NPC1L1 and cholesterol transport

The polytopic transmembrane protein, Niemann-Pick C1-Like 1 (NPC1L1), is enriched in the apical membrane of small intestine absorptive enterocytes where it mediates extracellular sterol transport across the brush border membrane. It is essential for intestinal sterol absorption and is the molecular target of ezetimibe, a potent cholesterol absorption inhibitor that lowers blood cholesterol in humans. NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 may be to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake seems to be a clathrin-mediated endocytic process and is regulated by cellular cholesterol content. Recently, NPC1L1 inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, and fatty liver, in addition to atherosclerosis.     

Anacetrapib promotes reverse cholesterol transport and bulk cholesterol excretion in Syrian golden hamsters

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol (HDL-C) and lowers LDL cholesterol in dyslipidemic patients; however, the effects of ANA on cholesterol/lipoprotein metabolism in a dyslipidemic hamster model have not been demonstrated. To test whether ANA (60 mg/kg/day, 2 weeks) promoted reverse cholesterol transport (RCT), 3H-cholesterol-loaded macrophages were injected and (3)H-tracer levels were measured in HDL, liver, and feces. Compared to controls, ANA inhibited CETP (94%) and increased HDL-C (47%). 3H-tracer in HDL increased by 69% in hamsters treated with ANA, suggesting increased cholesterol efflux from macrophages to HDL. 3H-tracer in fecal cholesterol and bile acids increased by 90% and 57%, respectively, indicating increased macrophage-to-feces RCT. Mass spectrometry analysis of HDL from ANA-treated hamsters revealed an increase in free unlabeled cholesterol and CE. Furthermore, bulk cholesterol and cholic acid were increased in feces from ANA-treated hamsters. Using two independent approaches to assess cholesterol metabolism, the current study demonstrates that CETP inhibition with ANA promotes macrophage-to-feces RCT and results in increased fecal cholesterol/bile acid excretion, further supporting its development as a novel lipid therapy for the treatment of dyslipidemia and atherosclerotic vascular disease.     

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.     

Ezetimibe blocks internalization of the NPC1L1/cholesterol complex

Niemann-Pick C1-like 1 (NPC1L1) is a target for ezetimibe, a drug that blocks intestinal cholesterol absorption. A new study by Ge et al. (2008) in this issue of Cell Metabolism shows that non-lipoprotein-bound cholesterol induces endocytosis of NPC1L1 and that ezetimibe blocks the internalization of the NPC1L1/cholesterol complex. The in vivo significance of these findings is discussed.     

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption.     

Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia

NPC1L1, a recently identified relative of Niemann-Pick C1, was characterized to determine its subcellular location and potential function(s). NPC1L1 was highly expressed in HepG2 cells and localized in a subcellular vesicular compartment rich in the small GTPase Rab5. mRNA expression profiling revealed significant differences between mouse and man with highest expression found in human liver and significant expression in the small intestine. In contrast, liver expression in mouse was extremely low with mouse small intestine exhibiting the highest NPC1L1 expression. A mouse knock-out model of NPC1L1 was generated and revealed that mice lacking a functional NPC1L1 have multiple lipid transport defects. Surprisingly, lack of NPC1L1 exerts a protective effect against diet-induced hyperlipidemia. Further characterization of cell lines generated from wild-type and knock-out mice revealed that in contrast to wild-type cells, NPC1L1 cells exhibit aberrant plasma membrane uptake and subsequent transport of various lipids, including cholesterol and sphingolipids. Furthermore, lack of NPC1L1 activity causes a deregulation of caveolin transport and localization, suggesting that the observed lipid transport defects may be the indirect result of an inability of NPC1L1 null cells to properly target and/or regulate caveolin expression.     

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.     

Molecular characterization of the NPC1L1 variants identified from cholesterol low absorbers

Niemann-Pick C1-like 1 (NPC1L1) is an essential protein for dietary cholesterol absorption. Nonsynonymous (NS) variants of NPC1L1 in humans have been suggested to associate with cholesterol absorption variations. However, information concerning the characteristics and mechanism of these variants in cholesterol uptake is limited. In this study, we analyzed the cholesterol uptake ability of the 19 reported NS variants of NPC1L1 identified from cholesterol low absorbers. Among these variants, L110F, R306C, A395V, G402S, T413M, R693C, R1214H, and R1268H could partially mediate cellular cholesterol uptake and were categorized as partially dysfunctional variants. The other 11 variants including T61M, N132S, D398G, R417W, G434R, T499M, S620C, I647N, G672R, S881L, and R1108W could barely facilitate cholesterol uptake, and were classified into the severely dysfunctional group. The partially dysfunctional variants showed mild defects in one or multiple aspects of cholesterol-regulated recycling, subcellular localization, glycosylation, and protein stability. The severely dysfunctional ones displayed remarkable defects in all these aspects and were rapidly degraded through the ER-associated degradation (ERAD) pathway. In vivo analyses using adenovirus-mediated expression in mouse liver confirmed that the S881L variant failed to localize to liver canalicular membrane, and the mice showed defects in biliary cholesterol re-absorption, while the G402S variant appeared to be similar to wild-type NPC1L1 in mouse liver. This study suggests that the dysfunction of the 19 variants on cholesterol absorption is due to the impairment of recycling, subcellular localization, glycosylation, or stability of NPC1L1.     

Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane

We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann-Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained.     

Membrane topology of human NPC1L1, a key protein in enterohepatic cholesterol absorption

The Niemann-Pick C1 Like 1 (NPC1L1) is a predicted polytopic membrane protein that is critical for cholesterol absorption. NPC1L1 takes up free cholesterol into cells through vesicular endocytosis. Ezetimibe, a clinically used cholesterol absorption inhibitor, blocks the endocytosis of NPC1L1 thereby inhibiting cholesterol uptake. Human NPC1L1 is a 1,332-amino acid protein with a putative sterol-sensing domain (SSD) that shows sequence homo­logy to HMG-CoA reductase (HMGCR), Niemann-Pick C1 (NPC1), and SREBP cleavage-activating protein (SCAP). Here, we use protease protection and immunofluorescence in selectively permeabilized cells to study the topology of human NPC1L1. Our data indicate that NPC1L1 contains 13 transmembrane helices. The NH₂-terminus of NPC1L1 is in the lumen while the COOH-terminus projects to the cytosol. human NPC1L1 contains seven small cytoplasmic loops—four small and three large luminal loops—one of which has been reported to bind ezetimibe. Ezetimibe-glucuronide, the major metabolite of ezetimibe in vivo, can block the internalization of NPC1L1 and cholesterol. The membrane topology of NPC1L1 is similar to that of NPC1, and the putative SSD of NPC1L1 is oriented in the same manner as those of HMGCR, NPC1, and SCAP. The defined topology of NPC1L1 provides necessary information for further dissecting the functions of the different domains of NPC1L1.     

Rapid transient absorption and biliary secretion of enantiomeric cholesterol in hamsters

To probe the pathway and specificity of cholesterol absorption, the synthetic enantiomer of cholesterol (ent-cholesterol) and cholesterol were labeled with deuterium, gavaged into hamsters, and measured by negative ion mass spectrometry. Initial uptake of both tracers into the intestinal mucosa at 30 min was similar but cholesterol was temporarily retained there, whereas mucosal ent-cholesterol declined rapidly with concomitantly increased enrichment in both the systemic circulation and the gut lumen. In a 3 day fecal recovery study, ent-cholesterol was quantitatively recovered in the stool, whereas cholesterol absorption was 53.2%. ent-Cholesterol given by intracardiac injection was selectively secreted into bile, and the ratio of ent-cholesterol to cholesterol tracers in the gut lumen increased down the length of the small bowel, with the largest value being found in stool. ent-Cholesterol is efficiently taken up by the intestinal mucosa and undergoes transient enterohepatic recirculation, but it is quantitatively eliminated over 3 days as a result of selective secretion into bile and selective enrichment within the lumen of the intestine. These findings suggest that cholesterol absorption is structurally specific and likely to be mediated by enantiospecific cellular proteins.     

Social defeat and footshock increase body mass and adiposity in male Syrian hamsters

Obesity is a world-wide epidemic, and many factors, including stress, have been linked to this growing trend. After social stress (i.e., defeat), subordinate laboratory rats and most laboratory mice become hypophagic and, subsequently, lose body mass; the opposite is true of subordinate Syrian hamsters. After social defeat, Syrian hamsters become hyperphagic and gain body mass compared with nonstressed controls. It is unknown whether this increase in body mass and food intake is limited to subordinate hamsters. In experiment 1, we asked, do dominant hamsters increase food intake, body mass, and adiposity after an agonistic encounter? Subordinate hamsters increased food intake and body mass compared with nonstressed controls. Although there was no difference in food intake or absolute body mass between dominant and nonstressed control animals, cumulative body mass gain was significantly higher in dominant than in nonstressed control animals. Total carcass lipid and white adipose tissue (WAT) (i.e., retroperitoneal and epididymal WAT) masses were significantly increased in subordinate, but not dominant, hamsters compared with nonstressed controls. In experiment 2, we asked, does footshock stress increase food intake, body mass, and adiposity. Hamsters exposed to defeat, but not footshock stress, increased food intake relative to nonstressed controls. In animals exposed to defeat or footshock stress, body mass, as well as mesenteric WAT mass, increased compared with nonstressed controls. Collectively, these data demonstrate that social and nonsocial stressors increase body and lipid mass in male hamsters, suggesting that this species may prove useful for studying the physiology of stress-induced obesity in some humans.     

Substituted oxazolidinones as novel NPC1L1 ligands for the inhibition of cholesterol absorption

Cholesterol absorption inhibition (CAI) represents an important treatment option for hypercholesterolemia. Herein, we report the design and evaluation of a series of substituted oxazolidinones as ligands for the Niemann Pick C1 Like 1 (NPC1L1) protein, a key mediator of cholesterol transport. Novel analogs were initially evaluated in a brush border membrane NPC1L1 binding assay; subsequently, promising compounds were evaluated in vivo for acute inhibition of cholesterol absorption. These studies identified analogs with low micromolar NPC1L1 binding affinity and acute in vivo efficacy of >50% absorption inhibition at 3mg/kg.     

Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake

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.