changes in cholesterol absorption efficiencywill affect equally cholesterol molecules derived from any source.Thus, we can infer that interventions aimed at inhibiting choles-terol absorption and so stimulating total RCT will likely promotethe m-RCT pathway as well, provided they do not have additionalspecific effects that would inhibit m-RCT initiation. This review ad-dresses the traffic of macrophage-derived cholesterol across theultimate RCT circuit connecting the enterocyte with the intestinallumen and the blood which, by determining the efficiency of fecalcholesterol excretion, can also affect the rate of the m-RCT path-

LiverLiverα-HDL α-HDCE SR-BIα HDL α-HD

CECE

LCAFCFC

BileVLDL

Bile

Lymph

VLDL

Lymph

VLDLrCM

apoA-I

? MTP

apoA-I

? apoB-48 CEMTP

4 apoB 48 CE

ACAT2FCFC

apoA-IC1 NPC1

FC FC

InIn

Fig. 2. Functional paths in the enterocyte involved in the net transfer of macrophage-decells promoted by the ABCA1/G1 transporters initiates m-RCT. The intimal fluid containscholesterol efflux. The minor preb-HDL subpopulation is converted in circulation into a-Hplasma. Part of the CE moiety of a-HDL is delivered via the SR-BI receptor to the liver, whacids. For simplicity the CETP-mediated delivery of cholesterol in apoB-lipoproteins to thincluded in this schematic representation. The enterocyte can handle cholesterol via diffebasolateral sides of the enterocyte and various intracellular enzymes participate. FC intransporter regulating the net absorption of sterols from the intestinal lumen. The NPC1postprandial phase. Once in the enterocyte, FC is also exposed to efflux mechanisms wforward to the interstitial fluid of the gut after ABCA1-mediated lipidation of intestinalHDL) (Path 3). An alternative non-biliary route for the transfer of cholesterol from the bloApoB-100-containing lipoproteins such as VLDL remnants and a solitary apoA-I fractioncholesterol to the enterocyte and facilitating its efflux to the lumen, respectively. Howevemost prominent paths for the maintenance of cholesterol homeostasis in the gut are Patrate in vivo (Table 1). Conceptually, of the presented pathways only Path 3 (HDL biogenecassette transporter (A1, G1, G5, G8); ACAT2, acyl CoA:cholesterol acyltransferase 2; CE,acyltransferase; MTP, microsomal triglyceride transfer protein; NPC1L1, Niemann–Pilipoproteins; VLDLr, VLDL remnants.

way. We further scrutinize the current experimental data targetingthe final step of the m-RCT in the gut (Table 1), and conceptualizefour distinct regulatory paths available for cholesterol in theenterocyte that affect the efficiency of the m-RCT rate in vivo(Fig. 2).

2. Inhibition of cholesterol absorption in the gut also hindersthe reabsorption of macrophage-derived cholesterol

Small intestine, notably the jejunum, as the primary site of die-tary cholesterol uptake, plays a non-disputable role in the wholebody cholesterol homeostasis [19]. Therefore, modulation of intes-tinal cholesterol absorption may have profound clinical implica-tions. In the Finnish population, a significant and positive

MacrophageMacrophagefoam cellfoam cell

ABCG1L ABCG1

FCL

FCCE

βT CEABCA1

Intimal preβFC fluidFC

Bloodpreβ

InterstitialInterstitialfluid

BASOLATERALpreβ

ABCA1BASOLATERAL

3 ABCA13

EnterocyteEnterocyte

FC

2 ABCG5/1L1 2 ABCG5/

ABCG8APICAL

FCT f

testinalTo feces

testinallumenlumen

rived cholesterol into feces. Efflux of FC to HDL particles from the macrophage foamnascent preb- and mature a-migrating HDL particles which stimulate macrophageDL particles via the enzyme LCAT, which esterifies cholesterol and remodels HDL in

ere it is secreted in the bile into the gut as FC as such or after its conversion into bilee liver and the flow of bile acids into the gut and its recirculation to the liver are notrent paths in which several cholesterol transporters localized either at the apical orthe lumen is absorbed by NPC1L1 (Path 1), which is the critical cholesterol influx

L1 pathway acts coordinately with ACAT2 and MTP to generate chylomicrons in thehich export FC backward to the lumen via the ABCG5/G8 heterodimer (Path 2) orapoA-I and subsequent generation of nascent HDL particles (here denoted as preb-od directly to the gut is also thought to contribute to fecal sterol excretion (Path 4).located in the intestinal microvilli have been proposed to participate by deliveringr, the identity of the specific mediators in this alternative route is still enigmatic. Thehs 1 and 2. These paths have also been experimentally proven to modify the m-RCTsis) could have a direct and specific stimulatory effect on m-RCT. ABC, ATP-bindingcholesteryl esters; CM, chylomicron; FC, free cholesterol; LCAT, lecithin-cholesterolck C1 Like 1; SR-BI, scavenger receptor class B type I; VLDL, very low density


correlation between the intestinal cholesterol absorption efficiencyand plasma LDL-cholesterol levels was observed [20]. Increasingdietary cholesterol mass decreases the percentage of cholesterolabsorbed indicating a saturable process [21,22]. Biliary cholesterolin humans constitutes �70% of cholesterol delivered to the gut perday and it was postulated to regulate dietary cholesterol absorp-tion, apparently by saturation of the intestinal micelles requiredfor the process [23].

Cholesterol absorption is a complex process at the enterocytelevel since it depends on various influx and efflux processes towhich luminal cholesterol molecules crossing the brush bordermembrane (BBM) of the enterocyte are exposed. We wish toemphasize that the term ‘‘absorption’’ implies the passage of cho-lesterol through the enterocyte and reflects the net transfer of cho-lesterol from the intestinal lumen to the intestinal interstitial fluidand ultimately to blood. In contrast, the term ‘‘uptake’’ has beenused to refer both the binding of luminal cholesterol to the BBMof the enterocyte and its actual entry into the enterocyte. Thus,not all the processes involved in cholesterol uptake by the entero-cyte lead to cholesterol absorption.

2.1. Niemann–Pick C1-like 1 (NPC1L1)

The understanding of the events involved in cholesterol absorp-tion was significantly enhanced by the recognition of several intes-tinal proteins, which act either at the apical or basolateral side ofthe plasma membrane or in the cytoplasm of the enterocyte andwhich coordinately regulate this process [19]. Among them, theNPC1L1 protein localized at the apical membrane of enterocyteswas identified as the key transporter for cholesterol absorptionin the intestine [24] (Fig. 2, Path 1). Shortly after its recognitionas the critical enterocyte cholesterol gatekeeper, NPC1L1 was alsoidentified as the molecular target of ezetimibe [25], the first drugspecifically inhibiting intestinal cholesterol absorption. Indeed,cholesterol absorption is markedly blunted in NPC1L1-KO mice,which are non-susceptible to ezetimibe [24]. In contrast to mice,which only express NPC1L1 in the intestine, humans also expressthis protein abundantly in the liver [26]. In cultured hepatomacells, NPC1L1 was found to traffic between the plasma membraneand intracellular compartments through an endocytic recyclingpathway regulated by cellular cholesterol availability [27]. Simi-larly, high cholesterol concentrations in the intestinal lumen stim-ulate NPC1L1 internalization with an ensuing reduction ofcholesterol absorption by the enterocyte [28,29]. NPC1L1-derivedcholesterol is believed to be sorted by a tightly regulated clath-rin-mediated endocytic process from the enterocyte BBM to theendoplasmic reticulum (ER) where cholesterol can be esterifiedby the ER-bound enzyme acetyl-CoA acetyltransferase 2 (ACAT2)[26]. This step is followed by packing of the esterified cholesterolwith apoB-48 into chylomicrons with the aid of the microsomal tri-glyceride transfer protein (MTP), secretion of the chylomicrons intothe basolateral extracellular fluid, and their passage into thelymph. Interestingly, the proprotein convertase subtilisin/kexintype 9 (PCSK9), an important regulator of the hepatic LDL receptorlevels that is also abundantly expressed in the intestine, was foundto significantly stimulate both NPC1L1 protein expression and theproduction of apoB-48-containing lipoproteins in the humanenterocyte Caco-2 cell line [30]. Thus, inhibition of PCSK9 couldpotentially result in an antiatherogenic effect also by decreasingcholesterol absorption. A higher NPC1L1 affinity [26] and ACAT2substrate specificity [31] for cholesterol than for plant sterols canpartially explain the inefficient absorption of the latter. Mere up-take of luminal cholesterol into the BBM by SR-BI and the scaven-ger receptor class B cluster determinant 36 (CD36) by theenterocytes is not rate-limiting for cholesterol absorption [32].Thus, NPC1L1-mediated cholesterol transfer into the interior of

the enterocyte appears to be essential for the efficiency of itsabsorption. Because specific inactivation of hepatic SR-BI withensuing impairment of HDL uptake by the liver also resulted in re-duced m-RCT [33], it appears that SR-BI is of major significance inthe liver-mediated rather than in the intestine-mediated step of m-RCT, at least in the mouse. Most of the enterocyte cholesterol is se-creted in chylomicrons, but part of it can be secreted back to theintestinal lumen or assembled together with apoA-I into HDL par-ticles by ATP-binding cassette (ABC) transporter-mediated pro-cesses, which will be described in Sections 3 and 4.

The efficiency of the human gut to absorb cholesterol is highlyvariable even within the range of normal dietary cholesterol con-sumption [23] and constitutes an inherited trait [34]. Indeed, stud-ies on individuals with a non-responsive phenotype of plasma LDL-cholesterol to ezetimibe treatment have identified carriers ofNPC1L1 gene variants [35,36]. Particularly, 19 multiple rare se-quence variants in human NPC1L1 gene have been shown to con-tribute to the efficiency of the gut to absorb cholesterol and toregulate plasma LDL levels [37]. Such naturally occurring variantsimpair with distinct efficiencies the recycling of NPC1L1 to theplasma membrane, its glycosylation and stability, and facilitateits degradation through the ER-associated degradation pathway[38]. Also, a single nucleotide polymorphism of NPC1L1 in the Jap-anese population was related to the efficiency of intestinal choles-terol absorption [39].

2.2. Effects of transcription factors on intestinal NPC1L1 expression

The effects on cholesterol absorption of transcription factorsthat regulate NPC1L1 gene expression have been reported in sev-eral animal models, and, in some of these studies, their effects onm-RCT have been evaluated, as well. Thus, in mice, NPC1L1 mRNAlevels are reduced by cholesterol feeding and increased by choles-terol deprivation [26]. The presence in the NPC1L1 gene of a sterolresponse element that binds to the sterol regulatory element-bind-ing protein 2 (SREBP2) transcription factor nicely explains themodulation of NPC1L1 promoter activity by cholesterol [40].NPC1L1 is also known to be downregulated via LXRa/b in Caco-2cells and in the mouse intestine [41]. This latter in vivo observationwas confirmed in mice in which selective activation of intestinalLXRa reduced cholesterol absorption, increased fecal neutral sterolexcretion and m-RCT, and reduced intestinal NPC1L1 mRNA levels[42]. Of note, it was found that the presence of both LXRa and LXRbin the enterocyte may compensate for each other in the transcrip-tional regulation of intestinal NPC1L1 [43]. Also, fenofibrate, anagonist of PPARa, impaired cholesterol absorption in chow-fedLXRa/b-double-knockout mice by acting at the level of intestinalNPC1L1 expression [44]. Moreover, PPARa gene has been identifiedas an intestine-specific LXR target, and a transcriptional cross talkbetween these two transcription factors was shown to regulate theexpression of several genes in the intestine [45], which seems alsoto apply to NPC1L1 [46]. PPARd activation has also been shown todecrease intestinal NPC1L1 expression and cholesterol absorptionefficiency in mice [47].

2.3. NPC1L1, m-RCT, and atherosclerosis

Because of the significance of NPC1L1 in the regulation of cho-lesterol absorption, targeting its functionality also affects fecalexcretion of macrophage-derived cholesterol. Indeed, ezetimibeadministration [48,49], as well as intestinal activation of PPARd[49] and PPARa [46], all have been found to increase excretion ofmacrophage-derived cholesterol in feces by reducing intestinalNPC1L1 expression in mice. LXR activation also increased m-RCTin mice, but the effect was attributed to upregulation of the ABCcholesterol transporters ABCA1/ABCG1 or ABCG5/ABCG8, the for-


mer ones facilitating cholesterol efflux from macrophages and thelatter ones facilitating cholesterol export from hepatocytes to bileand from enterocytes to the intestinal lumen [50,51]. However,LXRa/b activation in the hamster did stimulate m-RCT by reducingcholesterol absorption and increasing biliary cholesterol excretion,effects that were associated with reduced NPC1L1 and increasedABCG5/G8 mRNA expression in the gut [52]. Furthermore, intesti-nal downregulation of NPC1L1 and upregulation of ABCG5/ABCG8and ABCA1 expression were observed in mice in which intestinal-specific LXR activation had blunted cholesterol absorption andaccelerated m-RCT [42]. An increase in m-RCT was reported to re-sult from hepatic bile acid farnesoid X receptor (FXR) activationwhich, besides increasing hepatic SR-BI, also indirectly impairedcholesterol absorption, most likely by reducing the hydrophobicityof the bile acid pool [53].

Regarding the potential impact of NPC1L1 function modulatingthe pathogenesis of atherosclerosis, prolonged treatment with eze-timibe was found to dramatically reduce both plasma cholesteroland atherogenesis in the apoE-KO mouse atherosclerosis modeleven during a cholesterol-free diet, an effect that could be explainedby the ability of ezetimibe to limit the amount of biliary cholesterolrecycled from the intestine to the liver [54]. Similarly, NPC1L1 defi-ciency caused nearly complete protection from atherosclerosis inthe NPC1L1/apoE double-KO mice by reducing cholesterol absorp-tion, irrespective of whether the animals were fed a cholesterol-richor cholesterol-free diet [55]. These findings in a mouse model ofatherosclerosis strongly suggested that disruption of NPC1L1-med-iated cholesterol absorption may confer atheroprotection also inhumans. However, despite adding an extra LDL reduction to thatseen with statins alone, the clinical results with ezetimibe havebeen disappointing. Hence, a major still ongoing outcome trial ofezetimibe in patients with acute coronary syndromes will, hope-fully, provide long-awaited data on its clinical efficacy [56].

Regarding the effect of cholesterol absorption inhibition on ath-erosclerosis prevention, a potentially important observation wasmade by studying 14DKK-apoE-KO mice, which had been gener-ated by intercrossing apoE-KO mice with a chromosome 14DKKcongenic strain of mice with reduced cholesterol absorption [57].In this model even the moderate (�40%) reduction in cholesterolabsorption was found to profoundly (�70%) reduce atherosclerosis.Interestingly, RCT from subcutaneously injected bone marrowmacrophages was increased by 60% without any change in theirex vivo cholesterol efflux capacity. These findings suggested thatthe reduction of atherosclerotic burden associated with increasedm-RCT was due to inhibition of cholesterol absorption from thegut, rather than due to impaired initiation of m-RCT. However,the specific mechanism involved could not be defined in this study.Altogether, the findings further support a role for NPC1L1 as thekey intestinal cholesterol-influx transporter and strongly suggestthat the development of atherosclerosis can be potentially modu-lated by regulating the last step of RCT. It is important to recallthat, quantitatively, the mass of macrophage-derived cholesterolsecreted into the gut lumen and potentially reabsorbed is minimalwhen compared to the mass of cholesterol derived from other tis-sues (Fig. 1). Thus, inhibition of the reabsorption of this minutefraction must be only an insignificant player in atherogenesis,when compared to inhibiting the absorption of the bulk majorityof intestinal cholesterol.

3. Stimulation of cholesterol secretion from the enterocyte tothe intestinal lumen also favors fecal excretion of macrophage-derived cholesterol

The intracellular trafficking of cholesterol in the enterocyte iscritical for its ultimate fate. The ABC cholesterol efflux transportersin the apical (ABCG5/ABCG8) or basolateral side (ABCA1) of the

enterocyte contribute, respectively, to either exporting the inter-nalized cholesterol back to the intestinal lumen or forward to theinterstitial fluid and, ultimately, to the circulating blood. In princi-ple, the net cholesterol flux between the small intestinal lumenand the enterocyte compartment depends on the competing activ-ities at the apical membrane of cholesterol influx mediated byNPC1L1 and cholesterol efflux mediated by ABCG5/ABCG8. In thissection we will refer to the effect on m-RCT of the latter mecha-nism, which exports cholesterol and plant sterols to the intestinallumen (Fig. 2, Path 2).

3.1. ABCG5/G8 heterodimer

Unlike other ABC transporters, ABCG5 and ABCG8 operate as aunique heterodimer forming a 12-transmembrane competentcomplex for sterol transport activity [58]. This functional complexpromotes both hepatobiliary and intestinal sterol excretion andhas been proposed to limit intestinal absorption of sterols. It wasestimated that NPC1L1-KO mice secrete about 4 lmol of choles-terol/day via the ABCG5/G8 pathway [59]. In humans, loss-of-func-tion mutations in the ABCG5 and ABCG8 transporters cause theautosomal recessive disorder sitosterolemia [60]. Several SNPs ineither of these two genes have been associated with plasma cho-lesterol concentrations [61–66], and it appears that the geneticbackground, gender, and certain environmental factors may influ-ence the response. It was found that a missense mutation of ABCG5in Wistar Kyoto inbred rats increased the absorption of plant ster-ols without affecting that of cholesterol [67], whereas deletion ofthe ABCG8 gene in mice significantly increased the intestinalabsorption of both sitostanol and cholesterol [68]. In concordance,overexpression of ABCG5 and ABCG8 in Tg mice reduced choles-terol and sitosterol absorption and increased biliary neutral sterolsecretion by a compensatory increase in hepatic cholesterol syn-thesis [69]. Although ABCG5/G8-Tg mice show reduced suscepti-bility to atherosclerosis [70], the protective effect seems torequire the inhibition of intestinal cholesterol absorption since li-ver-specific ABCG5/G8 overexpression was not sufficient to en-hance cholesterol removal from the body or to alter theatherogenic burden [71], so highlighting a critical role of theABCG5/G8 transporters located in the gut. Decreasing the contentof sphingomyelin in the enterocyte membrane by inducing anintestinal-specific functional deficiency of serine palmitoyltrans-ferase (SPT) in mice increased intestinal ABCG5, and reducedNPC1L1 and ABCA1 protein levels [72]. These effects impairedabsorption of cholesterol, but not that of triglycerides, and so indi-cated the importance of sphingomyelin-containing lipid microdo-mains in the apical enterocyte surface in ensuring efficientintestinal cholesterol trafficking. In line with these findings, it ap-peared that it is the cholesterol contained within specializedmicrodomains or rafts in the apical membrane that traffics toABCA1 [73].

3.2. Effects of transcription factors on intestinal ABCG5/G8 expression

ABCG5 and ABCG8 genes appear to be direct targets of LXRa/b.In situ hybridization analyses of tissues from LXR agonist-treatedmice revealed that intestinal ABCG5/G8 and also ABCA1 mRNAswere increased upon LXR activation in wildtype but not in LXRa/b-deficient mice [74]. ABCG5 and ABCG8 expression was requiredfor the in vivo decrease of the fractional cholesterol absorptionand stimulation of cholesterol excretion induced by LXR agonistT0901317 administration [75]. Several studies have shown thatregulation of intestinal ABCG5/G8 expression is involved in theLXR agonist-mediated induction of m-RCT to feces in vivo. In-creased m-RCT induced by systemic LXR activation in mice wasattributed, at least in part, to upregulation of the intestinal


ABCG5/ABCG8 [51]. This LXR-dependent effect was also confirmedin CETP-expressing animals [52]. Moreover, the intestinal-specificLXR agonist GW6340 also enhanced m-RCT and this effect was clo-sely associated with ABCA1 and ABCG5/G8 upregulation ratherthan with NPC1L1expression [76]. Importantly, GW6340 increasedfecal cholesterol excretion in mice injected with LXR-deficientmacrophages, which indicated that the m-RCT stimulatory effectwas macrophage-independent and exerted at the intestinal level[76]. Further evidence underlining the critical role of intestinal,but not hepatic, LXR activation in m-RCT stimulation and athero-sclerosis protection was provided in mice expressing a constitu-tively activated form of LXR specifically in the intestine [42].These effects were related to increased ABCA1 and ABCG5/G8and reduced NPC1L1 mRNA intestinal levels, so suggesting thatboth increased HDL synthesis and reduced cholesterol absorptionin the intestine were responsible for the antiatherogenic effect[42].

4. Biogenesis of HDL particles and their export from theenterocyte to blood stimulate initiation of macrophage-RCT

In addition to the liver, the intestine also synthesizes substan-tial amounts of apoA-I, which for example in the rat accounts for>50% of its content in the plasma compartment [77]. Despite thewide distribution of ABCA1 in the body, only the liver and theintestine directly contribute to the biogenesis of HDL [78,79]. Thegut has been estimated to contribute �30% of the plasma HDL inchow-fed mice [79], and during LXR activation this can increaseto nearly 50% [80]. Early studies have shown that HDL in intestinallymph from fasting rats contained, in addition to the spherical HDLparticles typical of plasma, ‘‘CE-poor discoidal’’ nascent HDL parti-cles that comprised �50% of the total particle population [81].However, few studies have evaluated the composition of HDL par-ticles in the interstitial fluid of the small intestine, which may orig-inate from nascent HDL secreted by the enterocyte and matureHDL transcytosed from the circulation through the capillary endo-thelium into the interstitial fluid. In Caco-2 cells, induction ofABCA1 expression by LXR/RXR activation has been demonstratedto promote polarized basolateral, but not apical, efflux of choles-terol to apoA-I [82]. Interestingly, tissue-specific absence of intes-tinal ABCA1 in mice impaired the basolateral transport of theenterocyte cholesterol into plasma without affecting the transportof luminal cholesterol into lymph, which suggests that the intesti-nal-derived HDL are secreted directly into the circulation, whereasthe lymph HDL are derived from the plasma compartment [79].Such putative direct transfer of nascent (preb-migrating) HDL par-ticles from the intestine towards the circulation would facilitatetheir access to the arterial intima where they could promote initi-ation of RCT from local macrophages (Fig. 2, Path 3). The intestinalproduction of preb-HDL could potentially counteract for the spec-ulated loss of preb-HDL particles in the inflammatory extracellularfluid of the atherosclerotic intima [83]. Thus, the intestinal contri-bution to HDL biogenesis can be considered a direct antiatherogen-ic function of the gut. In support of the crucial contribution of theintestine to circulating nascent HDL particles, endurance trainingin rats induced the intestinal expression of ABCA1 mRNA and con-comitant elevations in plasma HDL-cholesterol, apoA-I, and preb-HDL [84]. Selective LXR-mediated upregulation of intestinal ABCA1also increased total HDL circulating particles and, importantly, thepreb-HDL subpopulation in mouse plasma which contributed to m-RCT stimulation [42].

Whether intestinal ABCA1 plays a direct role in cholesterolabsorption has been a controversial topic [85–87], however, it doesnot appear to be its primary function [73,79]. Of note, NPC1L1-mediated cholesterol absorption was found to regulate the intesti-

nal ABCA1-dependent increase in plasma HDL-cholesterol inducedby LXR agonists [88], which reflects the coordinated function of thetransporters involved in the vectorial transit of luminal cholesterolto circulation. This functional interplay is supported by the obser-vation that intestinal-specific activation of PPARa in human jejunalbiopsies and in Caco-2 cells reduced cholesterol esterification andalso increased ABCA1 and apoA-I expression [89]. Furthermore,intestinal LXR activation promoted m-RCT via upregulation ofABCA1 and ABCG5/G8 [76] indicating a concerted role of intestinalABC transporters in favoring the m-RCT pathway. Taken together,the available data suggest that intestinal ABCA1 participates inm-RCT mainly via HDL biogenesis rather than via cholesterolabsorption. Thus, also in the enterocyte ABCA1 can be assigned acentral role in cholesterol efflux like in macrophages and otherperipheral cells (Fig. 2, Path 3). This conclusion is supported bythe finding that increases in HDL levels and m-RCT occur in micein an ABCA1 gene dose-dependent fashion, but without any associ-ated effect on cholesterol absorption [90]. However, a specific roleof intestinal ABCA1 in m-RCT has not been evaluated so far.

5. Direct transfer of cholesterol from the blood to theenterocyte and the intestinal lumen may contribute tomacrophage-RCT

Besides diet and bile, the two major sources of cholesterol in theintestinal lumen, direct transfer of cholesterol from the blood tothe gut, an early and largely ignored observation, has recentlyemerged as a third source of intestinal cholesterol (Fig. 2, Path4). A recent description of a transintestinal cholesterol excretion(TICE) route has provided novel insights into the way the gut han-dles cholesterol (reviewed in [4,91,92]). TICE has been estimated toaccount for �33% of the total fecal neutral sterol excretion inC57Bl/6 J mice under physiological conditions [93]. In rats andother species, such as dogs and humans, TICE appears to also con-tribute to fecal cholesterol excretion [92]. However, little is knownabout the mechanisms bypassing the liver that govern the flow ofcholesterol from the interstitial fluid of the small intestine to theenterocyte and intestinal lumen, the potential molecular partici-pants in this route, and the source of cholesterol secreted in theprocess.

Recently, it has been proposed that VLDL remnants can delivercholesterol to the small intestine for secretion, whereas bile-de-rived phospholipids may act as luminal acceptors of cholesterol[92]. Moreover, depletion of hepatic ACAT2 in mice increased thesecretion rate of unesterified cholesterol by the liver that was pref-erentially delivered to the small intestine, and no alteration in bil-iary sterol secretion was observed, which also suggested that theliver might contribute to TICE [94]. TICE was impaired in ABCG5-deficient mice [93], thereby suggesting that the ABCG5/G8 hetero-dimer was involved in this pathway. However, this effect was notreproduced in ABCG8-deficient mice [95]. TICE increased 2-foldin SR-BI-KO mice [96], suggesting that the apoE-enriched HDL typ-ical of this mouse model would be one mediator. In contrast, TICEwas unaltered when ABCA1/SR-BI-double KO mice were infusedwith [3H]CO-labeled HDL [97], which suggested that HDL playsno significant role in TICE. Interestingly, it was recently shown thata fraction of the apically secreted apoA-I in the porcine small intes-tine was not released from the cell surface but instead deposited inthe BBM and associated with cholesterol [98]. Whether apoA-Imight contribute to TICE by mediating cholesterol efflux into thegut lumen is not known.

Of note, TICE has been reported to be markedly induced byeither nutritional manipulation [95] or pharmacological activationof LXR and PPARd [93,99]. Yet, the potential role of TICE in the m-RCT rate remains largely controversial. Concerning consumption of

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Fig. 3. Effect of plant sterols on cholesterol absorption by the gut and on the


saturated fat, it was shown that a high-fat/low-cholesterol, but nota low-fat/high-cholesterol diet, stimulated TICE in mice [100].However, this effect of the saturated fat has not translated itselfinto a beneficial effect on m-RCT. Thus, m-RCT was increased inmice fed with a high-fat/high-cholesterol diet but remained unaf-fected with a high-fat/low-cholesterol diet, the effect of cholesterolbeing related to hepatic ABCG5/G8 gene upregulation [101].

Direct evidence demonstrating that TICE can affect m-RCTin vivo was found by inhibiting the secretion of biliary cholesterolinto the small intestine of mice, either by overexpression of hepaticNPC1L1 or by a surgical procedure [102]. Thus, m-RCT remainednormal in NPC1L1Liver-Tg mice which exhibited normal fecal sterolloss even when biliary secretion was reduced by 90% and in micelacking the ability to secrete bile into the intestine [102]. In sharpcontrast, another study demonstrated that m-RCT was markedlydecreased or completely abolished in ABCA4-KO mice lacking bili-ary cholesterol secretion and also in wild-type mice after bile-ductligation [103]. The results of this study confirmed that biliary cho-lesterol secretion is the major relevant route for fecal sterol excre-tion via the RCT pathway. This study also demonstrated that thestimulatory effect of LXR agonists on m-RCT did require biliarycholesterol secretion [103], thus, the actual significance of TICEalternative route in RCT is still up for debate.

macrophage-RCT. C57BL/6 female mice were fed either a Western-type diet (21% fatand 0.2% cholesterol) (control group) or the Western diet supplemented with 2%plant sterol mixture (60% b-sitosterol, 15% b-sitostanol, 15% campesterol, 2%stigmasterol, and 5% campestanol) (plant sterols group). After 4 weeks of dietarytreatments, cholesterol absorption was measured using the dual isotope method(upper panel) and the transfer rate of intraperitoneally injected [3H]cholesterol-labeled J774 macrophages to plasma, liver, and feces was determined (bottompanels) over 48 h. The amount of [3H]tracer is expressed as a fraction of the injecteddose. Values are mean ± SEM (4–6 mice per group, ⁄P < 0.05). (A) The phytosterol-enriched diet markedly inhibited intestinal cholesterol absorption and this wasassociated with a significant reduction in plasma cholesterol levels in thephytosterol-treated group (2.7 ± 0.1 vs 3.4 ± 0.1 mM in phytosterol-treated andcontrol mice, respectively, P < 0.05). (B) Compared to the control group, [3H]cho-

6. Nutritional and physiological conditions may affectmacrophage-RCT in the intestinal compartment

Like pharmacological and genetic manipulations, dietary andphysiological conditions that modify the intestinal traffic of choles-terol may affect the m-RCT rate. In mice, aging enhances choles-terol absorption by upregulating NPC1L1 expression and bysuppressing ABCG5/G8 expression, whereas estrogen upregulatesthe expression of both transporters [104].

lesterol levels were moderately increased in plasma whereas levels of [3H]-tracer inthe liver were markedly reduced in the phytosterol-treated group, suggesting aslower catabolism of HDL in the phytosterol-treated mice. More importantly,despite �2-fold decrease in cholesterol absorption, the phytosterol-enriched dietresulted in a significant reduction of the transfer rate of the macrophage-derivedcholesterol to feces. This unexpected finding supports the notion that theantiatherosclerotic effect of high plant sterols consumption needs reappraisal.

6.1. Dietary components

The modulation of intestinal cholesterol absorption by dietarycomponents other than cholesterol, e.g. soluble fiber, saponins,phospholipids, and soy protein has been demonstrated [105]. Sim-ilarly to ezetimibe, also plant sterols and stanols, have been shownto efficiently inhibit cholesterol absorption [106]. However, themechanisms by which plant sterols reduce cholesterol absorptionand plasma cholesterol levels are not completely understood. Theincreased fecal sterol excretion induced by phytosterols is thoughtto result from competition with biliary and dietary cholesterol forthe formation of mixed micelles in the gut lumen required for cho-lesterol to be absorbed [107]. More recently, the phytosterol effecthas been attributed to TICE stimulation [108] or to the impairmentof LXRa-mediated induction of ABCA1 in the enterocyte [109]. Thislatter mechanism is, however, under debate since plant sterols andtheir derivatives were shown to act as LXR ligands in vitro andin vivo [106], and phytosterol-mediated inhibition of cholesterolabsorption was not altered in ABCA1-, ABCG5-, and ABCG5/G8-deficient mice [110–112]. Furthermore, no specific associationhas been observed between the phytosterol-dependent reductionin cholesterol absorption and intestinal gene expression levels ofABCA1, ABCG5, ABCG8, or NPC1L1 in C57Bl/6J mice [113]. Despitethe wide consumption of plant sterols and stanols as dietary sup-plements, their potential beneficial effects on atherosclerosis arestill controversial [114,115]. To evaluate the direct effect of phytos-terol consumption on m-RCT, we fed mice a Western diet supple-mented with plant sterols for 4 weeks, which markedly reducedcholesterol absorption. Surprisingly, in sharp contrast to ezetimibe[48,49], dietary phytosterols did not increase but rather reducedthe excretion of macrophage-derived cholesterol in feces (Fig. 3,

Escola-Gil JC, unpublished results). This effect may be related, atleast in part, to the increased content of phytosterols in HDLcaused by their high dietary levels [116], which may delay theselective uptake of HDL-derived cholesterol by the liver, as demon-strated with HDL derived from ABCG5/G8-KO mice [51].

The type of fat and other dietary components affecting theexpression of cholesterol transporters in the gut may also influencem-RCT. Thus, polyunsaturated fatty acids down-regulated theexpression of NPC1L1, while monounsaturated or saturated fattyacids did not [117], Similarly, a diet enriched in n-3 fatty acids de-creased gene expression of intestinal NPC1L1 in hamsters [118],and fish oil upregulated hepatic ABCG5/G8 and downregulatedintestinal NPC1L1 mRNA expression in mice, which altogether in-creased the m-RCT rate [119]. High levels of dietary calcium alsoenhanced the fecal cholesterol excretion in hamsters by a con-certed downregulation of intestinal NPC1L1 and MTP, and upregu-lation of ABCG5/G8 [120], but its effect on m-RCT has not beenevaluated.

6.2. Gut microbiota

Recent studies have identified specific effects beyond the intes-tine of the gut microbiota that may influence m-RCT and athero-sclerosis. The gut flora was recently found to be capable of


regulating bile acid metabolism not only by forming secondary bileacids within the intestine, but in other parts of the enterohepaticsystem as well [121]. Thus, bile acid synthesis in the liver wasinhibited by a complex FXR signaling pathway that was activatedby a mediator expressed in the ileum and was gut flora-dependent.The results suggested that, when compared to germ-free mice, thereduced bile acid pool size in control mice likely resulted from re-duced bile acid reabsorption in the distal ileum and concomitantlyincreased bile acid excretion in the feces.

Both pro- and antiatherosclerotic effects in the host have beenrelated to certain metabolites generated by the activity of the gutmicrobiota [5]. It was shown that the intestinal flora is able tometabolize phosphatidylcholine to the choline metabolite trimeth-ylamine N-oxide (TMAO) which promoted atherosclerosis in miceand also predicted risk for cardiovascular disease in a clinical co-hort [122]. In this study, the phenotype of peritoneal macrophagesisolated from apoE-KO mice which had been orally administeredwith choline or TMAO showed increased expression of the scaven-ger receptors CD36 and SR-A1. Moreover, increased formation ofmacrophage foam cells was observed when the mice were fed withdiets containing high levels of choline, an effect that was inhibitedwhen the intestinal flora was suppressed by pretreatment with or-ally administered antibiotics. Regarding the beneficial effects, thegut microbiota was found to metabolize dietary anthocyanincyanidin-3-0-b-glucoside to protocatechuic acid (PCA) which pro-moted m-RCT and atherosclerosis regression in apoE-KO mice, par-tially via upregulating ABCA1 and ABCG1 expression inmacrophages. These effects were mediated by inhibition of a novelmiRNA-10b signaling cascade [123].

Altogether, these findings reveal that the gut microbiota canpotentially affect both the size of the bile acid pool and, impor-tantly, the initiation of RCT in macrophages via formation ofmetabolites with systemic effects. Moreover, they disclosed a di-rect functional link between the gut and the peripheral macro-phages, suggesting that an altered gut microbial compositionmay directly modulate atherogenesis. Whether changes in themicroflora enterotype can also affect the expression of cholesteroltransporters in the intestine will require further evaluation.

6.3. Psychological stress and diabetes

Certain pathophysiological conditions associated with an in-creased risk of atherosclerotic cardiovascular diseases have beenfound to modify intestinal cholesterol absorption and affect the m-RCT pathway, yet, with unanticipated outcomes. We studied the rateof m-RCT in mice exposed to restraint stress which mimics psycho-logical stress in humans [46]. Despite the fact that stress is positivelyrelated to atherosclerosis, stress inhibited cholesterol absorptionwithout affecting macrophage cholesterol efflux so leading to stim-ulation of m-RCT in the stressed mice. Administration of corticoste-rone, the stress hormone in rodents, to non-stressed miceupregulated PPARa and downregulated NPC1L1 protein levels inthe small intestine, and fully reproduced the effect of stress on m-RCT, so providing a mechanistic explanation for the observed effectof stress [46]. Of note, we observed that the increase in m-RCT in-duced by stress in mice was maintained for 7 days of chronic expo-sure [46]. This novel effect of corticosterone calls for furtherevaluation of the potential effects of pharmaceutically active deriv-atives of glucocorticoids on cholesterol absorption and RCT.

Exposure to high concentrations of glucose has been shown toenhance NPC1L1 protein expression and stimulate cholesterol up-take in Caco-2 cells [124]. Importantly, analysis of human duode-nal biopsies has revealed more NPC1L1 and less ABCG5/G8mRNA expression in diabetic patients relative to control subjects[125]. Although enhanced cholesterol absorption has beenreported in diabetic patients [126], conflicting results have been

obtained by evaluating m-RCT in experimental models of diabetes.In hamsters exposed to a high-fat, high-fructose diet that inducesinsulin resistance and dyslipidemia, the fecal excretion of macro-phage-derived cholesterol paradoxically increased despite en-hanced intestinal cholesterol absorption [127]. In alloxan-treatedmice, a model of type I diabetes, enhanced cholesterol absorptiondid not modify fecal neutral sterol excretion because the secretionof bile acids increased in parallel [128]. In the cited study, m-RCTdecreased by 20%, which was attributed to reduced hepatic uptakeof HDL. In another study representing a model of obesity and insulinresistance, the effect on m-RCT of sitagliptin, a drug preventing deg-radation of the intestinal hormone glucagon-like peptide-1, wasevaluated in CETP-apoB100-Tg mice fed with a high-fat diet [129].Sitagliptin significantly reduced cholesterol absorption and in-creased the fecal excretion of macrophage-derived cholesterol by40%, but the molecular mechanism involved remained unexplained.

7. Conclusions and perspectives

Removal of cholesterol from the intimal macrophage foam cellsis critical for atherosclerosis prevention and regression and, thus,constitutes the essential atheroprotective RCT component. Theevents involved in the efflux of macrophage cholesterol and itstraffic along the RCT pathway are regulated in a compartment-spe-cific fashion. Extensive application of the macrophage-to-feces RCTassay has demonstrated that excretion of macrophage-derivedcholesterol can be modulated in the last step of this pathwaywhich occurs in the gut. By regulating cholesterol absorption, thecholesterol transporters NPC1L1 and ABCG5/G8 of the enterocyteare essential in maintaining the whole body cholesterol homeosta-sis. Multiple evidences derived from in vivo m-RCT studies indicatethat interventions that inhibit cholesterol absorption stimulate therate of the m-RCT pathway. Obviously, inhibitors of intestinal cho-lesterol absorption will affect total body RCT and m-RCT rates inparallel, unless they also specifically alter the efficiency of choles-terol release from macrophages. The enterocyte generates nascentHDL particles by an ABCA1-dependent mechanism, and so couldalso function as an important cell in the regulation of RCT initia-tion. Thus, stimulation of this function of the gut could be relevantin individuals having serum with a low cholesterol efflux capacityand who are likely also to have slow rates of m-RCT. Importantly,cholesterol balance in macrophages is highly responsive to varioussignaling pathways and recent studies have revealed that certaingut microbiota-dependent metabolites possess systemic effectsspecifically affecting the macrophage cholesterol cargo and therate of m-RCT initiation.

Further studies on the multifaceted functions of the small intes-tine as an absorptive and secretory organ regulating cholesterolfluxes across the enterocyte and out of the body can improve ourunderstanding of its specific role in the RCT originating in the cho-lesterol-filled intimal macrophages. Deeper understanding of theversatile role of the gut in this cardioprotective pathway will benecessary, when aiming at discovering additional molecular tar-gets as part of the development of gut-based therapeutic strategiesagainst atherosclerotic cardiovascular diseases.

Acknowledgments

Wihuri Research Institute is maintained by the Jenny and AnttiWihuri Foundation. This work was partly funded by EuropeanCooperation in Science and Technology (COST) action BM0904,Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III,FIS 11-0176 (to F.V-B.) and FIS 12-00291 (to J.C.E-G). CIBER deDiabetes y Enfermedades Metabólicas Asociadas is an Instituto deSalud Carlos III Project.


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The role of the gut in reverse cholesterol transport – Focus on the enterocyte

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