Niemann-Pick C1-Like 1 Mediates -Tocopherol Transport

Dietary lipids and fat-soluble micronutrients are solubilized in mixed micelles and absorbed in the small intestine. Based on an assumption that cholesterol and other fat-soluble molecules share a number of transport mechanisms and the fact that Niemann-Pick C1-like 1 (NPC1L1) is critical for intestinal cholesterol absorption, we hypothesized that some fat-soluble molecules may be transported by NPC1L1. To investigate this hypothesis, we compared the cellular uptake and inhibitory effects of ezetimibe, the molecular target of which is NPC1L1, between cholesterol and some fat-soluble molecules using rat NPC1L1-overexpressing Caco-2 cells. The in vitro analysis suggested that NPC1L1 mediates the uptake of -tocopherol (vitamin E) in an ezetimibe-sensitive manner as well as the uptake of cholesterol but does not mediate the uptake of retinol (vitamin A) or cyclosporin A. To confirm the ezetimibe-sensitive uptake of -tocopherol in vivo, we performed an in vivo absorption study using rats and the results suggested a physiologically significant role of NPC1L1-mediated -tocopherol absorption. Furthermore, using human NPC1L1 overexpression system, we demonstrated that both cholesterol and -tocopherol uptake was also significantly increased by the overexpression of human NPC1L1 and ezetimibe inhibited their uptake. Mutual inhibition studies of cholesterol and -tocopherol in human NPC1L1-mediated uptake revealed the inhibitory effect of cholesterol and the stimulatory effect of -tocopherol on the NPC1L1-mediated transport of both substrates. The present data suggest, for the first time, that NPC1L1 has the ability to transport -tocopherol and that ezetimibe is able to inhibit the intestinal absorption of -tocopherol. Intestinal absorption is an important process in the maintenance of cholesterol homeostasis in the body. Although the mechanism of cholesterol uptake from the intestinal lumen is poorly understood, the identification of ezetimibe as a potent selective inhibitor of intestinal cholesterol absorption suggests that this process is mediated by a specific transport system (van Heek et al., 2001; Patel et al., 2003). Through studies designed to understand the mechanism by which ezetimibe inhibits cholesterol absorption, Niemann-Pick C1like 1 (NPC1L1) was identified as a critical factor for intestinal cholesterol absorption (Altmann et al., 2004), because NPC1L1 knockout mice exhibited reduction in intestinal cholesterol absorption, and the low level of remaining cholesterol absorption was insensitive to ezetimibe treatment (Altmann et al., 2004; Davis et al., 2004). These results, together with the finding that NPC1L1 is expressed in the apical membrane of the small intestine, particularly in the jejunum, where most sterol absorption takes place (Altmann et al., 2004; Davis et al., 2004; Sané et al., 2006), suggest that NPC1L1 is involved in the intestinal absorption of cholesterol and is a target of ezetimibe. In our previous study, we established a Caco-2 cell line that stably overexpresses rat NPC1L1 (Yamanashi et al., 2007). Using these cells, we demonstrated that the uptake of cholesterol and plant sterols from micelles was increased by rat NPC1L1 overexpression and that the uptake was inhibited by ezetimibe. Under physiological conditions, cholesterol is present as mixed micelles formed by bile salts and phospholipids in the intestinal lumen. Other fat-soluble micronutrients, such as fat-soluble vitamins and parts of fat-soluble drugs, are also known to be solubilized in mixed micelles and then absorbed in the intestine. Cholesterol and other fatsoluble molecules, which also exhibit absorption from biliary micelles, may be taken up in the intestinal lumen via the shared pathway. In the current study, we focused on the possibility that NPC1L1 mediates the uptake of fat-soluble micronutrients, which are incorporated into micelles in addition to the uptake of cholesterol. To test this possibility, we examined the inhibitory effect of ezetimibe on the uptake of -tocopherol (vitamin E), retinol (vitamin A) and cyclosporin A as examples of fat-soluble micronutrients and drugs by using rat K.N. and T.T. contributed equally to this work. This work was supported by Grant-in-Aid 17081006 for Scientific Research on Priority Areas Transportsome from the Ministry of Education, Science, and Culture of Japan. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.043034. ABBREVIATIONS: NPC1L1, Niemann-Pick C1-like 1; BSA, bovine serum albumin; TBS-T, Tris-buffered saline/Tween 20; HA, hemagglutinin; PBS, phosphate-buffered saline; ANOVA, analysis of variance. 0026-895X/08/7401-42–49$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 1 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 43034/3353588 Mol Pharmacol 74:42–49, 2008 Printed in U.S.A. 42 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from NPC1L1-overexpressing Caco-2 cells. In addition, to confirm ezetimibe-sensitive uptake of -tocopherol in vivo, we performed an in vivo absorption study using rats. Furthermore, we constructed human NPC1L1 overexpressing Caco-2 cells and examined the transport activity of human NPC1L1 for cholesterol and -tocopherol. The present study suggests that the uptake of -tocopherol, the most relevant form of vitamin E, is partly mediated by NPC1L1 in enterocytes, and its transport is inhibited by ezetimibe. Vitamin E is used clinically for the prevention of arteriosclerosis, which involves cholesterol accumulation. NPC1L1, which is known to be a cholesterol transporter, might protect the body from the harmful effects of excess cholesterol by dual transport of cholesterol and -tocopherol. Materials and Methods Materials. [1 ,2 -H]Cholesterol (44.0 Ci/mmol), D-[5-methylC]tocopherol (57.0 mCi/mmol), and [N-methyl-butenyl-methylthreonine-H]cyclosporin A (7.00 Ci/mmol) were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). [4-C]Cholesterol (53 mCi/mmol) and [11,12-H]retinol (45.5 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Waltham, MA). Sodium taurocholate was obtained from Sigma Aldrich (St. Louis, MO), whereas ezetimibe was obtained from Sequoia Research Products Ltd (Pangbourne, UK). Caco-2 cells were obtained from Cell Bank, RIKEN BioResource Center (Ibaraki, Japan). pcDNA3.1( ) vector was obtained from Invitrogen (Carlsbad, CA). BamHI and EcoRI restriction enzymes were obtained from Takara (Shiga, Japan). Anti HA-tag antibody [HA-probe (Y-11)] was obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). All other chemicals used were commercially available and of reagent grade. Male Wistar rats were obtained from SLC Inc. (Shizuoka, Japan). All animals used in this study were housed in temperatureand humidity-controlled animal cages with a 12-h dark/light cycle and with free access to water and standard animal chow (MF, Oriental Yeast, Tokyo, Japan). All experiments involving rats were conducted by using protocols approved by the Animal Studies Committee of the University of Tokyo. Construction of Human NPC1L1-Overexpressed Caco-2 Cells. Human NPC1L1 cDNA was amplified by polymerase chain reaction from Caco-2 mRNA. The complete NPC1L1 cDNA (GenBank accession number AY437865) was amplified with the BamHI site at the 5 end, and with the EcoRI site and HA tag (YPYDVPDYA) sequence attached to the 3 -end by polymerase chain reaction and then inserted into pcDNA3.1( ) vector plasmid. NPC1L1 in pcDNA3.1( ) was transfected into Caco-2 cells grown on a six-well plate with FuGene 6 (Roche Diagnostics Corporation, Indianapolis, IN) according to the user’s manual. Then, Caco-2 cells were selected by culturing in the presence of 500 g/ml G418 sulfate (Nacalai Tesque, Osaka, Japan) and 16 g/ml ezetimibe. Cell Culture. Caco-2 cells stably transfected with rat NPC1L1-HA cDNA (rat NPC1L1 cells; Yamanashi et al., 2007), human NPC1L1-HA cDNA (human NPC1L1 cells), or pcDNA3.1( ) vector (control cells) were cultured in Eagle’s minimal essential medium (Nacalai Tesque) with 10% fetal bovine serum (Biological Industries, Beit Haemek, Israel), 100 units/ml penicillin and streptomycin (Nacalai Tesque), 1% nonessential amino acid (Invitrogen, Tokyo, Japan) and G418 sulfate (500 g/ml) at 37°C in an atmosphere supplemented with 5% CO2. Western Blot Analysis. For Western blotting, the cell pellet was resuspended in 1 ml of buffer A (50 mM Tris-HCl, pH 7.4, containing 2 mM EDTA, 2 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 1 g/ml pepstatin, and 5 g/ml aprotinin) with mild sonication. After centrifugation (1500g for 15 min), the supernatant was recentrifuged (20,000g for 60 min). The crude membrane fraction was resuspended in buffer A and stored at 80°C before being used for Western blot analysis. The protein concentrations were determined by the method of Lowry (Lowry et al., 1951), with bovine Cholesterol uptake (μl/mg protein) A