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.     

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.     

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.     

肝脏表达的Niemann-Pick C1-Like 1调节小鼠的胆汁胆固醇浓度

目的:探讨人(NPC1L1)在小鼠肝脏的过表达对胆汁胆固醇重吸收的影响。方法:通过杂交NPC1L1基因敲除小鼠(L1-KO)与肝脏过表达人的NPC1L1的转基因小鼠(L1-Tg),获得无小鼠自身NPC1L1基因表达、在肝脏过表达或不表达人NPC1L1基因的小鼠(L1-Tgliv+和L1-Tgliv-)。给予L1-Tgliv+小鼠(实验组)及L1-Tgliv-小鼠(对照组)含0.2%胆固醇的饮食并测定2组小鼠胆汁的脂质分泌、血浆的脂质水平和粪便核心固醇的分泌。结果:与L1-Tgliv-小鼠相比,L1-Tgliv+小鼠的胆汁胆固醇水平降低了90%,同时粪便核心固醇的分泌也降低了10%,而血浆总胆固醇、游离胆固醇和胆固醇酯水平分别升高了38.6%、47.6%和36.6%。结论:小鼠肝脏NPC1L1的过表达可提高肝脏对胆汁中胆固醇的重吸收。     

Niemann-Pick C1 (NPC1)/NPC1-like1 Chimeras Define Sequences Critical for NPC1’s Function as a Filovirus Entry Receptor

We recently demonstrated that Niemann-Pick C1 (NPC1), a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1), an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus receptor activity. We exploited the structural similarity between NPC1 and NPC1L1 to construct and analyze a panel of chimeras in which NPC1L1 sequences were replaced with cognate sequences from NPC1. Only one chimera, NPC1L1 containing the second luminal domain (C) of NPC1 in place of its own, bound to the viral glycoprotein, GP. This engineered protein mediated authentic filovirus infection nearly as well as wild-type NPC1, and more efficiently than did a minimal NPC1 domain C-based receptor recently described by us. A reciprocal chimera, NPC1 containing NPC1L1’s domain C, was completely inactive. Remarkably, an intra-domain NPC1L1-NPC1 chimera bearing only a ~130-amino acid N–terminal region of NPC1 domain C could confer substantial viral receptor activity on NPC1L1. Taken together, these findings account for the failure of NPC1L1 to serve as a filovirus receptor, highlight the central role of the luminal domain C of NPC1 in filovirus entry, and reveal the direct involvement of N–terminal domain C sequences in NPC1’s function as a filovirus receptor.     

肝脏表达Niemann-Pick C1-Like 1调节肝X受体诱导的小鼠胆固醇分泌

目的:探讨小鼠肝脏过表达人NPC1L1对LXR诱导的小鼠胆固醇分泌的影响。方法:给予野生型小鼠(WT)和肝脏过表达人NPC1L1的转基因小鼠(L1Tg)胃饲LXR激动剂(T0901317)7 d后,抽提小鼠粪便总脂质并检测核心甾醇的含量,检测小鼠的血浆脂质水平,分析肝脏ABCG5和ABCG8的mRNA表达水平。结果:L1Tg小鼠与WT小鼠相比,除血浆游离胆固醇含量显著升高外,粪便核心甾醇含量及肝脏ABCG5和G8的mRNA水平差异无统计学意义;在胃饲T0901317 1 w后,WT小鼠的粪便核心甾醇含量由[(3.22±0.44)升高到(28.68±1.05)μmol.d-1.100 g-1],血浆总胆固醇、游离胆固醇、胆固醇酯和磷脂的含量均显著升高,同时肝脏的ABCG5和G8的mRNA水平也分别上调了5倍和2倍;然而,L1Tg小鼠经T0901317处理后,与T0901317处理的WT小鼠相比,粪便核心甾醇的分泌减少了56%,血浆游离胆固醇含量升高了40%,肝脏的ABCG5和G8的mRNA水平分别降低了52.4%和40.6%。结论:肝脏特异表达人NPC1L1降低了LXR诱导的小鼠粪便核心甾醇的分泌,并升高了血浆游离胆固醇水平,可能与下调LXR诱导的肝脏ABCG5和G8 mRNA表述水平有一定关系。     

Niemann-Pick C1-Like 1表达缺陷缓解肝X受体激动剂诱导的小鼠肝脏脂肪性变

目的探讨Niemann-Pick C1-Like 1在肝X受体激动剂诱导的肝脏脂肪性变中的作用。方法给C57BL/6小鼠和Niemann-Pick C1-Like 1基因敲除小鼠喂食0.015%胆固醇饮食21天,胃饲溶剂或肝X受体激动剂T0901317一周后,收集小鼠肝脏,称重;抽提肝脏脂质并用酶法检测脂质含量;用实时定量聚合酶链反应法检测肝脏表达与脂肪合成相关基因固醇调节元件结合蛋白1c、脂肪酸合成酶和硬脂酰CoA去饱和酶1的mRNA水平。结果在胃饲T0901317一周后,C57BL/6小鼠肝脏由1.1±0.1g增大到2.8±0.3g,肝脏甘油三酯含量由34.2±18.1mg/g增至232.2±67.9mg/g,固醇调节元件结合蛋白1c、脂肪酸合成酶和硬脂酰CoA去饱和酶1mRNA水平在肝脏的表达也显著增高;而Niemann-Pick C1-Like 1基因敲除小鼠肝脏由0.9±0.1g增大到1.5±0.1g,肝脏甘油三酯含量由43.7±26.5mg/g增至104.9±62.1mg/g,脂肪酸合成酶和硬脂酰CoA去饱和酶1mRNA水平在肝脏的表达也显著增高。但在T0901317诱导下,Niemann-Pick C1-Like 1基因敲除小鼠肝脏脂肪酸合成酶mRNA水平仍比C57BL/6小鼠低63%。结论Niemann-Pick C1-Like 1表达缺陷降低肝X受体激动剂诱导的肝脏固醇调节元件结合蛋白1c和脂肪酸合成酶的表达,缓解小鼠肝脏脂肪性变。     

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.     

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.     

Identification of novel mutations in the NPC1 gene in German patients with Niemann-Pick C disease

Niemann–Pick disease type C (NPC) is an inherited neuro degenerative disorder associated with intracellular cholesterol trafficking defects. Mutations in two distinct genes, NPC1 and HE1, have recently been shown to cause this disease. We have analysed the NPC1 gene in five German patients with NPC from four unrelated families. We identified a total of five novel mutations in the coding region of the NPC1 gene (G231V, D874V, I642M, I1094T and R116stop). All affected individuals displayed compound heterozygosity. The mutated alleles were transmitted by the nonaffected parents with the exception of one patient, in whom a de novo mutation (G231V) had occurred. Interestingly, the G231V/P237S NPC1 genotype in this individual is associated with an early-onset form of NPC. In contrast, we found that the D874V/D948N genotype, observed in another NPC patient, is characterized by a late onset of clinical symptoms that presents with a pronounced white-matter disease. Our results will contribute to defining the association between the clinical phenotypes and the genetic abnormalities in Niemann–Pick C disease.     

Annexin 2-caveolin 1 complex is a target of ezetimibe and regulates intestinal cholesterol transport

Modulation of cholesterol absorption in the intestine, the primary site of dietary cholesterol uptake in humans, can have profound clinical implications. We have undertaken a reverse genetic approach by disrupting putative cholesterol processing genes in zebrafish larvae by using morpholino (MO) antisense oligonucleotides. By using targeted MO injections and immunoprecipitation (IP) experiments coupled with mass spectrometry, we determined that annexin (ANX)2 complexes with caveolin (CAV)1 in the zebrafish and mouse intestine. The complex is heat stable and unaffected by SDS or reducing conditions. MO targeting of anx2b or cav1, which are both strongly expressed in the larval and adult zebrafish intestinal epithelium, prevents formation of the protein heterocomplex. Furthermore, anx2b MO injection prevents processing of a fluorescent cholesterol reporter and results in reduced sterol mass. Pharmacological treatment of mice with ezetimibe disrupts the heterocomplex in only hypercholesterolemic animals. These data suggest that ANX2 and CAV1 are components of an intestinal sterol transport complex.     

An NPC1L1 gene promoter variant is associated with autosomal dominant hypercholesterolemia

Background and aimsA substantial number of subjects with autosomal dominant hypercholesterolemia (ADH) do not have LDL receptor (LDLR) or apolipoprotein B (APOB) mutations. Some ADH subjects appear to hyperabsorb sterols from the intestine, thus we hypothesized that they could have variants of the Niemann–Pick C1-Like 1 gene (NPC1L1). NPC1L1 encodes a crucial protein involved in intestinal sterol absorption.Methods and resultsFour NPC1L1 variants (?133A>G, ?18C>A, 1679C>G, 28650A>G) were analyzed in 271 (155 women and 116 men) ADH bearers without mutations in LDLR or APOB aged 30–70 years and 274 (180 women and 94 men) control subjects aged 25–65 years. The AC haplotype determined by the ?133A>G and ?18C>A variants was underrepresented in ADH subjects compared to controls (p = 0.01). In the ADH group, cholesterol absorption/synthesis markers were significantly lower in AC homozygotes that in all others haplotypes. Electrophoretic mobility shift assay (EMSA) results revealed that the ?133A-specific oligonucleotide produced a retarded band stronger than the ?133G allele. Luciferase activity with NPC1L1 ?133G variant was 2.5-fold higher than with the ?133A variant.ConclusionThe ?133A>G polymorphism exerts a significant effect on NPC1L1 promoter activity. NPC1L1 promoter variants might explain in part the hypercholesterolemic phenotype of some subjects with nonLDLR/nonAPOB ADH.     

NPC1L1基因多态性与冠心病的关系

目的:在上海地区汉族人群中探讨NPC1L1(Niemann-Pick C1-like1)基因启动子区-762T>C多态和编码区1679C>G多态与冠心病(CAD)的关系。方法:运用实时定量PCR基因分型技术,对经冠状动脉造影证实的361例CAD患者和421例对照者进行基因分型。结果:单位点分析发现,-762T>C多态的基因型分布在2组间有显著性差异(P=0.041),应用Logistic回归分析,在显性模式下,-762T>C多态增加CAD发病风险[比值比(OR)=0.74,95%可信区间(CI):0.56～0.99,P=0.042)]。1679C>G多态的基因型分布在2组间无显著差异,应用Logistic回归分析,在显性模式下,1679C>G多态与CAD发病风险之间亦关系密切(OR=0.81,95%CI:0.61～0.99,P=0.046)。单倍型分析发现,3种单倍型分布在2组间无统计学意义。结论:NPC1L1基因启动子区-762T>C多态可能与上海地区汉族人群的CAD发病相关,携带C等位基因者能显著降低CAD的发生风险。     

A defect in cholesterol esterification in Niemann-Pick disease (type C) patients.

The demonstration of a defect of cholesterol esterification in a mutant strain of BALB/c mice with an attendant reduction of sphingomyelinase activity [Pentchev, P. G., Boothe, A. D., Kruth, H.S., Weintroub, H., Stivers, J. & Brady, R. O. (1984) J. Biol. Chem. 259, 5784-5791] prompted us to examine the capacity of cultured human Niemann-Pick fibroblasts to esterify exogenously derived cholesterol. Cholesterol was supplied to cell cultures in the form of native or chemically modified, positively charged low density lipoprotein or as non-lipoprotein cholesterol. Cholesterol esterification was not impaired in cell cultures derived from patients with type A or B Niemann-Pick disease. However, esterification of exogenously administered cholesterol was deficient in 20 type C Niemann-Pick cell lines that were available for testing. Fluorescence histochemical staining of unesterified cholesterol in type C cells suggested that these cells were able to internalize and lysosomally process lipoprotein cholesterol. Acyl-CoA:cholesterol acyltransferase activity did not appear deficient in type C cell extracts. The error in cholesterol esterification may provide an opportunity for probing the molecular lesion in this disorder and may afford a useful and reliable means for establishing diagnosis.     

The efficacy of ezetimibe on nonalcoholic fatty liver disease (NAFLD)

The usefulness of ezetimibe was examined in 297 patients with dyslipidemia who did not achieve LDL-C target levels set in JAS 2007 Guidelines by lifestyle modification. The mean period of administration was 178.2±295.4 days. Ezetimibe significantly improved serum lipid levels in the patients with and without non-alcoholic fatty liver disease (NAFLD) (p<0.01). Significant improvement of AST, ALT and γ GTP levels were also observed in the patients with NAFLD (p<0.01, p<0.05, and p<0.01, respectively). Seventy of the patients with NAFLD who underwent abdominal ultrasound before and after administration of ezetimibe were followed. Of those, 38.6% of the patients showed disappearance of steatosis, indicating that administration of ezetimibe is useful in patients with NAFLD.