Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo. The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo. Dietary cholesterol absorption in small intestine is a major way for mammals to obtain cholesterol. In intestinal lumen, cholesterol incorporates into bile salt micelles and diffuses to the brush border membrane of enterocytes (1Wang D.Q. Regulation of intestinal cholesterol absorption.Annu. Rev. Physiol. 2007; 69: 221-248Crossref PubMed Scopus (230) Google Scholar, 2Nguyen T.M. Sawyer J.K. Kelley K.L. Davis M.A. Rudel L.L. Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation.J. Lipid Res. 2012; 53: 95-104Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 3Burrier R.E. Smith A.A. McGregor D.G. Hoos L.M. Zilli D.L. Davis Jr, H.R. The effect of acyl CoA: cholesterol acyltransferase inhibition on the uptake, esterification and secretion of cholesterol by the hamster small intestine.J. Pharmacol. Exp. Ther. 1995; 272: 156-163PubMed Google Scholar, 4Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese Jr, R.V. 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Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1426) Google Scholar). In rodents, NPC1L1 is selectively expressed in the small intestine and localizes on the brush border membrane (6Altmann S.W. Davis Jr, H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1426) Google Scholar, 7Davies J.P. Scott C. Oishi K. Liapis A. Ioannou Y.A. Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia.J. Biol. Chem. 2005; 280: 12710-12720Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar–8Davis Jr, H.R. Zhu L.J. Hoos L.M. Tetzloff G. Maguire M. Liu J. Yao X. Iyer S.P. Lam M.H. Lund E.G. et al.Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.J. Biol. Chem. 2004; 279: 33586-33592Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). It has been proposed that NPC1L1 mediates cholesterol movement into enterocytes in vivo. However, direct evidence is lacking. Studies in cultured cells indicate that NPC1L1 facilitates cholesterol entering cytoplasm by vesicular endocytosis (9Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 10Wang L.J. Song B.L. Niemann–Pick C1-Like 1 and cholesterol uptake..Biochim. Biophys. Acta. 2012; 1821: 964-972Crossref PubMed Scopus (62) Google Scholar). NPC1L1 forms cholesterol-enriched membrane microdomains on plasma membrane with lipid raft proteins Flotillin-1/-2 (11Ge L. Qi W. Wang L.J. Miao H.H. Qu Y.X. Li B.L. Song B.L. Flotillins play an essential role in Niemann-Pick C1-like 1-mediated cholesterol uptake.Proc. Natl. Acad. Sci. USA. 2011; 108: 551-556Crossref PubMed Scopus (17) Google Scholar). The NPC1L1-Flotillin-cholesterol microdomains are internalized via clathrin/AP2 pathway and to the (9Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, L. Qi W. Wang L.J. Miao H.H. Qu Y.X. Li B.L. Song B.L. Flotillins play an essential role in Niemann-Pick C1-like 1-mediated cholesterol uptake.Proc. Natl. Acad. Sci. USA. 2011; 108: 551-556Crossref PubMed Scopus (17) Google Scholar). The is a cholesterol and a (9Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, S. S. M. through the Biol. PubMed Scopus Google Scholar, L. C. Miao H.H. Wang J. Li B.L. Song B.L. of in of NPC1L1 to the Biol. Chem. Full Text Full Text PDF PubMed Scopus Google M. and transport in The is a major Biol. Chem. Full Text Full Text PDF PubMed Scopus (246) Google Scholar). the cholesterol NPC1L1 moves to plasma membrane to another of cholesterol transport (9Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, L. C. Miao H.H. Wang J. Li B.L. Song B.L. of in of NPC1L1 to the Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. Li Li B.L. Song B.L. The small with Niemann-Pick C1-like 1 (NPC1L1) and movement from to plasma membrane in a Biol. Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, L. S. J.M. W. Davis M.A. Liu Rudel L.L. of NPC1L1 to the facilitates cholesterol Biol. Chem. Full Text Full Text PDF PubMed Scopus Google L. Qi W. L. Miao H.H. Li B.L. M. Song B.L. The of NPC1L1 protein binds cholesterol and essential in cholesterol Biol. Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). a cholesterol absorption blocks the internalization of NPC1L1-Flotillin-cholesterol microdomains and decreases cholesterol uptake in cultured cells (9Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, L. Qi W. Wang L.J. 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Davis M.A. Rudel L.L. Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation.J. Lipid Res. 2012; 53: 95-104Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 4Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese Jr, R.V. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice.Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (296) Google Scholar, J. Kelley K.L. Davis M.A. Sawyer J.K. Farese Jr, R.V. J.M. Rudel L.L. of ACAT2 that intestinal is to diet-induced cholesterol in the and Lipid Res. 2012; 53: Full Text Full Text PDF PubMed Scopus Google Scholar). the of intestinal cholesterol absorption in vivo. The were to and that NPC1L1 localized to the brush border of enterocytes and that ACAT2 mainly localized to the of enterocytes. ACAT1 was in enterocytes and was in Paneth cells and mesenchymal Cholesterol induced the endocytosis of partially colocalized with the subapical beneath the brush ezetimibe the internalization of NPC1L1 and cholesterol, their retention on brush mice were from ACAT1 and ACAT2 mice were from and with mice to obtain a The mice and were by mice K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese Jr, R.V. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice.Nat. Med. 2000; 6: 1341-1347Crossref PubMed Scopus (296) Google Scholar, S. S. M. R.E. J. J. Farese Jr, R.V. of the acyltransferase in evidence multiple cholesterol esterification in Natl. Acad. Sci. USA. PubMed Scopus Google Scholar). mice were on a and in a in with a from to were by the and and and were from was from was from and were from was from and were from NPC1L1 was in with of ACAT1 and ACAT2 were in with of ACAT1 and of were with was in and by with to the cholesterol mice were with cholesterol. the mice were and the were and to ezetimibe mice were with ezetimibe by for (6Altmann S.W. Davis Jr, H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1426) Google Scholar). the mice were with ezetimibe the mice were with cholesterol. an the mice were and the were The and were M. K. W. G. M.A. in transporter the intestinal 2004; PubMed Scopus Google Scholar). The of the and were of the was used for lipid and the was to and were in and in for of the were in and in and to in and 1 were blocked and in and M.H. of in in a for the of by of Exp. Med. PubMed Scopus Google Scholar). of were in the with of the and and were and were and blocked with for with were with for were were in and in for and in in were with a The were in and with in for with and S. a to in and Niemann-Pick C1 Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). The was to cholesterol absorption in mice of the intestinal absorption and of cholesterol and in the Lipid Res. Full Text PDF PubMed Google Scholar, D.Q. of intestinal cholesterol absorption by plasma and and lymph in the mouse: an of direct Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). mice were with ezetimibe for the mice were with ezetimibe the mice were with and cholesterol. were for and and were to the cholesterol absorption. The cholesterol absorption was cholesterol absorption were with and in 2 with inhibitor 1 and and for The was with membrane protein and with J. Wang J. Qi W. Miao H.H. Wang J. L. J.J. Li B.L. Song B.L. is a of and a key role in cholesterol by the of Metab. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). of protein was to and by was L.J. Wang J. Li L. Li B.L. Song B.L. of the NPC1L1 from cholesterol Biol. Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). of small intestine in were to with and for the of and 2 were and the was for 1 Then the membrane protein and were into the were via and by were and in 2 was to obtain the for was with with a The of was to that of J.J. Li Qi W. Li Li B.L. Song B.L. of by a small and and Metab. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). The of in was to a of 1 The for NPC1L1 were and The for ACAT1 were and The for ACAT2 were and from the of were with and in 1 for 1 with and for and of was and to cholesterol and with J. M. for the and of from Biol. Chem. Full Text PDF PubMed Google Scholar). into the of NPC1L1 in mice we a against of with the NPC1L1 a in the small intestine than the of However, the NPC1L1 protein was in and NPC1L1 multiple and protein is essential for we this with small The NPC1L1 protein and that was in of small intestinal with and the were to the of The of NPC1L1 and that NPC1L1 was in brush border membrane of the small intestine were to the of NPC1L1 in villi and crypts. In NPC1L1 colocalized with a marker for cells that NPC1L1 localized in the brush border membrane of enterocytes. NPC1L1 was in the crypts. The intestinal crypts of Paneth and transit-amplifying cells and Paneth cells in the transit-amplifying cells to the of the crypts J. M. M. A. J. et of cells in small intestine and by marker 2007; PubMed Scopus Google Scholar). of cells NPC1L1 localized in the and were used for cells cells and transit-amplifying and Paneth NPC1L1 was in the apical of transit-amplifying cells that to the of crypts was in cells that NPC1L1 is expressed in small It in the enterocytes and transit-amplifying cells in small intestine and is in the apical membrane. entering cholesterol is esterified to cholesterol ester on by of and in ACAT1 and ACAT2 the the C. Davis M. M. Rudel L.L. of a of acyltransferase to and intestine in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.L. cholesterol acyltransferase 1 and and in PubMed Scopus Google Scholar, S. C. Li B.L. A. of and PubMed Scopus Google Li B.L. J. Physiol. Metab. PubMed Scopus Google Scholar). the of in small and were and we the by with mice ACAT2 is selectively expressed in and small intestine in In ACAT1 is expressed are of and in of and mice were of ACAT1 in small of small and from and mice were and to by with the of small intestine from and mice were with and ACAT1 localized to mesenchymal cells of the villi and Paneth cells of the crypts. intestinal were with and The of small intestine that ACAT2 was expressed in enterocytes that ACAT2 localized to the cytoplasm of enterocytes in the apical with NPC1L1 In ACAT1 was in Paneth cells of the crypts and mesenchymal cells of the villi was in cells of the intestinal layer indicate that the major for cholesterol esterification in enterocytes is the protein and of and ACAT2 of The of and from mice were to and The protein of NPC1L1 was in and in jejunum and in In that the NPC1L1 in small intestine was of that in The ACAT2 protein and was to NPC1L1 ACAT1 was expressed in the was in than in the small intestine that the of NPC1L1 and is the small studies have shown that cholesterol the endocytosis of NPC1L1 with cholesterol from the plasma membrane to the in cultured cells (9Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). the of NPC1L1 in we in small The mice were with cholesterol, and the intestinal were to the of In the absence of cholesterol, NPC1L1 localized mainly to brush border membrane and colocalized with in mice However, the mice were with cholesterol, of were found beneath the membrane and partially colocalized with a marker of was NPC1L1 and ACAT2 that the transport of cholesterol from to is the of cholesterol, we used to cholesterol in the In the the cholesterol was and mainly on the brush However, the cholesterol became in the intestine with cholesterol Cholesterol distributed the cells in the The cholesterol in apical cytoplasm was than that in the used the to cholesterol of intestinal with the intestinal cholesterol by 70% in the mice cholesterol that NPC1L1 was from the brush border to the cholesterol absorption in small intestine Ezetimibe is a inhibitor of NPC1L1 and cholesterol absorption (6Altmann S.W. Davis Jr, H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1426) Google Scholar, R.E. W. Rudel L.L. Ioannou Y.A. J.P. L.M. L. Niemann-Pick C1-like 1 cholesterol and is a of 2007; PubMed Scopus Google Scholar). studies in cultured cells have that ezetimibe blocked the uptake of cholesterol by the endocytosis of NPC1L1 (9Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, J. L. Li B.L. Song B.L. of a key protein in cholesterol Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). the endocytosis of NPC1L1 in enterocytes be blocked by ezetimibe was In mice ezetimibe NPC1L1 was to the layer beneath the brush NPC1L1 with that were in In the mice with NPC1L1 were the brush border that the endocytosis of NPC1L1 was blocked by ezetimibe. cholesterol enterocytes by of intestinal was The cholesterol However, the from mice that cholesterol the brush border and that cholesterol was of cholesterol and of intestinal cells that the cholesterol was with ezetimibe a to the effect of the cholesterol absorption in mice was by the Ezetimibe cholesterol absorption by that ezetimibe blocks the internalization of NPC1L1 and cholesterol in small This study that dietary cholesterol the endocytosis of NPC1L1 from brush border in small intestine and that ezetimibe blocks NPC1L1 and cholesterol from entering the The and of and ACAT1 in intestine were It has been that NPC1L1 is for intestinal cholesterol absorption (6Altmann S.W. Davis Jr, H.R. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1426) Google Scholar, Jr, H.R. Zhu L.J. Hoos L.M. Tetzloff G. Maguire M. Liu J. Yao X. Iyer S.P. Lam M.H. Lund E.G. et al.Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.J. Biol. Chem. 2004; 279: 33586-33592Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, M. and role of NPC1L1 in cholesterol absorption in intestine.J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). of the were in NPC1L1 mediates cholesterol brush border membrane and how have been in vivo. studies in cultured cells have shown that with proteins mediates cholesterol uptake in a (9Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. 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However, ezetimibe was in that In the of the cultured cells and are from the in vivo. we are to in the absence of intestinal cholesterol, NPC1L1 mainly on the brush border membrane of the enterocytes and that dietary cholesterol the internalization of NPC1L1 to the of the brush border and This can be with the of ezetimibe Ezetimibe dietary cholesterol from entering cytoplasm causes the of cholesterol in plasma membrane study is for the that NPC1L1 in cholesterol entering the cytoplasm of intestinal that ezetimibe caused of that cholesterol was from entering the enterocytes Jr, H.R. Zhu L.J. Hoos L.M. Tetzloff G. Maguire M. Liu J. Yao X. Iyer S.P. Lam M.H. Lund E.G. et al.Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.J. Biol. 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Full Text PDF PubMed Google mainly and are cholesterol and to efficient cholesterol absorption. the moves cholesterol is in the intestine to efficient of NPC1L1 in jejunum and the absorption In the of NPC1L1 in It is highly expressed in protein and play essential in M. M. J. of Metab. 2012; PubMed Scopus Google Scholar). are highly expressed in and jejunum are expressed in L.L. A. protein in Biol. 2005; PubMed Scopus Google Scholar). is from that of NPC1L1 and of was in the of the small intestine absorption of and cholesterol. Dietary and inhibition to 2000; Full Text Full Text PDF PubMed Scopus Google the of and in the of the intestine more chylomicrons to and In the of intestine is and the of and be for the of cholesterol and cholesterol ester into In the and were highly expressed in jejunum and Wang H.H. Wang D.Q. Cholesterol absorption is mainly by the and and in Lipid Res. 2004; Full Text Full Text PDF PubMed Scopus Google to NPC1L1 and that the major of cholesterol absorption be the and an in of dietary cholesterol absorption: The cholesterol in intestinal can to the brush border membrane by bile salt and NPC1L1 mediates cholesterol entering enterocytes via vesicular Cholesterol is to by and esterified by The cholesterol absorption inhibitor ezetimibe binds NPC1L1 Yao X. Davis Jr, H.R. S.W. In to ezetimibe with C1 (NPC1L1) of multiple NPC1L1 Pharmacol. 2007; PubMed Scopus (58) Google Scholar, M. Liu J. A. W. W. et of NPC1L1 is important for to Natl. Acad. Sci. USA. 2008; PubMed Scopus Google and blocks cholesterol from entering the cytoplasm of enterocytes. The and for and Qi for critical of the cholesterol acyltransferase endoplasmic reticulum protein Niemann-Pick C1 like1