The Cholesterol Absorption Inhibitor Ezetimibe Acts by Blocking the Sterol-Induced Internalization...

Cell Metabolism

Article

The Cholesterol Absorption Inhibitor EzetimibeActs by Blocking the Sterol-InducedInternalization of NPC1L1Liang Ge,1,3 Jing Wang,1,3 Wei Qi,2,3 Hong-Hua Miao,1 Jian Cao,1 Yu-Xiu Qu,1 Bo-Liang Li,1 and Bao-Liang Song1,*1State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology2Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences

Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China3These authors contributed equally to this work

*Correspondence: [email protected]

DOI 10.1016/j.cmet.2008.04.001

SUMMARY

Niemann-Pick C1-like 1 (NPC1L1) is a polytopictransmembrane protein that plays a critical role incholesterol absorption. Ezetimibe, a hypocholestero-lemic drug, has been reported to bind NPC1L1 andblock cholesterol absorption. However, the molecu-lar mechanism of NPC1L1-mediated cholesteroluptake and how ezetimibe inhibits this process arepoorly defined. Here we find that cholesterol specif-ically promotes the internalization of NPC1L1 andthat this process requires microfilaments and the cla-thrin/AP2 complex. Blocking NPC1L1 endocytosisdramatically decreases cholesterol internalization,indicating that NPC1L1 mediates cholesterol uptakevia its vesicular endocytosis. Ezetimibe preventsNPC1L1 from incorporating into clathrin-coated ves-icles and thus inhibits cholesterol uptake. Together,our data suggest a model wherein cholesterol is in-ternalized into cells with NPC1L1 through clathrin/AP2-mediated endocytosis and ezetimibe inhibitscholesterol absorption by blocking the internaliza-tion of NPC1L1.

INTRODUCTION

Cholesterol is an essential component of most biological mem-

branes and is the precursor for synthesis of steroid hormones

and bile acids. However, high levels of cholesterol cause severe

problems including coronary heart disease (CHD). In developed

societies, excessive dietary cholesterol uptake is a major risk

factor for CHD. Mammals obtain cholesterol by de novo synthe-

sis from acetyl-CoA and by absorption from diet and bile.

Although much is known about the enzymology and regulatory

pathways of cholesterol biosynthesis (Brown and Goldstein,

1986; Goldstein et al., 2006), far less is understood about the

molecular mechanisms by which cholesterol is absorbed.

Niemann-Pick C1-like 1 (NPC1L1) was recently identified as

a critical player in the intestinal absorption of dietary cholesterol

(Altmann et al., 2004). It is highly expressed in small intestine and

localized along the brush border in both human and mouse

(Davis et al., 2004; Altmann et al., 2004; Sane et al., 2006). How-

508 Cell Metabolism 7, 508–519, June 2008 ª2008 Elsevier Inc.

ever, significant expression of NPC1L1 is also observed in

human liver, but not in mouse liver (Davies et al., 2005; Altmann

et al., 2004). NPC1L1-deficient mice exhibit a drastic reduction

of dietary cholesterol absorption (Davies et al., 2005; Davis

et al., 2004; Altmann et al., 2004). Besides mediating intestinal

cholesterol uptake, overexpression of NPC1L1 in liver in a trans-

genic mouse model dramatically decreases biliary cholesterol

concentration and increases plasma cholesterol level, indicating

that liver NPC1L1 mediates biliary cholesterol reabsorption

(Temel et al., 2007). Taken together, these data indicate that

NPC1L1 plays key roles in dietary and biliary cholesterol absorp-

tion in both intestine and liver.

The NPC1L1 protein contains about 1300 residues with 13

predicted transmembrane domains (Davies et al., 2000). Inter-

estingly, the third to seventh transmembrane helices constitute

a sterol-sensing domain that is also present in other sterol-regu-

lated proteins including NPC1, HMG-CoA reductase, and SCAP

(Kuwabara and Labouesse, 2002). NPC1L1 has been reported to

localize on the plasma membrane (PM) (Altmann et al., 2004; Iyer

et al., 2005) or in intracellular compartments (Davies et al., 2005;

Sane et al., 2006). More recently, Yu et al. found that NPC1L1 is

transported from the endocytic recycling compartment (ERC) to

the PM after cholesterol depletion and that only when localized

on the PM can it promote cholesterol uptake (Brown et al.,

2007; Yu et al., 2006).

Ezetimibe (trademark name Zetia) is a potent cholesterol

absorption inhibitor that is being clinically used to treat hyper-

cholesterolemia (Davis and Veltri, 2007). NPC1L1 knockout

mice show no response to ezetimibe (Altmann et al., 2004),

and the phenotypes of NPC1L1 transgenic mice can be normal-

ized by ezetimibe (Temel et al., 2007). These findings indicate

that NPC1L1 and ezetimibe function in the same pathway. It

has been further found that ezetimibe binds to brush-border

membranes from wild-type animals but not those of NPC1L1

knockout mice, suggesting that ezetimibe interacts with

NPC1L1 and presumably inactivates it (Davis and Veltri, 2007;

Garcia-Calvo et al., 2005). However, how ezetimibe inhibits

NPC1L1-mediated cholesterol uptake is largely unknown.

In the present study, we find that NPC1L1 recycles between

ERC and PM: depletion of cholesterol causes the transport of

NPC1L1 from ERC to PM, whereas replenishment of cholesterol

results in the transportation of NPC1L1 from PM to ERC. Mean-

while, cholesterol is internalized together with NPC1L1. Through

disruption of microfilaments and knockdown of clathrin/AP2, we

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Cell Metabolism

NPC1L1 Mediates Cholesterol Uptake via Endocytosis

demonstrate that the endocytosis of NPC1L1 is dependent

on microfilaments and the clathrin/AP2 complex. Blocking

NPC1L1 endocytosis decreases cholesterol uptake, indicating

that NPC1L1 mediates cholesterol uptake through vesicular

endocytosis. We further find that ezetimibe blocks the internali-

zation of NPC1L1, thereby inhibiting cholesterol uptake. In sum-

mary, our results reveal the mechanism of NPC1L1-mediated

cholesterol uptake and how ezetimibe inhibits the process.

RESULTS

Cholesterol-Regulated Recycling of NPC1L1between the Endocytic RecyclingCompartment and Plasma MembraneTo gain insights into the mechanism of NPC1L1-mediated cho-

lesterol uptake, we established a cell line stably expressing

NPC1L1-EGFP fusion protein from rat CRL-1601 hepatocytes

and named it CRL-1601/NPC1L1-EGFP. Immunoblot analysis

showed that similar levels of NPC1L1 protein are expressed in

CRL-1601/NPC1L1-EGFP cells compared with human liver

cell lines including HepG2, HuH7, and L02 (see Figure S1A

available online). Therefore, this cell line was used as a model

system to study the localization of NPC1L1. We first examined

the localization of NPC1L1-EGFP at different time points after

cellular cholesterol levels were altered. In normal cholesterol-

rich medium, NPC1L1-EGFP protein was mainly present in

a perinuclear compartment (Figure 1B, time point �60 min).

Rab11a, an ERC marker (Ullrich et al., 1996), showed a similar

pattern and colocalized with NPC1L1-EGFP, indicating that

NPC1L1 is in the ERC under this condition (Figure S1B). Consis-

tent with the previous report of Yu et al. (2006), after cells were

refed with cholesterol-depleting medium, NPC1L1-EGFP grad-

ually moved to the PM (Figure 1B, time points �60 to 0 min).

Also, �85% of the cells showed PM localization of NPC1L1-

EGFP after depletion of cholesterol for 1 hr (Figure 1C, time

point 0 min). Interestingly, cholesterol replenishment induced

the internalization of NPC1L1-EGFP, which became evident af-

ter only 30 min (Figure 1B, time point 30 min). After 2 hr of cho-

lesterol replenishment, NPC1L1-EGFP was transported back

into the ERC and almost no cells showed surface localization

(Figure 1B, time points 30 to 120 min; Figure 1C, time point

120 min). This observation was confirmed using a cell surface

biotinylation assay under the same treatment condition

(Figure 1D). To further validate the localization of NPC1L1 and

rule out the possible interference caused by this stable cell

line, the subcellular localization of endogenous NPC1L1 protein

was examined in L02 (a human liver cell line) cells, and the re-

sults consistently showed that endogenous NPC1L1 is similarly

regulated by cholesterol (Figure 1E). These results demonstrate

that the behavior of NPC1L1-EGFP can faithfully represent that

of endogenous NPC1L1 protein.

Requirement of NPC1L1 for Free Cholesterol UptakeIn the process of low-density lipoprotein (LDL) cholesterol inter-

nalization, LDL binds to the extracellular domain of the LDL

receptor (LDLR), which triggers clathrin-dependent vesicular en-

docytosis. These vesicles are transported away from the PM and

fuse with the lysosome, where cholesterol esters are hydrolyzed

and LDLR is resorted and moved back to the PM (Brown and

Goldstein, 1986; Jeon and Blacklow, 2005). Inspired by this

mechanism, the observation of cholesterol-induced endocytosis

of NPC1L1 led us to hypothesize that cholesterol is absorbed via

the vesicular endocytosis of NPC1L1. To test this hypothesis, we

stained cells with filipin, a sterol-binding fluorescent dye (Severs,

1997), to indicate the amount and distribution of cellular choles-

terol after various treatments. As shown in Figure 2A, cholesterol

Figure 1. NPC1L1 Shuttles between the Plasma Membrane and

Endocytic Recycling Compartment According to Cholesterol Level

Alterations

(A) Diagram showing the procedure used to treat the cells. Briefly, the cells

were incubated in cholesterol-depleting medium to reduce cellular cholesterol.

The cells were then refed with cholesterol-replenishing medium to deliver

cholesterol.

(B) CRL-1601/NPC1L1-EGFP cells were treated as shown in (A). At various

time points, the cells were fixed and examined by confocal microscopy. Scale

bar = 10 mm.

(C) Quantification of cells in (B) with plasma membrane (PM) localization of

NPC1L1-EGFP. Error bars represent standard deviations.

(D and E) CRL-1601/NPC1L1-EGFP (D) or L02 (E) cells were treated as shown

in (A). At various time points, the cells were collected for cell surface biotinyla-

tion assay. IB, immunoblot; TnR, transferrin receptor.

Cell Metabolism 7, 508–519, June 2008 ª2008 Elsevier Inc. 509

Cell Metabolism


was barely observed in both CRL-1601 and CRL-1601/NPC1L1-

EGFP cells after incubation in cholesterol-depleting medium for

60 min, while the majority of NPC1L1-EGFP was localized on

the PM (Figure 2A, time point 0 min). When the cells were incu-

bated in cholesterol-replenishing medium, the intracellular cho-

lesterol gradually increased and the intensity of the filipin signal

in CRL-1601/NPC1L1-EGFP cells was much stronger than that

in control cells (Figure 2A, time points 30 to 120 min). Meanwhile,

NPC1L1-EGFP was gradually transported into the cytoplasm and

Figure 2. NPC1L1 Is Required for Cellular

Cholesterol Uptake

(A) CRL-1601 and CRL-1601/NPC1L1-EGFP cells

were treated as shown in Figure 1A. At various

time points, cells were fixed, stained with filipin,

and examined by two-photon confocal micros-

copy. Scale bar = 10 mm.

(B) Quantification of total cholesterol of cells in (A).

Error bars represent standard deviations.

(C) Retroviral vector-mediated RNAi was per-

formed in L02 cells as described in Experimental

Procedures. Lysates were separated by SDS-

PAGE and analyzed by western blot with the indi-

cated antibodies.

(D) After incubation incholesterol-depletingmedium

for 60 min, L02 cells treated with control or NPC1L1

RNAi were replenished with 15 mg/ml cholesterol.

After different time durations, cells were then fixed,

stained with filipin, and examined by two-photon

confocal microscopy. Scale bar = 10 mm.

(E) Quantification of total cholesterol of cells in (D).

Error bars represent standard deviations.

colocalized with cholesterol (Figure 2A,

time points 30 to 120 min). Fluorescence

quantification revealed that the CRL-

1601/NPC1L1-EGFP cells took up at least

2-fold more cholesterol than the control

cells (Figure 2B, timepoints 30 to 120min).

Similar results were obtained when the

cells were replenished with different

concentrations of cholesterol (Figures

S2A and S2B).

To exclude the variations of individual

stable cell clones and test the cell type spe-

cificity, we also examined HEK293T cells

transiently transfected with NPC1L1-

EGFP. We found that the subcellular lo-

calization of NPC1L1-EGFP was regu-

lated by cholesterol as well, and that cells

overexpressing NPC1L1-EGFP took up

more cholesterol than control cells

(Figure S2C). Next, we utilized a retroviral

vector-based RNA interference (RNAi)

technique to reduce the expression of

endogenous NPC1L1 in L02 cells (Fig-

ure 2C). After knockdown of NPC1L1,

the uptake of free cholesterol was de-

creased by �60% (Figures 2D and 2E).

Similar results were obtained when the endogenous NPC1L1

of HuH7 cells was knocked down by siRNA (data not shown).

We also examined the efflux of cholesterol and found no dra-

matic difference between control and NPC1L1-overexpressing

cells (Figure S3), indicating that NPC1L1 is not required for cho-

lesterol elimination and that the effect of NPC1L1 on cholesterol

uptake in our system is a specific event. Together, these data

demonstrate that NPC1L1 is necessary and sufficient for free

cholesterol uptake in our system.


Cell Metabolism


Specificity for NPC1L1-MediatedInternalization of SterolsMammals absorb about 50% of cholesterol from diet, compared

to less than 5% of available plant sterols (Glover and Morton,

1958; Schoenheimer, 1931). The structural differences between

cholesterol and plant sterols are very small (Figure 3A). To ex-

plore whether NPC1L1 is involved in selective cholesterol

absorption, we measured the sterol specificity of NPC1L1-medi-

ated uptake by redelivering different sterols into cholesterol-de-

pleted cells. As shown in Figure 3B, a massive amount of choles-

terol was internalized into the cytoplasm together with NPC1L1.

However, other sterols including ergosterol, b-sitosterol, cam-

pesterol, stigmasterol, and brassicasterol were poorly taken

up. In parallel, the majority of NPC1L1 was still residing on the

PM when cells were replenished with noncholesterol sterols

(Figure 3B, columns b–f). Fluorescence quantification revealed

that �80% of cholesterol was localized in the cytoplasm, com-

Figure 3. Sterol Specificity for NPC1L1-

Mediated Internalization

(A) Structures of sterols evaluated for NPC1L1-

mediated internalization in (B).

(B) After incubation in cholesterol-depleting me-

dium for 60 min, CRL-1601/NPC1L1-EGFP cells

were replenished with 15 mg/ml of different sterols

indicated in (A) for 60 min. The cells were then

fixed, stained with filipin, and examined by two-

photon confocal microscopy. Scale bar = 10 mm.

(C) Quantification of intracellular NPC1L1 and ste-

rol of the cells in (B). Error bars represent standard

deviations.

pared to 8%–13% of other sterols. For

NPC1L1 subcellular distribution, a similar

pattern was observed, with 85% intracel-

lular localization after cholesterol replen-

ishment and 12%–18% after other ste-

rols were replenished (Figure 3C).

These data show that cholesterol is the

most effective substrate of NPC1L1. Fur-

thermore, the correlative localization of

cholesterol and NPC1L1 suggests again

that the internalization of cholesterol is

cooperative with that of NPC1L1.

Disruption of MicrofilamentsBlocks NPC1L1 Endocytosisand Cholesterol UptakeIt isgenerally recognized that the cytoskel-

eton plays important roles in vesicular traf-

ficking (Schafer, 2002; Apodaca, 2001).

The intracellular transport of NPC1L1 is

likely reliant on the cytoskeleton network.

To test this possibility and dissect the rela-

tionship between NPC1L1 endocytosis

and cholesterol internalization, we utilized

cytoskeleton-disturbing drugs to block

the endocytosis of NPC1L1 and then mea-

sured cholesterol uptake. Cholesterol-

depleted cells were treated with nocodazole (to depolymerize

microtubules) (Lee et al., 1980) or cytochalasin D (to disrupt micro-

filaments) (Dubinsky et al., 1999) for 30 min. The cells were then

replenished with cholesterol for various durations. In control and

nocodazole-treated cells, NPC1L1 and cholesterol appeared in

the vesicles beneath the PM after 30 min of cholesterol replenish-

ment (Figure 4B, time point 30 min). After an additional 60 min of

treatment, NPC1L1 was completely endocytosed into intracellular

vesicles, although nocodazole treatment disrupted the organiza-

tion of the ERC (Vossenkamper et al., 2007; Yamashiro et al.,

1984) and caused a diffused pattern of NPC1L1 and cholesterol

in the cytoplasm (Figure 4B; Figure S4A). Along with the endocyto-

sis of NPC1L1, the cells took up a massive amount of cholesterol

mostly colocalized with NPC1L1 in this process. However, in cyto-

chalasin D-treated cells, the endocytosis of NPC1L1 was signifi-

cantly blocked, and the uptake of cholesterol was also inhibited

(Figure 4B, bottom three rows). Fluorescence quantifications of


Cell Metabolism


Figure 4. Disruption of Microfilaments, but Not Microtubules, Attenuates the Endocytosis of NPC1L1-EGFP and Cellular Uptake of Choles-

terol

(A) Diagram showing the procedure used to treat the cells. Briefly, the cells were depleted of cholesterol by incubation in cholesterol-depleting medium for 60 min.

The cells were then switched to medium A containing 5% lipoprotein-deficient serum (LPDS), 10 mM compactin, 50 mM mevalonate, and the indicated drugs. After

30 min, cholesterol was directly added to the medium, and the cells were subjected to staining at various time points.

(B) CRL-1601/NPC1L1-EGFP cells were treated as shown in (A). At different time points, cells were fixed, stained with filipin, and examined by two-photon

confocal microscopy. Scale bar = 10 mm.

(C) Quantification of intracellular NPC1L1 and cholesterol of the cells in (B). Error bars represent standard deviations.

(D) CRL-1601/NPC1L1-EGFP cells were treated as shown in (A). At various time points, the cells were collected for cell surface biotinylation assay.

the intracellular distribution of NPC1L1 and cholesterol are shown

in Figure 4C. Using a cell surfacebiotinylation assay, weconfirmed

again that cytochalasin D, but not nocodazole, inhibited the endo-

cytosis of NPC1L1 (Figure 4D, compare lanes 5 to 7).

We also examined the exocytosis of NPC1L1 and cholesterol

efflux after the cytoskeleton was disrupted. Similar to the endo-

cytosis of NPC1L1, its exocytosis was not affected by nocoda-

zole but was severely impaired by cytochalasin D, indicating

that microfilaments are required for bidirectional transport of


NPC1L1 (Figure S4). However, the efflux of cholesterol was

unaffected by either treatment (Figure S4). Thus, the endocytosis

of NPC1L1 is required for cholesterol uptake, whereas the efflux

of cholesterol is independent of the exocytosis of NPC1L1.

Loss of Clathrin/AP2 Attenuates NPC1L1 Endocytosisand Cholesterol UptakeTo identify proteins involved in NPC1L1 endocytosis, we

performed a large-scale immunoprecipitation with anti-EGFP

Cell Metabolism


agarose from detergent lysates of the membrane fraction of

CRL-1601/NPC1L1-EGFP cells. Specific bands were identified

by tandem mass spectrometry (Figure S5), one of which was

the m2 subunit of the AP2 complex. The AP2 adaptor complex,

which consists of a, b2, m2, and s2 subunits, mainly functions

to select cargo on the PM for inclusion into clathrin-coated

vesicles in the endocytic pathway (Owen et al., 2004). Coimmu-

noprecipitation experiments confirmed that NPC1L1-EGFP, m2,

and clathrin heavy chain (CHC) are present in the same protein

complex (Figure S5C). These data indicate that clathrin-medi-

ated endocytosis is involved in the transport of NPC1L1 protein.

This hypothesis was next addressed by diminishing CHC or m2

through RNAi.

CHC and m2 were substantially reduced by RNAi as revealed

by western blot (Figure 5A). Knockdown of CHC or m2 dramati-

cally inhibited the internalization of transferrin (Figure S6B),

indicating that the RNAi was effective. We then measured

NPC1L1-mediated cholesterol uptake following knockdown of

Figure 5. Knockdown of Clathrin Heavy

Chain or the m2 Subunit of the AP2 Complex

Inhibits the Internalization of NPC1L1 and

Cholesterol

(A) Lysates of CRL-1601/NPC1L1 cells trans-

fected with different siRNAs were blotted with

the indicated antibodies. CHC, clathrin heavy

chain.

(B) CRL-1601/NPC1L1-EGFP cells were treated

as shown in Figure S6A. After replenishment of

cholesterol for different time durations, cells

were fixed, stained with filipin, and examined by

two-photon confocal microscopy. Scale bar =

10 mm.

(C) Quantification of intracellular NPC1L1 and cho-

lesterol of the cells in (B). Error bars represent

standard deviations.

CHC or m2. Loss of CHC or m2 signifi-

cantly decreased the endocytosis of

NPC1L1 as well as the internalization of

cholesterol when cholesterol was replen-

ished (Figure 5B). Quantifications

showed that �50% of NPC1L1 endocy-

tosis and cholesterol uptake were elimi-

nated (Figure 5C). CHC knockdown also

decreased the endocytosis of NPC1L1-

EGFP in a surface biotinylation assay

(Figure S6C). Finally, the endocytosis of

NPC1L1-EGFP and the internalization of

cholesterol were increased in a choles-

terol concentration-dependent manner

in control cells but were significantly

attenuated in CHC or m2 RNAi cells (Fig-

ures S6D and S6E). As a control, knock-

down of caveolin-1 did not affect the

internalization of NPC1L1 or cholesterol

(Figure S7), which is consistent with a pre-

vious report that cholesterol absorption

is normal in caveolin-1-deficient mice

(Valasek et al., 2005).

Together, these data demonstrate that the NPC1L1 protein is

internalized through clathrin/AP2-mediated endocytosis. When

the transport of NPC1L1 from the PM is directly inhibited by

loss of clathrin/AP2, the internalization of cholesterol is also

decreased, indicating again that the endocytosis of NPC1L1 is

required for cholesterol uptake.

Ezetimibe Blocks NPC1L1 Endocytosisand Cholesterol UptakeEzetimibe is a potent inhibitor of cholesterol absorption and has

been approved as a novel hypocholesterolemic drug (Davis and

Veltri, 2007). NPC1L1 has been shown to bind ezetimibe and is

the target of ezetimibe (Field et al., 2007; Hawes et al., 2007;

Pfefferkorn et al., 2008; Weinglass et al., 2008; Garcia-Calvo

et al., 2005). However, how ezetimibe inhibits NPC1L1’s function

and blocks cholesterol absorption has not been fully understood.

Since our data demonstrated that the endocytosis of NPC1L1

is required for cholesterol uptake, we then tested whether


Cell Metabolism


Figure 6. Ezetimibe Blocks Both NPC1L1 Endocytosis and Cholesterol Uptake by Inhibiting NPC1L1 Incorporation into Clathrin-Coated

Vesicles

(A) Diagram showing the procedure used to treat the cells. Briefly, the cells were depleted of cholesterol by incubation in cholesterol-depleting medium for 60 min.

The cells were then switched to medium A containing 5% LPDS, 10 mM compactin, 50 mM mevalonate, and the indicated drugs. After 60 min, cholesterol was

directly added to the medium, and the cells were subjected to staining at various time points.

(B) CRL-1601/NPC1L1-EGFP cells were treated as shown in (A). After replenishment of cholesterol for 60 min, cells were fixed, stained with filipin, and examined

by two-photon confocal microscopy. Scale bar = 10 mm.

(C) Quantification of intracellular NPC1L1 and cholesterol of the cells in (B). Error bars represent standard deviations.

(D and F) CRL-1601/NPC1L1-EGFP (D) or L02 (F) cells were treated as shown in (A). At various time points, cells were collected for cell surface biotinylation

assay.


Cell Metabolism


ezetimibe inhibits cholesterol uptake by blocking the endocyto-

sis of NPC1L1. Cholesterol-depleted cells were treated with

various concentrations of ezetimibe followed by cholesterol re-

plenishment. Notably, ezetimibe inhibited the endocytosis of

NPC1L1-EGFP in a concentration-dependent manner (Figures

6B and 6C). The internalization of cholesterol was blocked as

well (Figures 6B and 6C). As a control, U18666A, a compound

that interferes with LDL-derived cholesterol transport from endo-

somes/lysosomes to the ER likely by inhibiting NPC1 (Neufeld

et al., 1999), had no effect on NPC1L1-mediated cholesterol

absorption. The results of a cell surface biotinylation assay

confirmed that ezetimibe inhibited the endocytosis of NPC1L1-

EGFP but not that of the transferrin receptor (Figure 6D, compare

lanes 4 and 5), suggesting that the effect of ezetimibe on

NPC1L1 transport is specific.

We showed earlier that the CHC protein can be coimmunopre-

cipitated together with NPC1L1-EGFP protein when cholesterol

is replenished (Figure S5C). We next performed a time-course

study using the coimmunoprecipitation assay and found that

cholesterol promoted the NPC1L1-clathrin complex formation

(Figure 6E). The peak of the NPC1L1-clathrin interaction was

observed after 20 min of cholesterol replenishment, and the in-

teraction then decreased (Figure 6E). This phenomenon can be

explained by the fact that CHC is only involved in the early stage

of vesicular endocytosis and dissociates from cargo vesicles

before the vesicles can fuse with the distal endocytic compart-

ment. However, this cholesterol-induced NPC1L1-CHC com-

plex was dramatically reduced in the presence of ezetimibe

(Figure 6E). Similarly, ezetimibe inhibited the endocytosis of

endogenous NPC1L1 as well as the internalization of cholesterol

in human L02 hepatocytes (Figures 6F and 6H). Similar effects

were observed in HuH7 cells (data not shown). On the other

hand, ezetimibe did not show any significant effect on the exocy-

tosis of NPC1L1, the efflux of cholesterol (Figure S8), or the

uptake of fluorescently labeled LDL (DiI-LDL) (Figure S9).

In summary, these data indicate that ezetimibe specifically

blocks the incorporation of NPC1L1 into clathrin-coated vesi-

cles, thereby inhibiting cholesterol absorption.

DISCUSSION

In this study, we observed cholesterol-regulated NPC1L1 shut-

tling: depletion of cholesterol causes the transport of NPC1L1

from ERC to PM, whereas replenishment of cholesterol results

in the internalization of NPC1L1 (Figure 1). Furthermore, the

endocytosis of NPC1L1 requires microfilaments and the cla-

thrin/AP2 complex (Figure 4; Figure 5; Figure S6). More impor-

tantly, we propose that NPC1L1 mediates cellular cholesterol

uptake through vesicular endocytosis, which is supported by

five lines of evidence:

(1) NPC1L1 is required for free cholesterol uptake (Figure 2;

Figure S2).

(2) Replenishment of cholesterol, but not plant sterols,

promotes the endocytosis of NPC1L1; meanwhile, cho-

lesterol is internalized more efficiently than plant sterols

(Figure 3).

(3) Disruption of microfilaments blocks the endocytosis of

NPC1L1 as well as the uptake of cholesterol (Figure 4).

(4) siRNA-mediated knockdown of the clathrin/AP2 complex

attenuates the endocytosis of NPC1L1 and decreases

cholesterol uptake (Figure 5; Figure S6).

(5) A cholesterol absorption inhibitor, ezetimibe, blocks

NPC1L1 endocytosis and cholesterol internalization

(Figure 6).

Our data show that ezetimibe inhibits cholesterol internaliza-

tion by blocking the endocytosis of NPC1L1 (Figure 6). Ezetimibe

does so through preventing the association between NPC1L1

and clathrin/AP2, which is stimulated by cholesterol

(Figure 6E). Interestingly, ezetimibe does not impede the inter-

nalization of the transferrin receptor or LDL receptor. These

results are consistent with the previous finding that ezetimibe

binds to NPC1L1 and is a highly selective inhibitor of cholesterol

absorption (Garcia-Calvo et al., 2005; Davis et al., 2004). How

ezetimibe inhibits NPC1L1-clathrin complex formation is still

unknown. To answer this question, we need to understand the

endocytosis process of NPC1L1 and how cholesterol initiates

this process. An interesting feature revealed by the sequence

of the NPC1L1 protein is the presence of a sterol-sensing do-

main (SSD). The human genome encodes six SSD-containing

proteins: HMGCR, SCAP, NPC1, Patched, TRC8, and NPC1L1

(Kuwabara and Labouesse, 2002). It has been demonstrated

that the SSDs of SCAP and NPC1 can directly bind cholesterol

(Radhakrishnan et al., 2004; Ohgami et al., 2004). Moreover,

the binding of cholesterol causes a conformational change in

SCAP that leads to the association of SCAP with an endoplasmic

reticulum protein called Insig (Brown et al., 2002; Goldstein et al.,

2006). Conceivably, it is likely that cholesterol directly binds the

SSD of NPC1L1, causes the conformational change of NPC1L1,

and promotes the interaction between NPC1L1 and the clathrin/

AP2 complex. If this is the case, ezetimibe may compete with

cholesterol for binding NPC1L1. Another possibility is that the in-

teraction between ezetimibe and NPC1L1 may inhibit the choles-

terol-induced conformational change of the NPC1L1 protein. It is

also possible that ezetimibe interferes with the local distribution

of cholesterol in the plasma membrane, which impedes choles-

terol binding to NPC1L1. Further experiments will be required to

test these possibilities.

Humans obtain cholesterol from de novo synthesis as well as

through dietary and biliary cholesterol absorption. Compared

with the abundantly available inhibitors of cholesterol

(E) CRL-1601/NPC1L1-EGFP cells were treated as shown in (A). After replenishment with cholesterol for 0, 20, 40, and 60 min, cells were collected and sub-

jected to coimmunoprecipitation with anti-EGFP antibodies. The immunoprecipitated proteins were separated by SDS-PAGE and analyzed by western blot

with the indicated antibodies.

(G) L02 cells were first treated with cholesterol-depleting medium in the absence or presence of 30 mM ezetimibe for 60 min. After being switched to cho-

lesterol-replenishing medium for an additional 60 min, cells were washed, fixed, stained with filipin, and examined by two-photon confocal microscopy. Scale

bar = 10 mm.

(H) Quantification of intracellular cholesterol of the cells in (G). Error bars represent standard deviations.


Cell Metabolism


Figure 7. A Working Model for NPC1L1-Mediated Cholesterol Uptake

NPC1L1 protein recycles between the plasma membrane (PM) and endocytic recycling compartment (ERC). When the extracellular cholesterol concentration is

high, cholesterol is incorporated into the PM and is sensed by cell surface-localized NPC1L1. NPC1L1 and cholesterol are then internalized together through

clathrin/AP2-mediated endocytosis and transported along microfilaments to the ERC in vesicles. The ERC is where massive amounts of cholesterol and

NPC1L1 are stored. When the intracellular cholesterol level is low, ERC-localized NPC1L1 moves back to the PM along microfilaments in order to absorb

cholesterol. Notably, ezetimibe prevents NPC1L1 from entering the AP2-mediated clathrin-coated vesicles, thus inhibiting the endocytosis of NPC1L1 and

eventually decreasing cholesterol absorption.

biosynthesis, such as molecules of the statin family, ezetimibe is

the only cholesterol absorption inhibitor known to target the

NPC1L1 pathway thus far. One potential problem is that the re-

sponsiveness of different individuals to ezetimibe varies sub-

stantially. Ezetimibe can dramatically decrease plasma choles-

terol concentrations in some individuals, while it is barely

effective in others (Pisciotta et al., 2007). Therefore, there is an

urgent need for more cholesterol uptake inhibitory drugs. Our

work provides the molecular basis for developing additional cho-

lesterol absorption inhibitors. Moreover, the cell-based assay

that we have established can potentially be used to screen for

novel inhibitors of NPC1L1 endocytosis, which will block choles-

terol uptake eventually.

Almost every kind of mammalian cell is capable of synthesizing

cholesterol; however, de novo cholesterol synthesis is an energy-

consuming process. It costs about 18 ATP, 27 NADPH, and 11 O2

to generate a molecule of cholesterol from acetyl-CoA. Therefore,

mammals obtain significant amounts of cholesterol from diet.

Both animal sterol (cholesterol) and plant sterols are present in

the intestinal lumen. Despite the structural similarity, cholesterol

and plant sterols differ in the nature of their side chains (Figure 3A),

and the functions of cholesterol cannot be completely replaced


by plant sterols (Xu et al., 2005). In fact, humans and animals se-

lectively absorb cholesterol from diet. This important phenome-

non is partly explained by the substrate specificity of ABCG5/

G8. Previous studies have shown that ABCG5/G8 may preferen-

tially secrete plant sterols out of cells (Yu et al., 2004), thereby

ensuring the selective uptake of cholesterol. However, a massive

amount of cholesterol is also pumped out by ABCG5/G8 (Yu et al.,

2002b). Meanwhile, the rank order of the efficiency of absorption

of different sterols is maintained in sitosterolemic patients and

ABCG5/G8-deficient mice (Yu et al., 2002a; Lutjohann et al.,

1995; Iyer et al., 2005), indicating that substrate specificity of

ABCG5/G8 is not the sole mechanism for selective cholesterol

absorption. We have observed that NPC1L1 efficiently mediates

the internalization of cholesterol, but not other sterols (Figure 3).

Brown et al. (2007) have also reported consistent results. These

data indicate that besides ABCG5/G8-mediated selective excre-

tion of plant sterols, NPC1L1 represents another layer of choles-

terol absorption regulation: selective cholesterol internalization.

Although it has been reported that NPC1L1 is responsible for

the absorption of both cholesterol and sitosterol (Yamanashi

et al., 2007; Davis et al., 2004), our data suggest that cholesterol

is the most efficient substrate for NPC1L1.

Cell Metabolism


According to our results, we propose a model illustrating the

pathway of dietary or biliary cholesterol absorption, depicted in

Figure 7. After being incorporated into the PM of enterocytes

or hepatocytes, free cholesterol is then sensed by PM-localized

NPC1L1, and they are internalized together through clathrin/

AP2-mediated endocytosis. These NPC1L1-positive and cho-

lesterol-rich vesicles move along microfilaments to the ERC,

where massive amounts of cholesterol and NPC1L1 are stored.

When intracellular cholesterol levels are low, NPC1L1 moves

back to the PM along the microfilaments in order to absorb

cholesterol. Notably, ezetimibe prevents NPC1L1 from entering

the AP2-mediated clathrin-coated vesicles, thus inhibiting the

endocytosis of NPC1L1 and eventually decreasing cholesterol

absorption.

EXPERIMENTAL PROCEDURES

A complete description of primary antibodies and other methods are available

in the Supplemental Experimental Procedures.

Materials and Plasmids

We obtained horseradish peroxidase-conjugated donkey anti-mouse and

anti-rabbit IgG from Jackson ImmunoResearch Laboratories; filipin, nocoda-

zole, and cytochalasin D from Sigma; Alexa Fluor 555 donkey anti-rabbit IgG

and iron-loaded transferrin-rhodamine conjugate from Invitrogen; sulfosucci-

nimidyl 6-(biotinamido) hexanoate and NeutrAvidin agarose from Pierce;

methyl-b-cyclodextrin (CDX) from Cyclodextrin Technologies Development,

Inc.; all sterols from Steraloids, Inc.; and other reagents from previously

described sources (Cao et al., 2007). The purity of cholesterol was above

99% as measured by gas chromatography/mass spectrometry (GC/MS) in

the F.-J. Nan laboratory (Shanghai Institute of Materia Medica, Shanghai,

China). Lipoprotein-deficient serum (LPDS, density > 1.215 g/ml) was pre-

pared from newborn calf serum by ultracentrifugation (Goldstein et al., 1983).

The coding region of human NPC1L1 was amplified from human liver cDNA

by standard PCR and cloned into pEGFP-N1 vector. The resulting plasmid

encodes full-length human NPC1L1 fused with EGFP at the COOH terminus.

The coding region of Rab11a was amplified from HEK293 cell cDNA and

cloned into pDsRed-monomer-C1 vector. The resulting construct encodes

DsRed-Rab11a.

Cell Culture

CRL-1601 (a McArdle RH7777 rat hepatoma cell line), L02 (a human liver cell

line), and HuH7 (a human hepatoma cell line) cells were grown in monolayer

at 37�C in 5% CO2. The cells were maintained in medium A (Dulbecco’s

modified Eagle’s medium containing 100 units/ml penicillin and 100 mg/ml

streptomycin sulfate) supplemented with 10% FBS. Cholesterol-depleting

medium was medium A supplemented with 5% LPDS, 10 mM compactin,

50 mM mevalonate, and 1.5% CDX. Cholesterol-replenishing medium con-

tained medium A supplemented with 5% LPDS, 10 mM compactin, 50 mM

mevalonate, and different concentrations of cholesterol/CDX. The sterol/

CDX inclusion complexes were prepared as described previously (Brown

et al., 2002).

Cell Surface Biotinylation Assay

After being washed twice with ice-cold PBS and treated with 1mg/ml sulfosuc-

cinimidyl 6-(biotinamido) hexanoate in PBS for 40 min at 4�C, cells were

washed with ice-cold buffer A (20 mM Tris-HCl [pH 8.0] and 150 mM NaCl)

and incubated in buffer A for 15 min at room temperature. Cells were then col-

lected by scraping and lysed in buffer B (10 mM Tris-HCl [pH 8.0], 150 mM

NaCl, and 1% NP-40). Each sample of cell lysates was incubated with

100 ml 50% (v/v) NeutrAvidin agarose and rotated overnight at 4�C. The aga-

rose beads were then washed three times with buffer B. Biotinylated proteins

binding to the beads were eluted by incubation with SDS-PAGE sample buffer

at 37�C for 30 min and analyzed by SDS-PAGE followed by western blot.

RNA Interference

Duplexes of siRNA were synthesized by Genepharma (Shanghai, China). The

siRNA targeting rat clathrin heavy chain (CHC) was described previously

(Radulescu et al., 2007). The sequences of siRNAs targeting rat AP2 m2 and

caveolin-1 were 50-AAGGCATGAAGGAATCACAGA-30 and 50-AAGCAAGTG

TACGACGCGCAC-30, respectively. The siRNA against vesicular stomatitis

virus G protein (VSV-G), used as a control, was described previously (Song

et al., 2005). Transfection of siRNAs was carried out as described previously

(Sever et al., 2003).

Immunofluorescence Microscopy

Cells were fixed with 4% paraformaldehyde in PBS and permeabilized with

0.2% Triton X-100 in PBS. Cells were then blocked with 1% BSA in PBS

and labeled with primary antibodies and fluorescent secondary antibodies

as described in the figure legends. Confocal immunofluorescence microscopy

was performed using a Leica TCS SP2 laser scanning microscope. In each

experiment, images of the same channel were acquired at identical laser

output, gain, and offset.

Filipin Staining

A fresh 5 mg/ml stock solution of filipin was prepared in ethanol. After fixation

as described earlier, cells were washed twice with PBS and stained with 50 mg/

ml filipin in the dark for 30 min at room temperature followed by three washes

with PBS. Filipin signals of stained cells were analyzed with a Zeiss LSM 510

confocal microscope equipped with a two-photon laser using an excitatory

wavelength of 720 nm. Red pseudocolor was assigned to show the filipin sig-

nal. In each experiment, images were acquired at identical laser output, gain,

and offset.

Fluorescence Quantification

For quantification of intracellular NPC1L1 or cholesterol abundance, two

circles, one outlining the whole cell and the other beneath the plasma mem-

brane, were drawn manually. After subtraction of the background, the fluores-

cence intensities in each circle were measured by Image-Pro Plus 5.02 and

regarded as whole-cell and intracellular fluorescence intensity, respectively.

The intensity of each cell was arbitrarily defined as 1, against which the intra-

cellular intensity was normalized. For each time point, 50 cells were randomly

selected and calculated. The data shown in the figures are representative of

four or more independent experiments.

For quantification of total cellular cholesterol, a similar method was em-

ployed except that the intracellular circles were omitted. The total cholesterol

of control cells at time point �60 min was defined as 1, against which the total

cellular cholesterol at other time points was normalized.

For quantification of cells with NPC1L1-EGFP PM localization, �100 cells in

each treatment were randomly selected and counted. The results are pre-

sented as the percentage of cells with NPC1L1-EGFP PM localization. Three

independent experiments were performed, and one representative is pre-

sented in the figures.

SUPPLEMENTAL DATA

Supplemental Data include Supplemental Experimental Procedures, Supple-

mental References, and nine figures and can be found with this article online

at http://www.cellmetabolism.org/cgi/content/full/7/6/508/DC1/.

ACKNOWLEDGMENTS

B.-L. Song is deeply grateful to R.A. DeBose-Boyd, J. Goldstein, and M. Brown

(University of Texas Southwestern Medical Center at Dallas) for training. We

thank S.-Z. Pan and Q. Li for their excellent technical assistance, K. Liao

and G. Wang for providing materials, F.-J. Nan for GC/MS analysis, and

D.-S. Li for critical reading of the manuscript. This work was supported by grants

from the Ministry of Science and Technology of China (2006CB910600), the

National Natural Science Foundation of China (30670432 and 90713025), the

Chinese Academy of Sciences (KSCX2-YW-R-109), and the Shanghai Science

and Technology Committee (06QA14057).


http://www.cellmetabolism.org/cgi/content/full/7/6/508/DC1/

Cell Metabolism


Received: December 16, 2007

Revised: March 6, 2008

Accepted: April 1, 2008

Published: June 3, 2008

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