Cholesterol homeostasis by the intestine: lessons from Niemann-Pick C1 Like 1 [NPC1L1)

Ezetimibe is a selective cholesterol absorption inhibitor, which potently inhibits the uptake and absorption of biliary and dietary cholesterol from the small intestine without affecting the absorption of fat-soluble vitamins, triglycerides or bile acids. Identification and characterization of Niemann-Pick C1 Like 1 (NPC1L1) has established NPC1L1 as an essential protein in the intestinal cholesterol absorption process. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that ezetimibe specifically binds to NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice were resistant to diet-induced hypercholesterolemia, and when crossed with apoE null mice, were completely resistant to the development of atherosclerosis. In Npc1l1/apoE null mice or apoE null mice treated with ezetimibe plasma cholesterol levels were reduced primarily in the apoB48 containing chylomicron remnant lipoproteins relative to untreated apoE null mice. SR-B1 has been proposed to play a role in intestinal cholesterol uptake, but in Npc1l1/SR-B1 double null mice intestinal cholesterol absorption was not different than Npc1l1 null alone mice. Therefore, NPC1L1 is the critical intestinal sterol transporter which influences whole body cholesterol homeostasis, and is the molecular target of ezetimibe.     

Extracellular loop C of NPC1L1 is important for binding to ezetimibe

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 [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.     

Zetia: inhibition of Niemann-Pick C1 Like 1 (NPC1L1) to reduce intestinal cholesterol absorption and treat hyperlipidemia

Zetia (ezetimibe) is a selective cholesterol absorption inhibitor, which potently inhibits the absorption of biliary and dietary cholesterol from the small intestine without affecting the absorption of fat-soluble vitamins, triglycerides or bile acids. Ezetimibe reduces the small intestinal enterocyte uptake and absorption of cholesterol by binding to Niemann-Pick C1 Like 1 (NPC1L1), which keeps cholesterol in the intestinal lumen for excretion. Ezetimibe undergoes glucuronidation to a single metabolite and localizes at the intestinal wall, where it binds with higher affinity for NPC1L1 than ezetimibe to prevent cholesterol absorption. Enterohepatic recirculation of ezetimibe and/or its glucuronide ensures repeated delivery to the intestinal site of action and limited peripheral exposure. Ezetimibe has no effect on the activity of major drug metabolizing enzymes (CYP450), which reduces any potential drug-drug interactions with other medications. Ezetimibe (10 mg/day) was found to inhibit cholesterol absorption by an average of 54% in hypercholesterolemic individuals and by 58% in vegetarians. Ezetimibe alone reduced plasma total and LDL-Cholesterol (18%) levels in patients with primary hypercholesterolemia. When ezetimibe was added to on-going statin treatment, an additional 25% reduction in LDL-C was found in patients with primary hypercholesterolemia and an additional 21% reduction in LDL-C in homozygous familial hypercholesterolemia. Ezetimibe in combination with statins produces additional reductions in plasma cholesterol levels and allows for more patients to achieve their LDL-C goals.     

NPC1L1: evolution from pharmacological target to physiological sterol transporter

Niemann-Pick C1-like 1 protein (NPC1L1) was recently shown to be the molecular target of the cholesterol absorption inhibitor class of drugs, of which ezetimibe is the first widely used member. Since its discovery, NPC1L1 has also been shown to play a focal physiological role in intestinal absorption of sterols, including plant sterols and cholesterol. Evidence in support of this new metabolic pathway has been garnered not only through human, animal, and cell studies of function but also through the use of human genetics as an approach to study the association of NPC1L1 sequence variation with metabolic and drug-response phenotypes. The example of NPC1L1 shows how the elucidation of a pharmacological target can serve as a means to gain understanding of a key physiological pathway.     

Genetic demonstration of intestinal NPC1L1 as a major determinant of hepatic cholesterol and blood atherogenic lipoprotein levels

Objective: The correlation between intestinal cholesterol absorption values and plasma low-density lipoprotein-cholesterol (LDL-C) levels remains controversial. Niemann-Pick-C1-Like 1 (NPC1L1) is essential for intestinal cholesterol absorption, and is the target of ezetimibe, a cholesterol absorption inhibitor. However, studies with NPC1L1 knockout mice or ezetimibe cannot definitively clarify this correlation because NPC1L1 expression is not restricted to intestine in humans and mice. In this study we sought to genetically address this issue. Methods and results: We developed a mouse model that lacks endogenous (NPC1L1) and LDL receptor (LDLR) (DKO), but transgenically expresses human NPC1L1 in gastrointestinal tract only (DKO/L1^IntOnly mice). Our novel model eliminated potential effects of non-intestinal NPC1L1 on cholesterol homeostasis. We found that human NPC1L1 was localized at the intestinal brush border membrane of DKO/L1^IntOnly mice. Cholesterol feeding induced formation of NPC1L1-positive vesicles beneath this membrane in an ezetimibe-sensitive manner. Compared to DKO mice, DKO/L1^IntOnly mice showed significant increases in cholesterol absorption and blood/hepatic/biliary cholesterol. Increased blood cholesterol was restricted to very low-density lipoprotein (VLDL) and LDL fractions, which was associated with increased secretion and plasma levels of apolipoproteins B100 and B48. Additionally, DKO/L1^IntOnly mice displayed decreased fecal cholesterol excretion and hepatic/intestinal expression of cholesterologenic genes. Ezetimibe treatment virtually reversed all of the transgene-related phenotypes in DKO/L1^IntOnly mice. Conclusion: Our findings from DKO/L1^IntOnly mice clearly demonstrate that NPC1L1-mediated cholesterol absorption is a major determinant of blood levels of apolipoprotein B-containing atherogenic lipoproteins, at least in mice.     

Ezetimibe, a selective inhibitor of the transport of cholesterol

Niemann-Pick C1-like 1 (NPC1L1) has recently been identified and has been shown to have features of a plasma membrane transporter, including a secretion signal, 13 predicted transmembrane domains, extensive N-linked glycosylation sites and a sterol-sensing domain. It is highly expressed on the surface of absorptive jejunal enterocytes. NPC1L1 has been shown to be a direct target of ezetimibe, and an ezetimibe-sensitive pathway plays a role in intestinal cholesterol absorption. Ezetimibe-based therapy represents an exciting new area in the treatment of dyslipidemia.     

Flotillins play an essential role in Niemann-Pick C1-like 1-mediated cholesterol uptake

Dietary absorption is a major way for mammals to obtain cholesterol, which is mediated by Niemann-Pick C1-like 1 (NPC1L1) via vesicular endocytosis. One fundamental question in this process is how free cholesterol is efficiently taken up through the internalization of NPC1L1. Using exogenously expressed NPC1L1-EGFP, we show that the lipid raft proteins flotillins associate with NPC1L1 and their localization is regulated by NPC1L1 during intracellular trafficking. Furthermore, flotillins are essential for NPC1L1-mediated cellular cholesterol uptake, biliary cholesterol reabsorption, and the regulation of lipid levels in mice. Together with NPC1L1, they form cholesterol-enriched membrane microdomains, which function as carriers for bulk of cholesterol. The hypocholesterolemic drug ezetimibe disrupts the association between NPC1L1 and flotillins, which blocks the formation of the cholesterol-enriched microdomains. Our findings reveal a functional role of flotillins in NPC1L1-mediated cholesterol uptake and elucidate the formation of NPC1L1-flotillins-postive cholesterol-enriched membrane microdomains as a mechanism for efficient cholesterol absorption.     

Niemann-Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding

Niemann-Pick type C1 (NPC1) protein is needed for cellular utilization of low-density lipoprotein-derived cholesterol that has been delivered to lysosomes. The protein has 13 transmembrane domains, three large lumenal domains, and a cytoplasmic tail. NPC1's lumenally oriented, N-terminal domain binds cholesterol and has been proposed to receive cholesterol from NPC2 protein as part of the process by which cholesterol is exported from lysosomes into the cytosol. Using surface plasmon resonance and affinity chromatography, we show here that the second lumenal domain of NPC1 binds directly to NPC2 protein. For these experiments, a soluble NPC1 lumenal domain 2 was engineered by replacing adjacent transmembrane domains with antiparallel coiled-coil sequences. Interaction of NPC2 with NPC1 lumenal domain 2 is only detected at acidic pH, conditions that are optimal for cholesterol binding to NPC2 and transfer to NPC1; the pH is also appropriate for the acidic environment where binding would take place. Binding to NPC1 domain 2 requires the presence of cholesterol on NPC2 protein, a finding that supports directional transfer of cholesterol from NPC2 onto NPC1's N-terminal domain. Finally, human disease-causing mutations in NPC1 domain 2 decrease NPC2 binding, suggesting that NPC2 binding is necessary for NPC1 function in humans. These data support a model in which NPC1 domain 2 holds NPC2 in position to facilitate directional cholesterol transfer from NPC2 onto NPC1 protein for export from lysosomes.     

Effect of ezetimibe coadministered with statins in genotype-confirmed heterozygous FH patients

We investigated the effect of statins and statins plus ezetimibe in 65 FH heterozygotes carrying LDLR-defective or LDLR-negative mutations as well as the effect of ezetimibe monotherapy in 50 hypercholesterolemic (HCH) patients intolerant to statins. PCSK9 and NPC1L1 genes were analysed to assess the role of genetic variants in response to therapy. In FH patients combined therapy reduced LDL-C by 57%, irrespective of the type of LDLR mutation. The additional decrease of plasma LDL-C induced by ezetimibe showed wide inter-individual variability (from -39% to -4.7%) and was negatively correlated with percent LDL-C decrease due to statin alone (r=-0.713, P<0.001). The variable response to statins was not due to PCSK9 gene variants associated with statin hyper-sensitivity. The highest response to ezetimibe was observed in a carrier of R174H substitution in NPC1L1, which had been found to be associated with high cholesterol absorption. In HCH patients, ezetimibe monotherapy induced a variable decrease of plasma LDL-C (from -47.7% to -13.4%). To investigate this variability, we sequenced NPC1L1 gene in patients with the highest and the lowest response to ezetimibe. This analysis showed a higher prevalence of the G allele of the c.816 C>G polymorphism (L272L) in hyper-responders, an observation confirmed also in FH patients hyper-responders to ezetimibe. In both FH and HCH patients, the G allele carriers tended to have a higher LDL-C reduction in response to ezetimibe. These observations suggest that in FH heterozygotes LDL-C reduction following combined therapy reflects a complex interplay between hepatic synthesis and intestinal absorption of cholesterol.     

Effect of ezetimibe on plasma cholesterol levels, cholesterol absorption, and secretion of biliary cholesterol in laboratory opossums with high and low responses to dietary cholesterol

Partially inbred lines of laboratory opossums differ in plasma low-density lipoprotein cholesterol concentration and cholesterol absorption on a high-cholesterol diet. The aim of the present studies was to determine whether ezetimibe inhibits cholesterol absorption and eliminates the differences in plasma cholesterol and hepatic cholesterol metabolism between high and low responders on a high-cholesterol diet. Initially, we determined that the optimum dose of ezetimibe was 5 mg/(kg d) and treated 6 high- and 6 low-responding opossums with this dose (with equal numbers of controls) for 3 weeks while the opossums consumed a high-cholesterol and low-fat diet. Plasma and low-density lipoprotein cholesterol concentrations decreased significantly (P < .05) in treated but not in untreated high-responding opossums. Plasma cholesterol concentrations increased slightly (P < .05) in untreated low responders but not in treated low responders. The percentage of cholesterol absorption was significantly higher in untreated high responders than in other groups. Livers from high responders with or without treatment were significantly (P < .01) heavier than livers from low responders with or without treatment. Hepatic cholesterol concentrations in untreated high responders were significantly (P < .05) higher than those in low responders with or without treatment (P < .001). The gall bladder bile cholesterol concentrations in untreated high responders were significantly (P < .05) lower than those in other groups. A decrease in biliary cholesterol in low responders treated with ezetimibe was associated with a decrease in hepatic expression of ABCG5 and ABCG8. These studies suggest that ezetimibe decreases plasma cholesterol levels in high responders mainly by decreasing cholesterol absorption and increasing biliary cholesterol concentrations. Because ezetimibe's target is NPC1L1 and NPC1L1 is expressed in the intestine of opossums, its effect on cholesterol absorption may be mediated by inhibiting NPC1L1 function in the intestine.     

Role of ezetimibe in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) encompasses a histological spectrum ranging from simple steatosis to steatohepatitis,advanced fibrosis and inflammatory changes.Ezetimibe inhibits cholesterol absorption from the intestinal lumen into enterocytes.The molecular target of ezetimibe is the sterol transporter Niemann-Pick C1-like 1 protein (NPC1L1).Human NPC1L1 is abundantly expressed in the liver and may facilitate the hepatic accumulation of cholesterol.Ezetimibe ex-erts beneficial effects on several metabolic variables.Ezetimibe treatment attenuates hepatic steatosis and is beneficial in terms of NAFLD biochemical markers.The combination of ezetimibe with other interventions may also be beneficial in NAFLD patients.Our group inves-tigated the ezetimibe-orlistat combination treatment in overweight and obese patients with hypercholeste-rolemia,with beneficial effects on NAFLD biochemical markers.These results are promising for patients with NAFLD,who usually have increased cardiovascular disease risk and need a multifactorial treatment.How-ever,it should be mentioned that most results are from animal studies and,although modest elevation of liver function tests may raise the suspicion of NAFLD,none of these tests are sensitive to establish the diagnosis of NAFLD with great accuracy.     

New insights into the genetic regulation of intestinal cholesterol absorption

The small intestine is a unique organ providing dietary and reabsorbed biliary cholesterol to the body. However, the molecular mechanisms whereby cholesterol is absorbed have not yet been fully understood. Recent research suggests that the newly identified Niemann-Pick C1-like 1 protein (NPC1L1) is expressed at the apical surface of enterocytes and plays a critical role in the absorption of intestinal cholesterol. Furthermore, adenosine triphosphate (ATP)-binding cassette (ABC) transporters ABCG5 and ABCG8 represent apical sterol export pumps that promote active efflux of cholesterol and plant sterols from enterocytes back into the intestinal lumen for excretion. This provides an explanation why cholesterol absorption is a selective process, with plant sterols and other noncholesterol sterols being absorbed poorly or not at all. These findings strongly support the concept that cholesterol absorption is a multistep process, which is regulated by multiple genes at the enterocyte level. The absorption efficiency of cholesterol is most likely determined by the net effect between influx and efflux of intraluminal cholesterol molecules across the brush border of the enterocyte. Combination therapy using a novel, specific, and potent cholesterol absorption (NPC1L1) inhibitor (ezetimibe) and HMG-CoA reductase inhibitors (statins) offers an efficacious new approach to the prevention and treatment of hypercholesterolemia.     

Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain

Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 by using [(3)H]7,7-azocholestanol ([(3)H]AC). After immunoprecipitation, (3)H-labeled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, we show that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [(3)H]AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin A1, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. We also developed a procedure to label the NPC1-YFP by [(3)H]AC in vitro and showed that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-alpha-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1.     

NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann-Pick C1 (NPC1) and soluble Niemann-Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25-264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [(3)H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.     

Beneficial effects of ezetimibe-based therapy in patients with dyslipidemia

Treatment of dyslipidemia is important for the primary and secondary prevention of cardiovascular events. Although statins induce the intensive lowering of low-density lipoprotein (LDL) cholesterol levels, two-thirds of cardiovascular events are not prevented. Ezetimibe has been shown to be a selective inhibitor of the Niemann-Pick C1-like 1 (NPC1L1) transporter of cholesterol across the intestinal wall. Ezetimibe-based therapy may hold the promise of more intensive lowering of LDL cholesterol. This review will address the beneficial effects of ezetimibe in patients with dyslipidemia.     

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Lysosomes are lined with a glycocalyx that protects the limiting membrane from the action of degradative enzymes. We tested the hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer of low density lipoprotein-derived cholesterol across this glycocalyx. A prediction of this model is that cells will be less dependent upon NPC1 if their glycocalyx is decreased in density. Lysosome cholesterol content was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside, an inhibitor of O-linked glycosylation. Direct biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient fibroblasts contained at least 30% less cholesterol when O-linked glycosylation was blocked. As an independent means to modify protein glycosylation, we used Chinese hamster ovary ldl-D cells defective in UDP-Gal/UDP-GalNAc 4-epimerase in which N- and O-linked glycosylation can be controlled. CRISPR generated, NPC1-deficient ldl-D cells supplemented with galactose accumulated more cholesterol than those in which sugar addition was blocked. In the absence of galactose supplementation, NPC1-deficient ldl-D cells also transported more cholesterol from lysosomes to the endoplasmic reticulum, as monitored by an increase in cholesteryl [¹⁴C]-oleate levels. These experiments support a model in which NPC1 protein functions to transfer cholesterol past a lysosomal glycocalyx.Low-density lipoprotein-derived cholesterol is delivered to cells by receptor-mediated endocytosis and transport to lysosomes. Within lysosomes, cholesterol esters are hydrolyzed and cholesterol is exported to the cytoplasm for cellular use (1). Cholesterol export requires two lysosomal glycoproteins: NPC1 and NPC2 (2, 3). Patients carrying homozygous mutations in either of these proteins present with Niemann-Pick type C (NPC) disease, a neurological disorder that is associated with massive accumulation of unesterified cholesterol and glycosphingolipids in lysosomes (2–4).NPC2 is a small, soluble, cholesterol binding protein that is thought to pick up cholesterol both from lipoprotein lipase and from the abundant, intralumenal membranes present within lysosomes (5, 6). NPC1 is a much larger glycoprotein that contains 13 transmembrane domains and three, relatively large, lumenally oriented domains (2). NPC1’s first (N-terminal), luminal domain binds cholesterol directly (7, 8); the second domain can bind NPC2 in a cholesterol-dependent manner and has been proposed to facilitate transfer of cholesterol from NPC2 onto NPC1’s N-terminal domain (9).The requirement for NPC1 and NPC2 proteins for cholesterol export from lysosomes is somewhat enigmatic because cholesterol can generally partition freely into membrane bilayers (10). The limiting membrane of lysosomes is lined predominantly by densely packed, highly glycosylated, lysosomal membrane proteins; their oligosaccharides are thought to protect the phospholipid bilayer from hydrolytic destruction (11, 12). This glycoprotein coat can be visualized by electron microscopy and has an average thickness of about 8 nm (13). It has been proposed that NPC1 is needed to help cholesterol traverse this glycocalyx (8). A direct prediction of this model is that decreasing the lysosomal glycocalyx density should make cells less dependent on NPC1 function.In this paper, we have used two approaches to modify the glycocalyx. Alteration of glycosylation decreases lysosomal cholesterol levels in NPC1-deficient Chinese hamster ovary (CHO) and human cells. These experiments support a model in which NPC1 protein functions to help cholesterol traverse the glycocalyx that lines the interior of lysosome limiting membranes.     

Potential therapeutic uses for ezetimibe beyond lowering LDL‐c to decrease cardiovascular events

Ezetimibe is a relatively new drug that inhibits the absorption of dietary cholesterol in the small intestine. It is a low density lipoprotein‐cholesterol (LDL‐C) lowering medication that acts directly on the intestine by inhibiting Niemann‐Pick C1 Like1 (NPC1L1). Recently, results of the ARBITER 6–HALTS trial (Arterial Biology for the Investigation of the Treatment Effects of Reducing Cholesterol 6–HDL and LDL Treatment Strategies) and the ENHANCE trial (Ezetimibe and Simvastatin in Hypercholesterolemia Enhances Atherosclerosis Regression) showed that ezetimibe had no effect on atherosclerosis despite producing a marked decrease in LDL‐C. Recent studies show a potential benefit of ezetimibe in treating insulin resistance, non‐alcoholic fatty liver disease (NAFLD), gallstones and dyslipidaemia associated with chronic renal failure and organ transplantation. All of these conditions are known to be associated with an increase in risk of cardiovascular disease (CVD) and further studies are needed to assess the potential benefits of ezetimibe in these therapeutics areas.