Niemann-Pick C1 Like 1 (NPC1L1) Is the Intestinal Phytosterol andCholesterol Transporter and a Key Modulator of Whole-bodyCholesterol Homeostasis*Received for publication, May 25, 2004Published, JBC Papers in Press, June 1, 2004, DOI 10.1074/jbc.M405817200Harry R. Davis, Jr.‡§, Li-ji Zhu‡, Lizbeth M. Hoos‡, Glen Tetzloff‡, Maureen Maguire¶,Jianjun Liu¶, Xiaorui Yao‡, Sai Prasad N. Iyer‡, My-Hanh Lam储, Erik G. Lund储,Patricia A. Detmers**, Michael P. Graziano‡, and Scott W. Altmann‡From the ‡Department of Cardiovascular/Metabolic Disease and¶Department of Discovery Technologies, Schering-PloughResearch Institute, Kenilworth, New Jersey 07033 and储Departments of Cardiovascular Diseases and **Immunology,Merck Research Laboratories, P. O. Box 2000, Rahway, New Jersey 07065Niemann-Pick C1 Like 1 (NPC1L1) is a protein localizedin jejunal enterocytes that is critical for intestinal choles-terol absorption. The uptake of intestinal phytosterolsand cholesterol into absorptive enterocytes in the intes-tine is not fully defined on a molecular level, and the roleof NPC1L1 in maintaining whole body cholesterol home-ostasis is not known. NPC1L1 null mice had substantiallyreduced intestinal uptake of cholesterol and sitosterol,with dramatically reduced plasma phytosterol levels. TheNPC1L1 null mice were completely resistant to diet-in-duced hypercholesterolemia, with plasma lipoproteinand hepatic cholesterol profiles similar to those of wildtype mice treated with the cholesterol absorption inhibi-tor ezetimibe. Cholesterol/cholate feeding resulted indown-regulation of intestinal NPC1L1 mRNA expressionin wild type mice. NPC1L1 deficiency resulted in up-reg-ulation of intestinal hydroxymethylglutaryl-CoA syn-thase mRNA and an increase in intestinal cholesterol syn-thesis, down-regulation of ABCA1 mRNA, and no changein ABCG5 and ABCG8 mRNA expression. NPC1L1 is re-quired for intestinal uptake of both cholesterol and phy-tosterols and plays a major role in cholesterol homeosta-sis. Thus, NPC1L1 may be a useful drug target for thetreatment of hypercholesterolemia and sitosterolemia.Cholesterol absorption of both dietary cholesterol and cho-lesterol cleared from the liver through biliary secretion contrib-utes along with regulation of cholesterol biosynthesis to main-tain a tight control of cholesterol homeostasis. The mechanismby which cholesterol moves from the intestinal lumen into theabsorptive enterocytes lining the proximal small intestine ispoorly understood. The identification of ezetimibe as a potentselective inhibitor of intestinal cholesterol uptake and absorp-tion confirmed this mechanism as a key point of therapeuticintervention for lowering plasma cholesterol levels and indi-cated that this process is mediated by a specific transporter(1– 4). Based on the properties of ezetimibe in animal models ofcholesterol uptake, it was predicted that such a transporterwould be expressed in jejunal enterocytes and localized to thebrush border membrane, which forms the interface betweenthe intestinal lumen and the intracellular compartments re-sponsible for cholesterol esterification and packaging intochylomicrons.Through studies designed to understand the mechanism bywhich ezetimibe inhibits cholesterol absorption, we recentlyidentified Niemann-Pick C1 Like 1 (NPC1L1)1as a criticalprotein for the intestinal absorption of dietary and biliary cho-lesterol (5). NPC1L1 was identified through a genomics-bioin-formatics approach by sequencing an expression sequence tagslibrary from rat jejunum, annotating the sequences, andsearching databases for intestinal proteins with features of acholesterol transporter (5). NPC1L1 was found to be highlyexpressed in the jejunum and localized on the surface of theabsorptive enterocytes. Mice deficient in NPC1L1 exhibited asignificant reduction in cholesterol absorption, and the lowlevel of residual cholesterol absorption was insensitive toezetimibe treatment. These previous results suggested thatNPC1L1 resides in an ezetimibe-sensitive pathway responsiblefor cholesterol absorption (5).In addition to inhibiting cholesterol absorption, ezetimibereduces plasma phytosterol levels in patients with hypercho-lesterolemia, although a molecular mechanism for intestinalphytosterol uptake and absorption has yet to be established (4).Ezetimibe also effectively reduces phytosterol levels in patientswith sitosterolemia, which is caused by a mutation in the ATPbinding cassette (ABC) co-transporters, either ABCG5 orABCG8 (7). ABCG5 and ABCG8 are expressed on the apicalsurface of hepatocytes and enterocytes, and their function isrequired to export phytosterols into the bile and intestinallumen, respectively. The present study describes the role ofNPC1L1 in the selective intestinal uptake and absorption ofcholesterol and phytosterols, regulation of the expression ofintestinal NPC1L1, and other proteins related to sterol metab-olism and resistance to diet-induced hypercholesterolemia inNPC1L1-deficient mice.EXPERIMENTAL PROCEDURESMaterials—All solvents, chemicals, and sterols were purchased fromSigma-Aldrich Co. Campesterol was purchased from Supelco (Belle-fonte, PA). Platinum quantitative PCR SuperMix-UDG (catalog no.11730-017) was purchased from Invitrogen. [14C]Cholesterol ([4-14C]cholesterol, 45– 60 mCi/mmol, catalog no. NEC018) and [14C]tri-* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.§ To whom correspondence should be addressed: Schering-PloughResearch Institute, K15-2-2600, 2015 Galloping Hill Rd., Kenilworth,NJ 07033. Tel.: 908-740-3242; Fax: 908-740-3294; E-mail: [email protected] abbreviations used are: NPC1L1, Niemann-Pick C1 Like 1;HMG, hydroxymethylglutaryl; ACAT, acyl-CoA:cholesterol acyltrans-ferase; ABC, ATP binding cassette; GC, gas chromatography; MS, massspectrometry; LDL, low density lipoprotein; VLDL, very low densitylipoprotein.THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 32, Issue of August 6, pp. 33586 –33592, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.This paper is available on line at http://www.jbc.org33586 at Stanford University on November 12, 2007 www.jbc.orgDownloaded fromglyceride ([carboxy-14C]triolen, 100 mCi/mmol, catalog no. NEC 674)were purchased from PerkinElmer