Cholesterol-regulated Translocation of NPC1L1 to the Cell Surface Facilitates Free Cholesterol Uptake

University of Texas at Dallas, Richardson, Texas, United States
Journal of Biological Chemistry (Impact Factor: 4.6). 04/2006; 281(10):6616-24. DOI: 10.1074/jbc.M511123200
Source: PubMed

ABSTRACT Although NPC1L1 is required for intestinal cholesterol absorption, data demonstrating mechanisms by which this protein facilitates the process are few. In this study, a hepatoma cell line stably expressing human NPC1L1 was established, and cholesterol uptake was studied. A relationship between NPC1L1 intracellular trafficking and cholesterol uptake was apparent. At steady state, NPC1L1 proteins localized predominantly to the transferrin-positive endocytic recycling compartment, where free cholesterol also accumulated as revealed by filipin staining. Interestingly, acute cholesterol depletion induced with methyl-beta-cyclodextrin stimulated relocation of NPC1L1 to the plasma membrane, preferentially to a newly formed "apical-like" subdomain. This translocation was associated with a remarkable increase in cellular cholesterol uptake, which in turn was dose-dependently inhibited by ezetimibe, a novel cholesterol absorption inhibitor that specifically binds to NPC1L1. These findings define a cholesterol-regulated endocytic recycling of NPC1L1 as a novel mechanism regulating cellular cholesterol uptake.

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    • "These findings, however, are in agreement with a previous study reported the relocation of NPC1L1 to the cell membrane after cholesterol depletion by methyl-β-cyclodextrin (Yu et al., 2006). This translocation was associated with an increase in cellular cholesterol uptake which was inhibited by ezetimibe (Yu et al., 2006). "
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    ABSTRACT: The known mechanism by which orlistat decreases the absorption of dietary cholesterol is by inhibition of intestinal lipases. The aim of this study was to investigate the ability of orlistat to limit cholesterol absorption by inhibition of the cholesterol transport protein Niemann-Pick C1-like 1 (NPC1L1) as another mechanism of action. In situ rat intestinal perfusion studies were conducted to study the effect of orlistat on jejunal cholesterol absorption. Inhibition kinetic parameters were calculated from in vitro inhibition studies using Caco2 and NPC1L1 transfected cell lines. The in situ studies demonstrated that intestinal perfusion of orlistat (100µM) was able to reduce cholesterol absorption by three-fold when compared to control (i.e. in the absence of orlistat, P<0.01). In vitro studies using Caco2 cells demonstrated orlistat to reduce the cellular uptake of cholesterol by 30%. Additionally, orlistat reduced the cellular uptake of cholesterol in dose dependent manner in NPC1L1 transfected cell line with an IC50=1.2µM. Lineweaver-Burk plot indicated a noncompetitive inhibition of NPC1L1 by orlistat. Beside the already established mechanism by which orlistat reduces the absorption of cholesterol, we demonstrated for the first time that orlistat limits cholesterol absorption by the inhibition of NPC1L1 transport protein. Copyright © 2015. Published by Elsevier B.V.
    European journal of pharmacology 06/2015; 762. DOI:10.1016/j.ejphar.2015.05.060 · 2.68 Impact Factor
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    • "Studies on hepatoma cells have revealed that NPC1L1 is predominantly localized to intracellular components but relocated to the plasma membrane when acute cholesterol depletion via MβCD occurred (Yu et al. 2006). The NPC1L1-mediated cholesterol uptake seems to occur via a cholesterol-regulated clathrin-dependent endocytosis (Betters and Yu 2010; Yu et al. 2006). NPC1L1 mRNA has been found with highest values in the intestine with variant expression in rodent and human liver indicating species differences; other tissues such as the lung or brain also expressed NPC1L1 but at a relatively low level (Davies et al. 2000, 2005; Pramfalk et al. 2011). "
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    • "NPC1L1 is a transporter protein, which mediates cholesterol and plant sterol absorption from intestinal micelles into the enterocyte [5]. It was believed to reside strictly at the apical intestinal membrane, although it is becoming clear that NPC1L1 might have additional intracellular locations and functions [6] [7] [8]. NPC1L1 is the molecular target of ezetimibe [5], which is used as a cholesterol lowering agent, although its exact function and targets are not completely elucidated [9] [10]. "
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