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Niemann-Pick C1 Like 1 Protein Is Critical for Intestinal Cholesterol Absorption

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Science  20 Feb 2004:
Vol. 303, Issue 5661, pp. 1201-1204
DOI: 10.1126/science.1093131

Abstract

Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.

Whole-body cholesterol homeostasis is a highly regulated balance of de novo synthesis, dietary cholesterol absorption, and biliary clearance and excretion. The cholesterol biosynthetic and clearance pathways are well defined. An understanding of the biosynthetic pathway has led to the development of the statin class of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors for the management of hypercholesterolemia and associated cardiovascular disease (1). In contrast, the mechanisms controlling cholesterol absorption are less well understood. The adenosine triphosphate (ATP)–binding cassette (ABC) A1 transporter and scavenger receptor type B1 (SR-BI) were postulated to play a role in intestinal cholesterol absorption (24), but targeted inactivation of these genes in mice had no effect on cholesterol uptake (57). The ABCG5 and ABCG8 transporters, which are defective in the sterol absorption disease sitosterolemia, provide an apparatus for efficient shunting of sterols away from the transfer pathway directing the production of cholesteryl esters by acyl-CoA cholesterol acyl transferase 2 (ACAT2) (810). However, none of these mechanisms appear to be involved in the initial uptake of cholesterol.

In humans, cholesterol absorption occurs in the proximal jejunum of the small intestine, where both dietary cholesterol and biliary cholesterol are available for uptake from the intestinal lumen. The second-order kinetics of cholesterol absorption (11), its sterol specificity (12, 13), and its inhibition by drugs such as ezetimibe (14) all suggest that this process is mediated by a specific transport protein(s). However, the identity of this putative cholesterol transporter has remained elusive.

To identify genes involved in cholesterol uptake, we used a genomic-bioinformatics approach. Because sequences from gastrointestinal tissues are poorly represented in the public sequence databases, we prepared two cDNA libraries for sequencing, one from rat jejunum mucosal scrapings and the second from jejunum enterocytes isolated by laser capture microdissection (15). The ∼16,500 expressed sequence tags (ESTs) derived from these libraries were combined with all available public rat ESTs and were annotated by cross-referencing the rat sequences with both mouse and human data. This sequence database was analyzed for all transcripts containing features anticipated in a cholesterol transporter, i.e., sequences predictive of transmembrane domains, extracellular signal peptides, and N-linked glycosylation sites as well as known cholesterol interacting motifs such as sterol-sensing domain (1618). Only one credible candidate gene emerged from this analysis: the rat homolog of NPC1L1 (19).

Human NPC1L1 has several of the predicted features of a plasma membrane–expressed transporter including a secretion signal, 13 putative transmembrane domains and extensive potential N-linked glycosylation sites located within the extracellular loops of the protein. Furthermore, this protein contains a sterol-sensing domain (fig. S1), which is present in other key regulators of cholesterol homeostasis including HMG-CoA reductase (20), SREBP cleavage-activating protein (SCAP) (16), PATCHED (21), and Niemann-Pick C1 (NPC1) (22).

NPC1L1 has ∼50% amino acid homology to NPC1 (19), a gene that is defective in the cholesterol storage disease Niemann-Pick type C (NP-C) and functions in intracellular cholesterol trafficking (22). However, in contrast to NPC1 mRNA, which is abundantly expressed and widely distributed in many tissues (23), NPC1L1 mRNA is expressed at low levels and is enriched in the gastrointestinal tract. BlastN and E-Northern analyses of the rat, mouse, and human EST databases were used to estimate the abundance and tissue distribution of the NPC1L1 mRNA (GenBank accession numbers AY437865, AY437866, and AY437867). In the rat, only 4 of 289,849 available ESTs— three of which were from the jejunum libraries described above—encoded NPC1L1. In the mouse, 10 of 2,170,430 ESTs encoded NPC1L1, 6 of which were from intestine, 2 from cecum, and 2 from embryo. The human EST database contained 6 NPC1L1 ESTs out of 9,274,986 total (2 from pancreas, 2 colon, 1 gallbladder, and 1 fetal liver or spleen).

We assessed NPC1L1 mRNA expression in 15 tissues from rat, mouse, and human using both quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) and microarray hybridization (Fig. 1A). Northern blot analysis was inconclusive because of the low levels of NPC1L1 mRNA and because NPC1 mRNA cross-reacted with the labeled probe (24). In all three species, small intestine showed the highest level of mRNA expression. Expression in other tissues including gallbladder, liver, testis, and stomach was also observed. Because cholesterol absorption occurs predominantly in the duodenum and proximal jejunum, with little absorption from ileal segments of the intestine (25, 26), we investigated NPC1L1 mRNA expression along the duodenum-ileum axis. The levels of NPC1L1 mRNA varied in different segments of rat intestine, with peak expression in the proximal jejunum (Fig. 1B). Comparison of relative levels of NPC1L1 protein by Western blot reiterated the distribution pattern seen with the mRNA (Fig. 1B). Furthermore, NPC1L1 mRNA and protein expression in the jejunum was confined to the enterocyte, as demonstrated by in situ hybridization (Fig. 2A) and immunohistochemistry (Fig. 2B). Both techniques revealed discrete localization of NPC1L1 to the epithelial layer bordering the luminal space (arrows) along the crypt-villus axis. Under high magnification, NPC1L1 protein expression in the enterocyte was observed closest to the luminal space (Fig. 2B, arrow). Western blot analysis of whole enterocytes prepared from discrete segments of the intestine showed NPC1L1 expression in enterocytes from the proximal (jejunum) but not in the distal (ileum) region (Fig. 2C). Subfractionation of brush border membranes from proximal enterocytes suggested considerable association with the apical membrane fraction (Fig. 2C).

Fig. 1.

(A) Tissue distribution of NPC1L1 mRNA. Rat and mouse tissue samples were evaluated by quantitative RT-PCR; human mRNA was measured by quantitative microarray using multiple tissue samples hybridization to the Affymetrix HG-U95 chip (Gene Logic, Gaithersburg, MD). Tissue with the most abundant signal was set to a normalized value of 100 arbitrary units. (B) Localization of mRNA and protein along the duodenum-ileum axis of the rat small intestine. Separate animals were used for each set of measurements. The small intestine from the pyloric valve to the ileacolic valve was removed, flushed, and divided into 10 segments of equal length (∼10 cm) for purification of mucosal mRNA or isolation of epithelial cells (31). Equal amounts of mRNA from each segment were subjected to quantitative RT-PCR (top), and NPC1L1 signal was normalized with the enterocyte-specific marker villin. Equal amounts of total protein from each segment were analyzed by Western blot analysis (bottom).

Fig. 2.

(A) Cell-specific expression of NPC1L1 mRNA in the rat jejunum. Bright field micrographs of tissue sections labeled with rat NPC1L1 anti-sense (top panels) and sense (bottom panels) cRNA probes. Arrows indicate luminal space. Bars, 200 μm (left panels) and 100 μm (right panels). (B) Cell-specific expression of NPC1L1 protein in the rat jejunum. Immunohistochemistry using immune serum A0715 (top panels) and pre-immune control (bottom panels). NPC1L1 protein staining (red) and counterstained with hematoxylin (blue). Bars: left, 100 μm; right, 25 μm. (C) Localization of NPC1L1 protein expression by Western blot analysis using immune serum A1801. Isolated enterocytes from proximal 20 cm (P) and distal 20 cm (D) segments of rat small intestine as well as brush border membranes (BB) purified from proximal enterocytes. Equal amounts of total protein from all three samples are used for this comparison.

To investigate the role of NPC1L1 in cholesterol absorption in vivo, we characterized NPC1L1-null (–/–) mice [produced by Deltagen Inc. (Palo Alto, CA)]. Embryonic stem cells derived from the 129/OlaHsd mouse sub-strain were used to generate chimeric mice. F1 mice were generated by breeding with C57BL/6 females, and F2 homozygous (–/–) mutant mice were produced by intercrossing F1 heterozygous (+/–) males and females. Both wild-type (+/+) and (+/–) littermates exhibited NPC1L1 mRNA and protein expression in jejunal enterocytes, whereas (–/–) mice showed no detectable NPC1L1 expression (Fig. 3). Physical examination of the (–/–) mice indicated that NPC1L1 deletion had no detrimental effect on development, fertility, or any hematological or plasma parameters. At necropsy, the (–/–) mice were macroscopically and histologically normal. Intestinal morphology was normal in the (–/–) mice, with no lipid accumulation in the enterocytes. No differences were found in plasma cholesterol and triglyceride levels among the (+/+), (+/–), and (–/–) mice (table S1). Hepatic lipids were also similar, with the exception of significantly reduced cholesteryl ester levels in the (–/–) mice compared to (+/+) (table S1).

Fig. 3.

Intestinal expression of NPC1L1 mRNA and protein in (+/+), (+/–), and (–/–) mice. (A) Small intestine mRNA was isolated and pooled from mucosal scrapings from each genotype (n = 2 mice). Equal amounts of mRNA were subjected to oligo-dT primed reverse-transcriptase followed by 40 cycles of PCR using mouse NPC1L1-specific oligomers. Reaction products analyzed by agarose gel electrophoresis are compared with a full-length mouse NPC1L1 cDNA control plasmid (C). (B) Jejunal enterocytes were isolated from two mice of each genotype (31). Total protein from each sample was subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot with immune serum A0868 or with antibody to α-tubulin control monoclonal B-5-2-1 (Sigma-Aldrich, Milwaukee, WI).

We next compared the uptake of orally administered radiolabeled cholesterol in NPC1L1 (–/–), (+/–), and age-matched (+/+) mice (Fig. 4). The fraction of cholesterol absorbed, as evaluated by the fecal dual isotope technique, was similar in (+/+) and (+/–) mice fed a chow diet (Fig. 4A). The (+/–) mice absorbed 45 ± 4% and (+/+) mice absorbed 51 ± 3% of an oral dose of 14C-cholesterol. In contrast, the NPC1L1 (–/–) mice absorbed only 15.6 ± 0.4% of the 14C-cholesterol, a 69% reduction in comparison to (+/+) mice (P < 0.001). A similar reduction has been observed in mice lacking the bile acid synthetic enzymes cholesterol 7 alpha-hydroxylase (Cyp7α) or sterol 27-hydroxylase (Cyp27) (27, 28); however, dietary supplementation of bile salts (via cholic acid) restored cholesterol absorption in these mutant mice to wild-type levels. To determine whether the reduction in cholesterol absorption in the NPC1L1 (–/–) mice involved alterations in the bile acid pathway, we measured cholesterol absorption in mice fed a diet containing 0.1% cholic acid (Fig. 4C). As expected from the bile salt addition to the diet, the fraction of cholesterol absorbed was increased in cholic acidfed (+/+) (66.2 ± 3%) and (+/–) mice (52.5 ± 4%) compared with mice fed a chow diet (P < 0.05) (Fig. 4, A versus C). In contrast, cholic acid supplementation of the diet had no effect on 14C-cholesterol absorption in NPC1L1 (–/–) mice (Fig. 4, A versus C), indicating that the reduced cholesterol absorption observed in these mice was not caused by an alteration in the bile acid pathway. The 15% residual cholesterol absorption remaining in the NPC1L1 (–/–) mice may be the result of a nonspecific adsorption in the gastrointestinal tract or of a minor uptake mechanism exposed by elimination of the principal mechanism.

Fig. 4.

Cholesterol absorption in NPC1L1 knockout mice. NPC1L1 (–/–) (white bars), (+/–) (hatched bars), and age-matched (+/+) (black bars) mice were fed a standard chow diet (A and B) or a diet containing 0.1% sodium cholate (C and D) for 5 days, then orally gavaged with [14C]-cholesterol and [3H]-sitostanol in 0.1 ml corn oil. Groups of NPC1L1 (–/–) (gray bars) and (+/+) (striped bars) [(B) and (D)] mice were also treated with ezetimibe (10 mg/kg) (32) once daily (n = 4 or 5 mice per group, with a minimum of 2 male and female mice per group). Cholesterol absorption was determined by the fecal ratio method (48-hour fecal collection), as previously described (7). Acute 2-hour [14C]-cholesterol intestinal uptake and absorption was determined in female NPC1L1 (–/–) and age-matched (+/+) mice (n = 5 mice per group) (E and F). Values are mean ± SEM. *P < 0.001 compared with wild-type mice (+/+) and heterozygous (+/–) mice groups; **P < 0.001 and ***P < 0.05 compared with +/+ only.

The azetidinone drug, ezetimibe, inhibits the absorption of dietary and biliary cholesterol in rodents and humans and has recently been approved for cholesterol lowering in patients with hypercholesterolemia. In (+/+) mice, ezetimibe lowered cholesterol absorption by 68 to 73% (Fig. 4, B and D). This level of cholesterol absorption was similar to that seen in NPC1L1 (–/–) mice not treated with ezetimibe (Fig. 4A). Interestingly, ezetimibe caused no further reduction in cholesterol absorption in the NPC1L1 (–/–) mice (Fig. 4, B and D), suggesting that NPC1L1 plays an essential role in the ezetimibe-sensitive cholesterol absorption pathway. Efforts to demonstrate direct binding of ezetimibe to NPC1L1 using radiolabeled (29) and fluorescent (7, 30) analogs have not been successful, however.

Because ezetimibe inhibits the uptake of cholesterol into enterocytes and its absorption, we also measured acute intestinal cholesterol uptake in the NPC1L1 (–/–) mice. In this 2-hour assay, cholesterol absorption was reduced by 86% and intestinal cholesterol uptake inhibited by 72%, with substantially more cholesterol remaining in the lumen of the intestine for excretion in the (–/–) mice compared with (+/+) mice (Fig. 4, E and F). Triglyceride absorption was not changed in the NPC1L1 (–/–) mice (table S1). The selective reduction in enterocyte cholesterol uptake resulted in a compensatory 3.3-fold up-regulation of the mRNA encoding the cholesterol synthesis enzyme HMG CoA synthase in the intestine, without significantly affecting ABCG5 or ABCG8 mRNA expression (fig. S2). The reduced delivery of intestinal cholesterol to liver also caused an up-regulation of hepatic HMG CoA synthase mRNA by ∼3.8-fold (fig. S2), suggesting that increased hepatic cholesterol synthesis is responsible for the normal plasma cholesterol levels in these mice (table S1).

These data indicate that NPC1L1 is critical for the uptake of cholesterol across the plasma membrane of the intestinal enterocyte. Our initial attempts to reconstitute cholesterol transport in nonenterocyte cells by overexpression of NPC1L1 have been unsuccessful, suggesting that additional subunits or cofactors in the jejunal enterocyte may be required to reconstitute an active cholesterol transporter. The complete insensitivity of NPC1L1-null mice to the cholesterol absorption inhibitor ezetimibe suggests that NPC1L1 or an associated protein may be the molecular target of this drug.

Supporting Online Material

www.sciencemag.org/cgi/content/full/303/5661/1201/DC1

Materials and Methods

Figs. S1 to S3

Table S1

References

References and Notes

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