Special Contribution

Yonago Acta medica 2010;53:29–36

Niemann-Pick C1-Like 1:
A Key Player in Intestinal Cholesterol Absorption
Haruaki Ninomiya
Department of Biological Regulations, School of Health Science, Tottori University Faculty of Medicine, Yonago 683-8503, Japan

Niemann-Pick C1-Like 1 (NPC1-L1), as its name indicates, was identified in 2000 as ahomolog of NPC1. Its major physiological function was clarified by a research group in
Shöring’s laboratory who had long been searching for a target of ezetimibe, a cholesterollowering drug. They published a paper on 2004 in Science, reporting a reduction of intestinal cholesterol absorption and a lack of effects of ezetimibe in NPC1-L1 knockout mice.
With subsequent studies that confirmed their findings, it is now clear that NPC1-L1 is a
key player in one of the major pathways of intestinal cholesterol absorption and that it is
the target of ezetimibe. This review summarizes what has been shown up to now about thestructure and function of NPC1-L1. This review also refers to ABCG5/G8, a member ofABC family transporters, which co-localizes with NPC1-L1 on the intestinal epithelial cells
and appears to work in a closely related manner.
Key words: ABCG5/G8; cholesterol; low-density lipoprotein; Niemann-Pick; Niemann-Pick disease type C1-like 1
have shown that cholesterol directly binds to thisdomain (Infante et al., 2008). The middle part ofthis molecule (transmembrane domain 5–8) constitutes a sterol-sensing domain (SSD). The SSD isshared by other molecules involved in the control
of cholesterol homeostasis, including hydroxymethyl-glutaryl-CoA reductase and sterol regulatory element-binding protein cleavage-activating
protein. NPC1 is one of the house-keeping genes,
expressed in every tissue and cell.
Loss of function of NPC1 results in NPC, anautosomal recessive lipid storage disease (Patterson
et al., 2001). Although heterogeneous, NPC is typically a childhood disease with an onset in the infancy and an eventual demise in the second decadeof life. The NPC kids exhibit a variety of neurological symptoms including ataxia, cataplexy, vertical gaze palsy and so on. At the cellular level, themost prominent feature of NPC is accumulation of

Structures of NPC1 and NPC1-L1
Let us start with Niemann-Pick disease type C1(NPC1) which was identified on 1997 (Fig. 1), because it is the prototype of these two proteins andhence much of what we know about NPC1-Like 1(NPC1-L1) originates in studies on NPC1. It is a
glycoprotein with a molecular mass of 170 to 200
kDa and is predicted to contain 13 transmembrane
domains. Its primary structure is featured by a signal peptide on its N-terminus and a dileucine motifon the C-terminus, the latter of which is believed to
be a targeting signal to the endosome (Davies et al.,2000). The N-terminal extracellular loop contains
an amino acid sequence of approximately 100 amino acids long (55–165), which is of high conservation between species, and is called an NiemannPick disease type C (NPC) domain. Recent studies

Abbreviations: ABC, ATP-binding Cassette; GFP, Green Fluorescent Protein; LDL, low-density lipoprotein; NPC, NiemannPick disease type C; NPC1, Niemann-Pick disease type C1; NPC1-L1, NPC1-Like 1; SSD, sterol-sensing domain

29

H. Ninomiya

Fig. 1. Structure and function of NPC1 and NPC1-L1. See text for details. NPC, Niemann-Pick disease type C; NPC1,
Niemann-Pick disease type C1; NPC2, Niemann-Pick disease type C2; NPC1-L1, NPC1-Like 1; SSD, sterol-sensing domain.

free cholesterol in the endosome/lysosome. Cholesterol is supplied to peripheral cells mainly by
low-density lipoprotein (LDL). Lysosomal hydolyses convert cholesterol esters in LDL to the freeform, which then must be delivered to other cellular compartments such as the plasma membrane
and the endoplasmic reticulum. NPC1 localizes
mainly in the late endosomes. Cells without NPC1
function fail to transport cholesterol out of the endosomes and develop hybrid structures containing
both the marker molecules of late endosomes andlysosomes. These abnormal endosomes containmultivesicular bodies within their lumen. Becauseof these loss-of-function phenotypes, it is clear that
NPC1 takes part in the cholesterol transport out of
the endosomal system. Although the precise mechanism of action remains to be clarified, it has beenshown that the protein “moves” within the cell, being contained in transport vesicles that shuttle between cellular compartments (Zhang et al., 2001).

NPC1-L1 has the amino acid sequence that is
40% identical to that of NPC1 and is predicted tohave a similar secondary structure. Like NPC1,
it has a signal peptide on its N-terminus, an NPC
domain on its N-terminal extracellular loop, and an
SSD in its middle part. A clear difference is that it
lacks the C-terminal dileucine motif (Davies et al.,2000). The tissue distribution of NPC1-L1 is alsoquite different from that of NPC1; both in mice andhumans, its expression is tissue-specific and is specifically high in the small intestine. According toan analysis in mice, its expression is heterogeneousin the intestine, being high in the duodenum andjejunum, and low in the ileum (Davis et al., 2004).
This distribution provides a good explanation for
the fact that dietary cholesterol is absorbed mainlyin the duodenum and jejunum. It should be notedthat its expression level in the liver is clearly different between mice and humans; in humans, its levelin the liver is as high as in the intestine whereas it

30

NPC1-L1 and cholesterol absorption

is barely expressed in mouse livers. Histochemical studies using human tissues demonstrated its
sequestration on the brush border membrane of
the intestinal epithelial cells and on the bile canaliculi of hepatocytes (Sané et al., 2006; Temel et al.,2007). Figure 2 depicts its cellular localization in ascheme.
Intestinal absorption of dietary cholesterolin NPC1-L1 knockout mice is reduced to approximately half the level of that in wild-type mice
(Altmann et al., 2004). Feeding of the knockout
mice with a high-cholesterol diet failed to induce
hypercholesterolemia and resultant fatty liver(Davies et al., 2005).
Its selective tissue distribution and the phenotypes of gene knockout mice made it clear that
NPC1-L1 is required for intestinal absorption of dietary cholesterol. Following studies focused on how
this protein takes part in the absorption process at
the molecular level. Although its localization in thehepatic canaliculi suggests its role in cholesterol uptake from bile, the following discussion focuses on
its role in the intestine, because of space limitation.

Fig. 2. Cholesterol circulation and NPC1-L1 localization.
In humans, NPC1-L1 resides on the apical membrane ofintestinal absorptive cells and in bile canaliculi of hepatocytes, where it facilitates cholesterol uptake. ABCG5/G8
resides on the same sites and facilitates cholesterol flow inthe opposite direction. NPC1-L1, Niemann-Pick diseasetype C1-Like 1.

A later report appeared to reconcile the discrepancy of these initial studies. Yu et al. studied
intracellular localization of GFP-tagged NPC1-L1
in rat hepatoma cells and observed that this protein
shuttles between the cell surface and cytosolic vesicles (Yu et al., 2006). This movement appeared tobe regulated by the cellular level of cholesterol: theprotein resided on the cell surface in a low-cholesterol condition and on the cytosolic vesicles in a highcholesterol condition. The cytosolic vesicles appeared to be recycling endosomes because they contained fluorescent transferrin. Subsequently, several
groups made a similar observation that NPC1-L1“moves” within the cell in a cholesterol-dependent
manner (Ge et al., 2008; Petersen et al., 2008). Our
observation using a confocal microscopy is depicted
in Fig. 3. It has also been shown that the movementfrom the cell surface to the endosome depends onclathrin/AP2 complexes (Ge et al., 2008).
This intracellular movement of NPC1-L1 isreminiscent of that of NPC1. In the case of NPC1,
it has been shown that a steroid-derivative U18666A
that inhibits its function apparently blocks its intracellular movement. Likewise, the intracellular
movement of NPC1-Like1 is blocked by its inhibitor

Intracellular sequestrationof NPC1-L1 and cholesterol transport:
findings in cultured cells
Immediately following its identification, the intracellular localization of NPC1-L1 was examinedusing cultured cells and epitope-tagged recombinant proteins. Initial two reports were obviously
contradictory to each other. Shöring’s group reported localization of NPC1-L1 tagged with Flag
epitope or Green Fluorescent Protein (GFP) on thecell surface of Chinese hamster ovary cells (Iyer etal., 2005) whereas Mt. Sinai’s group demonstrated
localization of Yellow Fluorescent Protein-tagged
NPC1-L1 in the intracellular vesicles of COS cells(Davies et al., 2005). The latter group also showed
sequestration of endogenous NPC1-L1 in cytosolic
vesicles of HepG2 cells. They claimed that thesevesicles represent early endosomes and recycling
endosomes that emanate from them.
31

H. Ninomiya

A

B

Fig. 3. Intracellular localization of Yellow Fluorescent Protein-tagged NPC1-L1 expressed in Human Embryonic Kidney 293 cells.
A: Low-cholesterol condition. Cells cultured in lipoprotein-deficient serum were treated with methyl- -cyclodextrin
to reduce cellular cholesterol contents.
B: High-cholesterol condition. Cells in condition A were loaded with free cholesterol.
NPC1-L1, Niemann-Pick disease type C1-Like 1.

ezetimibe. These pharmacological findings suggestthat these proteins must move between the cellularcompartments to operate cholesterol transport.

in the free form whereas they are in the esterified
form in chylomicron. These conversion and transport processes consist of multiple steps as shown in
Fig. 4. Depending on the observations in cultured
cells, the site of action of NPC1-L1 is thought to be
step 2, in which the cholesterol-enriched plasma
membrane domains are internalized to the endosomal compartment.
The total cholesterol content of intestinal
epithelial cells from NPC1-L1 knockout mice was
reduced compared with wild-type mice, which
does not contradict the role of this protein in step2 (Davies et al., 2005). Knopfel et al. found nodifference in the rate of in vitro cholesterol uptake
between the apical membrane vesicles preparedform the wild-type and knockout mice (Knopfel etal., 2007). Accordingly, ezetimibe failed to reducethe capacity of these vesicles to take up cholesterol.
These negative data indicate that step 1 (i.e., transfer of micellar cholesterol to the apical membrane)
does not depend on NPC1-L1 function.
As described above, the cholesterol transport
out of the endosomal system (steps 3 and 4 in Fig.
4) depends on NPC1 function and its deficiency

Function of NPC1-L1
in intestinal epithelial cells
There has been no report yet that examined intracellular localization of NPC1-L1 in the intestinal epithelium in situ. On ordinary immunohistochemical examinations of fixed tissues, it appears to besequestrated on the apical membrane of absorptive cells; however, immunoelectron microscopy
revealed that in addition to the apical membrane,
it was also localized on the limiting membraneof intracellular vesicles (Sané et al., 2006). Frommorphological features, these vesicles appear to belysosomes.
The intestinal absorptive cells receive cholesterol from biliary micelle on its apical membraneand package it in chylomicron, which in turn is released from the basolateral membrane. Almost allof the cholesterol molecules in biliary micelle are32

NPC1-L1 and cholesterol absorption

leads to endosomal free cholesterol accumulation.
This is also true for intestinal epithelial cells, asevidenced by positive filipin staining of these cells
from NPC1 knockout mice. However, the rate ofintestinal cholesterol absorption was not at all reduced in these mice compared with wild-type mice(Dixit et al., 2007). Thus it appears that cholesterol
molecules derived either from biliary micelle orfrom LDL take different transport pathways inthese cells.

Cholesterol molecules take either free or esterified form in our body. Biochemically, the freeform possesses both hydrophilic and hydrophobic
parts and is thus amphiphilic whereas the esterified form is totally hydrophobic. Owing to its
amphiphilic nature, the free form always resides
within a membrane, i.e., on a two-dimensionalsurface, whereas the esterified can form a threedimensional particle.
Most of the cholesterol molecules delivered
to peripheral cells by LDL are in the esterified
form. To utilize these molecules for structural or
metabolic purposes, cells hydrolyze them to thefree form in the endosomes and lysosomes. This
conversion requires a massive amount of membranes, because the molecules tightly packed in athree-dimensional particle must be spread to a twodimensional membrane. In the endosomal system,
this amount of membranes is supplied by multivesicular bodies, which form three-dimensional particles of stratified membranes. NPC1 is required
to transport these membrane structures out of theendosomal system.
Cholesterol molecules are delivered to theintestinal epithelial cells mostly in their free form,
because of hydrolysis of dietary cholesterol by digestive enzymes. These molecules enter the apicalside of the cells where a massive amount of membranes is equipped by forming microvilli. NPC1L1 is required to transport the membranes fromhere to the endosomal system. Thus in both cases,
an elaborate membrane system was prepared todeal with free cholesterol. Both NPC1 and NPCL1 work as a chaperon to transport the membranes
out of these compartments.

NPC1 and NPC1-L1 as chaperons
for cholesterol-enriched membranes
NPC1 and NPC1-L1 share the amino acid sequences of no more than 40% identity and they havequite different tissue specificity and intracellularsites of action. However, the above-mentioned
studies have shed light to some common aspects ofthe functions of these two proteins.

Cholesterol-specificity of
NPC1-L1 function

Fig. 4. Intracellular cholesterol flow in intestinal absorptive cells. Free cholesterol (FC) contained in biliary micelle is incorporated into the apical side membrane ( ) andthen transported to the endosomes ( ). A part of internalized FC is recycled back to the apical side and is excretedby ABCG5/G8 ( ). Another part is transported to theendoplasmic reticulum ( ) where it is esterified and packaged in chylomicron. FC is also supplied by endogenous
synthesis from acetylCoA. Step depends on NiemannPick disease type C1-Like 1 function while others do not.

Neither the serum concentrations of free fatty acids
nor lipid-soluble vitamins were altered in NPC1-L1
knockout mice (Altmann et al., 2004). When administered to healthy humans, ezetimibe reduced
the serum concentration of LDL but failed to alter33

H. Ninomiya

range, the net intake of intestinal cholesterol is determined by summation of addition by NPC1-L1
and subtraction by ABCG5/G8. As related to the
speed, NPC1-L1 appears to be the major determinant. This is because upon cellular loading of cholesterol, NPC1-L1 is internalized within minutes,
whereas there is no known regulatory mechanism
for ABCG5/G8 that operates within this time lag.
Apart from the efficiency of regulation, theset of NPC1-L1 and ABCG5/G8 is implicated inselection of sterol species. Our diet contains sterols
from both animals and plants, the latter of which(plant sterols or phytosterols) cannot be utilized byour body. Although NPC1-L1 facilitates absorption of phytosterols (Yamanashi et al., 2007), it
is relatively selective for animal-derived sterols.
ABCG5/G8 has clear preference to phytosterols.
Thus for the selective absorption of animal-derived
sterols, the intestinal cells adopt a dual filteringsystem that preferentially takes in useful moleculesand discards useless ones. NPC1-L1 gene knockout in ABCG5/G8-deficient mice prevented themfrom developing hyperlipidemia (Tang et al., 2009),
suggesting that a major portion of phytosterols
discarded by ABCG5/G8 is taken up by NPC1-L1.
This is in accordance with the observed effects ofezetimbe to suppress symptoms and signs of patients with hereditary sitosterolemia.

those of other lipid molecules including triglyceride. These findings suggested that the function of
NPC1-L1 was specifically required for the transportof cholesterol but not for that of other lipids. Given
the role as chaperones for membrane transport,
however, a speculation persists that NPC1-L1 is involved in the transport of other membrane lipids.
Narushima et al., by using an expression
system in Coca2 cells, found that NPC1-L1 facilitated cellular absorption of -tocofenol (vitamin E)(Narushima et al., 2008). Labonte et al. reported
that in mice, NPC1-L1 facilitated intestinal absorption of saturated fatty acids, and that its inhibition
lead to suppression of insulin resistance and weightgain (Labonte et al., 2008). If the latter findings
are also true for humans, eztimibe may have dreameffects in prevention of diabetes mellitus and obesity. It is of regret to note that in humans, there
has been no report yet of weight loss caused byadministration of this drug. In addition, the rate ofintestinal cholesterol absorption had no relation to
the body mass index values (Cohen et al., 2006).

Co-operation by
NPC1-L1 and ABCG5/G8
ABCG5/G8, a heterodimer of G5 and G8 subunits,
is a member of the ATP-binding Cassette (ABC)transporter family. Similar to NPC1-L1, this protein localizes on the apical membrane of intestinal
absorptive cells, but in quite contrary to NPC1-L1,
it operates cholesterol transport from the cells to
the intestinal lumen: thus NPC1-L1 and ABCG5/
G8 work in an opposite direction in the regulationof intestinal cholesterol flow. In humans, these two
proteins also co-localize in bile canaliculi. Loss offunction of either G5 or G8 proteins results in hereditary sitosterolemia characterized by increased
serum and tissue levels of sterols derived fromplants.
In general, co-operation of two proteins thatfunction in an opposite direction to and independently from each other enables a regulation ina wider range and in a faster speed. As for the

Human genetics of NPC1-L1
Besides basic studies on cultured cells or experimental animals, studies on humans are in progress.
One of the major subjects of these studies is the
genotype-phenotype relationship of NPC1-L1. The
efficacy of intestinal cholesterol absorption varies
between individuals, in a range from as low as 30
up to 80%. Given the role of NPC1-L1 as a critical player in this process, one would expect to see
whether the genomic variation of this gene could
explain the individual variation of the absorption
efficacy. Studies are going on to analyze this relationship (Cohen et al., 2006). Clinically, one ofthe disadvantages of ezetimibe is the individual34

NPC1-L1 and cholesterol absorption

variation in responsiveness to this drug. Studies
are also going on to reveal a relationship between
ezetimibe efficacy and NPC1-L1 genotype (Simon
et al., 2005). These lines of studies may in the
future make it possible to treat patients with hyperlipidemia in an “order-made” fashion, dependingon their genetic haplotype of NPC1-L1.

1075.
11 Iyer SP, Yao X, Crona JH, Hoos LM, Tetzloff G, DavisHR Jr, et al. Characterization of the putative nativeand recombinant rat sterol transporter Niemann-Pick
C1 Like 1 (NPC1L1) protein. Biochim Biophys Acta
2005;1722:282–292.
12 Knopfel M, Davies JP, Duong PT, Kvaerno L, CarreiraEM, Phillips MC, et al. Multiple plasma membrane
receptors but not NPC1L1 mediate high-affinity,
ezetimibe-sensitive cholesterol uptake into the intestinal
brush border membrane. Biochim Biophys Acta 2007;
1771:1140–1147.
13 Labonte ED, Camarota LM, Rojas JC, Jandacek RJ,
Gilham DE, Davies JP, et al. Reduced absorption of saturated fatty acids and resistance to diet-induced obesityand diabetes by ezetimibe-treated and Npc1l1–/– mice.
Am J Physiol Gastrointest Liver Physiol 2008;295:
G776–G783.
14 Narushima K, Takada T, Yamanashi Y, Suzuki H.
Niemann-pick C1-like 1 mediates alpha-tocopheroltransport. Mol Pharmacol 2008;74:42–49.
15 Patterson MC, Vanier MT, Suzuki K, Morris JA,
Carstea E, Neufeld EB, et al. Niemann-Pick diseasetype C: a lipid trafficking disorder. In: Scriver CR, etal., eds. The metabolic and molecular bases of inherited
diseases. New York: McGrawHill; 2001. p. 3611–3634.
16 Petersen NH, Faergeman NJ, Yu L, Wustner D. Kinetic
imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma
cells. J Lipid Res 2008;49:2023–2037.
17 Sané AT, Sinnett D, Delvin E, Bendayan M, Marcil V,
Ménard D, et al. Localization and role of NPC1L1 incholesterol absorption in human intestine. J Lipid Res
2006;47:2112–2120.
18 Simon JS, Karnoub MC, Devlin DJ, Arreaza MG, Qiu
P, Monks SA, et al. Sequence variation in NPC1L1
and association with improved LDL-cholesterol lowering in response to ezetimibe treatment. Genomics
2005;86:648–656.
19 Tang W, Ma Y, Jia L, Ioannou YA, Davies JP, Yu L.
Genetic inactivation of NPC1L1 protects against sitosterolemia in mice lacking ABCG5/ABCG8. J Lipid Res
2009;50:293–300.
20 Temel RE, Tang W, Ma Y, Rudel LL, Willingham MC,
Ioannou YA, et al. Hepatic Niemann-Pick C1-like 1
regulates biliary cholesterol concentration and is a targetof ezetimibe. J Clin Invest 2007;117:1968–1978.
21 Yamanashi Y, Takada T, Suzuki H. Niemann-Pick C1like 1 overexpression facilitates ezetimibe-sensitivecholesterol and beta-sitosterol uptake in CaCo-2 cells. J
Pharmacol Exp Ther 2007;320:559–564.
22 Yu L, Bharadwaj S, Brown JM, Ma Y, Du W, DavisMA, et al. Cholesterol-regulated translocation of Niemann-pick C1-like 1 to the cell surface facilitates freecholesterol uptake. J Biol Chem 2006;281:6616–6624.
23 Zhang M, Dwyer NK, Love DC, Cooney A, Comly

References
1 Altmann SW, Davis HR Jr, Zhu LJ, Yao X, Hoos LM,
Tetzloff G, et al. Niemann-Pick C1 Like 1 protein is
critical for intestinal cholesterol absorption. Science
2004;303:1201–1204.
2 Cohen JC, Pertsemlidis A, Fahmi S, Esmail S, Vega
GL, Grundy SM, et al. Multiple rare variants in
NPC1L1 associated with reduced sterol absorption andplasma low-density lipoprotein levels. Proc Natl Acad
Sci USA 2006;103:1810–1815.
3 Davies JP, Ioannou YA. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identicalto those of 3-hydroxy-3-methylglutaryl-CoA reductase
and sterol regulatory element binding protein cleavageactivating protein. J Biol Chem 2000;275:24367–24374.
4 Davies JP, Levy B, Ioannou YA. Evidence for a
Niemann-pick C (NPC) gene family: identification andcharacterization of NPC1L1. Genomics 2000;65:137–
145.
5 Davies JP, Scott C, Oishi K, Liapis A, Ioannou YA.
Inactivation of NPC1L1 causes multiple lipid transport
defects and protects against diet-induced hypercholesterolemia. J Biol Chem 2005;280:12710–12720.
6 Davis HR Jr, Zhu LJ, Hoos LM, Tetzloff G, Maguire M,
Liu J, et al. Niemann-Pick C1 Like 1 (NPC1L1) is theintestinal phytosterol and cholesterol transporter and a
key modulator of whole-body cholesterol homeostasis.
J Biol Chem 2004;279:33586–33592.
7 Dixit SS, Sleat DE, Stock AM, Lobel P. Do mammalian NPC1 and NPC2 play a role in intestinal cholesterol
absorption? Biochem J 2007;408:1–5.
8 Garcia-Calvo M, Lisnock J, Bull HG, Hawes BE,
Burnett DA, Braun MP, et al. The target of ezetimibe
is Niemann-Pick C1-Like 1 (NPC1L1). Proc Natl Acad
Sci USA 2005;102:8132–8137.
9 Ge L, Wang J, Qi W, Miao HH, Cao J, Qu YX, et al.
The cholesterol absorption inhibitor ezetimibe acts byblocking the sterol-induced internalization of NPC1L1.
Cell Metab 2008;7:508–619.
10 Infante RE, Radhakrishnan A, Abi-Mosleh L, Kinch
LN, Wang ML, Grishin NV, et al. Purified NPC1 protein: II. Localization of sterol binding to a 240-aminoacid soluble luminal loop. J Biol Chem 2008;283:1064–

35

H. Ninomiya

M, Neufeld E, et al. Cessation of rapid late endosomal
tubulovesicular trafficking in Niemann-Pick type C1
disease. Proc Natl Acad Sci USA 2001;98:4466–4471.

Received and accepted, April 1, 2010
Corresponding author: Haruaki Ninomiya, MD, PhD

36