Niemann-Pick C1-Like 1 Overexpression Facilitates Ezetimibe-Sensitive Cholesterol and β-Sitosterol Uptake in CaCo-2 Cells

Yamanashi, Yoshihide; Takada, Tappei; Suzuki, Hiroshi

doi:10.1124/jpet.106.114181

Cited by 119 publications

(119 citation statements)

References 20 publications

(29 reference statements)

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“…As we described, it was reported that NPC1L1 was identified as a critical factor for cholesterol absorption. 14) We then next determined the effects of various concentrations of ezetimibe on uptake of cholesterol by Caco-2 cells (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

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An Approach to Improve Intestinal Absorption of Poorly Absorbed Water-Insoluble Components <i>via</i> Niemann–Pick C1-Like 1

Takekawa

Sato

Yamaki

et al. 2016

Biological & Pharmaceutical Bulletin

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Dietary and biliary cholesterol absorption contributes to the maintenance of tight control of cholesterol homeostasis. Cholesterol is present as mixed micelles formed by bile salts and phospholipids in the intestinal lumen. Recently, Niemann-Pick C1-Like 1 (NPC1L1) transporter was identified as being critical for cholesterol absorption. However, the uptake mechanism of an enveloped substrate of NPC1L1 in whole lipid emulsion particles remains unclear. In this study, we investigated the uptake mechanism of a substrate of NPC1L1 in lipid emulsion particles. We also investigated whether these particles containing cholesterol can improve the intestinal absorption of other lipophilic components via NPC1L1. The uptake of lysophosphatidylcholine (LPC)-4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid saccinimidyl ester (BODIPY), a fluorescently labeled phospholipid, in lipid emulsion particles containing cholesterol (1 µM) was significantly increased compared to that without cholesterol in Caco-2 cells. On the other hand, its increased uptake was significantly inhibited by ezetimibe, a selective inhibitor of NPC1L1. These results suggested that not only cholesterol but also some components in lipid emulsion particles are taken up into enterocytes via NPC1L1. We also examined an approach to improve intestinal absorption of a poorly absorbed water-insoluble component, coenzyme Q10 (CoQ10), by this mechanism. The uptake of CoQ10 in lipid emulsion particles containing cholesterol was significantly increased compared to that without cholesterol. Its increased uptake was significantly inhibited by ezetimibe. Though it is still not clear whether CoQ10 is a substrate of NPC1L1, there is a potential for improvement of the absorption of poorly absorbed components by lipid emulsion particles containing cholesterol.Key words Niemann-Pick C1-Like 1; absorption; cholesterol; emulsion; coenzyme Q10Intestinal absorption is an important process in the maintenance of homeostasis in the body. Cholesterol homeostasis is a highly regulated balance of de novo synthesis, dietary cholesterol absorption, and biliary clearance and excretion. Although the cholesterol uptake mechanism in the intestinal lumen is not fully understood, the identification of ezetimibe as a selective inhibitor of intestinal cholesterol absorption suggested that these processes are mediated by a specific transport system rather than by passive diffusion.1) In 2004, Altmann et al. identified Niemann-Pick C1-Like 1 (NPC1L1) as an apically localized sterol transporter in the small intestine.2) NPC1L1 is highly expressed in the small intestine, particularly in the upper intestine.2,3) NPC1L1 protein has 13 predicted transmembrane domains and extensive N-linked glycosylation sites located within the extracellular loops and it contains a sterol sensing domain (SSD).4) It has predicted that the substrates of NPC1L1 have sterol domains and have been mainly thought to transport the substance involving the sterol structure.Cholesterol is pre...

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Section: Resultsmentioning

confidence: 99%

“…This result is roughly consistent with previous reports. 14,15) We set that ezetimibe at a concentration of 25 µM inhibited the uptake of a substrate of NPC1L1. We also assessed the presence of NPC1L1 in Caco-2 cells.…”

Section: Resultsmentioning

confidence: 99%

An Approach to Improve Intestinal Absorption of Poorly Absorbed Water-Insoluble Components <i>via</i> Niemann–Pick C1-Like 1

Takekawa

Sato

Yamaki

et al. 2016

Biological & Pharmaceutical Bulletin

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“…[2][3][4][5][6][7][8] It is also the molecule target for the new cholesterol-lowering agent, ezetimibe. 9 Human NPC1L1 is expressed mainly in the intestine and liver and is responsible for transporting cholesterol from intestinal lumen to enterocytes.…”

Section: Introductionmentioning

confidence: 99%

The g.−762T>C polymorphism of the NPC1L1 gene is common in Chinese and contributes to a higher promoter activity and higher serum cholesterol levels

et al. 2009

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Niemann-Pick type C1-like 1 (NPC1L1) protein is responsible for intestinal cholesterol absorption. The aim of the study was to identify genetic polymorphisms of the NPC1L1 gene as well as their functional significance. The method involved screening of promoter and coding regions of the NPC1L1 gene for genetic polymorphisms by direct DNA sequencing of genomic DNA from 50 individuals. Functional studies on promoter polymorphisms were performed using luciferase assay. Association between the polymorphisms and serum cholesterol levels were investigated in 224 individuals. The results showed that in total, 11 single nucleotide polymorphisms were identified. Among them, a promoter polymorphism, g.À762T4C, and a synonymous polymorphism, g.1679C4G, were common (34 and 36%, respectively). These two polymorphisms were highly linked (D¢ value¼0.7459, P-value o0.00001). For the g.À762T4C promoter polymorphism, luciferase assay in HepG2 cell line demonstrated that the À762C allele had a significantly higher promoter activity than the À762T allele (1.30 ± 0.22 vs 0.37 ± 0.06, 3.5-fold, Po0.05). We also showed that the NPC1L1 promoter activity was downregulated by cholesterol content in both genotypes. When association studies were performed, we found that À762C allele was associated with significantly higher serum total cholesterol and LDL-cholesterol content levels in a recessive model (LDL-cholesterol value¼131.2 ± 8.1 vs 116.4 ± 2.2 mg dl -1 ; total cholesterol value¼214.7 ± 9.0 mg dl -1 vs 196.9 ± 2.6, P-value o0.05, n¼224). In conclusion, the C allele at À762 position of the NPC1L1 gene was common in people of Chinese ethnicity. The À762C allele had a higher promoter activity and was associated with a higher serum total cholesterol and LDL-cholesterol level.

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“…However, although EZE-sensitive cholesterol transport correlates well with the amount of NPC1L1 expression (7,8), it is not possible to determine whether NPC1L1 functions alone or as part of a multiprotein complex [SR-B1 (9-15), CD36 (14), CD13 (9), caveolin-1/annexin-2 (16)], facilitating the transfer of cholesterol from outside the cell to internal cholesterol pools (9,15). Furthermore, it has been speculated that EZE may inhibit cholesterol transfer by binding to some of these other targets, in addition to its inhibition of NPC1L1 (9).…”

mentioning

confidence: 99%

Extracellular loop C of NPC1L1 is important for binding to ezetimibe

Weinglass¹,

Köhler²,

Schulte

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

107

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Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [ 3 H]AS. Chimeric and mutational studies indicate that high-affinity binding of W hole-body cholesterol homeostasis is maintained through three major pathways: de novo synthesis, intestinal absorption, and biliary excretion. Absorption of dietary and biliary cholesterol occurs in the proximal jejunum of the small intestine (1), and this process is blocked by ezetimibe (EZE), a drug used for the treatment of hypercholesterolemia. EZE, a potent cholesterol and phytosterol uptake inhibitor, effectively lowers circulating plasma cholesterol in humans by 15-20% (2), and its coadministration with 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitors (statins), inhibitors of cholesterol synthesis, leads to further reductions in cholesterol plasma levels (3).By searching expressed sequence tag databases for the presence of a sterol-sensing domain (SSD), a plasma membrane secretion signal, and enriched expression in intestinal enterocytes, the Niemann-Pick C1-Like 1 (NPC1L1) protein was identified in 2004 as a potential candidate gene for the EZE-sensitive pathway of cholesterol absorption (4). Mice deficient in NPC1L1 were found to have Ϸ70% reduction in sterol absorption, with the residual component being insensitive to EZE (4), suggesting that NPC1L1 is a critical component of cholesterol uptake in enterocytes (4). The use of enterocyte brush border membranes (BBMs) from several species, including NPC1L1 KO mice (5), or membranes derived from cells expressing recombinant NPC1L1, has provided strong evidence for NPC1L1 being the target to which EZE binds (5).More recently, in vitro studies have demonstrated EZE-sensitive cholesterol transport into McArdles RH7777 hepatoma (6), CaCo-2 (7), and MDCKII cells (8) overexpressing NPC1L1. However, although EZE-sensitive cholesterol transport correlates well with the amount of NPC1L1 expression (7, 8), it is not possible to determine whether NPC1L1 functions alone or as part of a multiprotein complex [SR-B1 (9-15), CD36 (14), CD13 (9), caveolin-1/annexin-2 (16)], facilitating the transfer of cholesterol from outside the cell to internal cholesterol pools (9, 15). Furthermore, it has been speculated that EZE may inhibit cholesterol transfer by binding to some of these other targets, in addition to its inhibition of NPC1L1 (9).In the present study, we attempted to determine whether EZE binds to NPC1L1 directly by purifying an EZE-NPC1L1 complex and analyzing its constituents by mass spectrometry. Quantitative analysis unambiguously demonstrated that NPC1L1 is the only prote...

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Niemann-Pick C1-Like 1 Overexpression Facilitates Ezetimibe-Sensitive Cholesterol and β-Sitosterol Uptake in CaCo-2 Cells

Cited by 119 publications

References 20 publications

An Approach to Improve Intestinal Absorption of Poorly Absorbed Water-Insoluble Components <i>via</i> Niemann–Pick C1-Like 1

An Approach to Improve Intestinal Absorption of Poorly Absorbed Water-Insoluble Components <i>via</i> Niemann–Pick C1-Like 1

The g.−762T>C polymorphism of the NPC1L1 gene is common in Chinese and contributes to a higher promoter activity and higher serum cholesterol levels

Extracellular loop C of NPC1L1 is important for binding to ezetimibe

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