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.     

Do mammalian NPC1 and NPC2 play a role in intestinal cholesterol absorption?

NPC1L1 (Niemann-Pick C1-like 1), the pharmacological target of the cholesterol-uptake inhibitor ezetimibe, is a transporter localized on the brush border of enterocytes. Although this protein plays a key role in intestinal uptake of sterols, multiple molecular events that underlie intestinal cholesterol absorption have not been fully characterized. Two proteins that might be involved in this process are NPC1 and NPC2 (Niemann-Pick disease type C proteins 1 and 2), which function in the endosomal/lysosomal cholesterol egress pathway and whose deficiency results in NPC (Niemann-Pick type C) disease. The involvement of these proteins in intestinal cholesterol absorption was examined in mutant mice lacking either NPC1 or NPC2. Our data indicate that deficiencies in either protein do not have an effect on cholesterol uptake or absorption. This contrasts with recent results obtained for the fruitfly Drosophila melanogaster, which indicate that a deficiency of NPC1 (dNPC1a being its Drosophila homologue) leads to activation of an NPC1L1 (Drosophila homologue dNPC1b)-independent cholesterol uptake pathway, underscoring fundamental differences in mammalian and non-mammalian cholesterol metabolism.     

Niemann-Pick C1-Like 1 and cholesterol uptake

Niemann-Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.     

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.     

Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein

The exact mechanistic pathway of cholesterol absorption in the jejunum of the small intestines is a poorly understood process. Recently, a relatively novel gene, Niemann-Pick C1 Like 1 (NPC1L1), was identified as being critical for intestinal sterol absorption in a pathway which is sensitive to sterol absorption inhibitors such as ezetimibe. NPC1L1 is a multi-transmembrane protein, with a putative sterol sensing domain. Very little else is known about the NPC1L1 protein. In this report, we characterize the native and recombinant rat NPC1L1 protein. We show that NPC1L1 is a 145 kDa membrane protein, enriched in the brush border membrane of the intestinal enterocyte and is highly glycosylated. In addition, sequential detergent extraction of enterocytes result in highly enriched preparations of NPC1L1. An engineered Flag epitope tagged rat NPC1L1 cDNA was expressed as recombinant protein in CHO cells and demonstrated cell surface expression, similar to the native rat protein. These biochemical data indicate that NPC1L1 exists as a predominantly cell surface membrane expressed protein, consistent with its proposed role as the putative intestinal sterol transporter.     

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.     

Standardization of PCR-RFLP analysis of nsSNP rs1468384 of NPC1L1 gene

Niemann-Pick C1-like 1 (NPC1L1) protein, a newly identified sterol influx transporter, located at the apical membrane of the enterocyte, which may actively facilitate the uptake of cholesterol by promoting the passage of sterols across the brush border membrane of the enterocyte. It effects intestinal cholesterol absorption and intracellular transport and as such is an integral part of complex process of cholesterol homeostasis. The study of population data for the distribution of these single nucleotide polymorphisms (SNP) of NPC1L1 has lead to the identification of six non-synonymous single nucleotide polymorphisms (nsSNP). The in vitro analysis using the software MuPro and StructureSNP shows that nsSNP M510I (rs1468384), which involves A→G base pair change leads to decrease in the stability of the protein. A reproducible and a cost-effective PCR-RFLP based assay was developed to screen for the SNP among population data. This SNP has been studied in Caucasian, Asian, and African American populations. Till date, no data is available on Indian population. The distribution of M510I NPC1L1 genotype was estimated in the North Western Indian Population as a test case. The allele distribution in Indian Population differs significantly from that of other populations. The methodology thus proved to be robust enough to bring out these differences.     

Niemann–Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter

Niemann–Pick C1 Like 1 (NPC1L1) has been identified and characterized as an essential protein in the intestinal cholesterol absorption process. NPC1L1 localizes to the brush border membrane of absorptive enterocytes in the small intestine. Intestinal expression of NPC1L1 is down regulated by diets containing high levels of cholesterol. 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 cholesterol absorption inhibitor ezetimibe specifically binds to an extracellular loop of NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice are resistant to diet-induced hypercholesterolemia, and when crossed with apo E null mice, are completely resistant to the development of atherosclerosis. Intestinal gene expression studies in Npc1l1 null mice indicated that no exogenous cholesterol was entering enterocytes lacking NPC1L1, which resulted in an upregulation of intestinal and hepatic LDL receptor and cholesterol biosynthetic gene expression. Polymorphisms in the human NPC1L1 gene have been found to influence cholesterol absorption and plasma low density lipoprotein levels. Therefore, NPC1L1 is a critical intestinal sterol uptake transporter which influences whole body cholesterol homeostasis.     

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.     

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.     

Cholesterol-binding molecules MLN64 and ORP1L mark distinct late endosomes with transporters ABCA3 and NPC1

Cholesterol is an essential lipid in eukaryotic cells and is present in membranes of all intracellular compartments. A major source for cellular cholesterol is internalized lipoprotein particles that are transported toward acidic late endosomes (LE) and lysosomes. Here the lipoprotein particles are hydrolyzed, and free cholesterol is redistributed to other organelles. The LE can contain over half of the cellular cholesterol and, as a major sorting station, can contain many cholesterol-binding proteins from the ABCA, STARD, and ORP families. Here, we show that metastatic lymph node 64 (MLN64, STARD3) and oxysterol-binding protein-related protein 1L (ORP1L) define two subpopulations of LE. MLN64 is present on a LE containing the cholesterol transporter ABCA3, whereas ORP1L localizes to another population of LE containing Niemann Pick type C1 (NPC1), a cholesterol exporter. Endocytosed cargo passes through MLN64/ABCA3-positive compartments before it reaches ORP1L/NPC1-positive LE. The MLN64/ABCA3 compartments cycle between LE and plasma membrane and frequently contact “later” ORP1L/NPC1-containing LE. We propose two stages of cholesterol handling in late endosomal compartments: first, cholesterol enters MLN64/ABCA3-positive compartments from where it can be recycled to the plasma membrane, and later, cholesterol enters ORP1L/NPC1 endosomes that mediate cholesterol export to the endoplasmic reticulum.     

Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells

The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann-Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.     

The Niemann-Pick C proteins and trafficking of cholesterol through the late endosomal/lysosomal system

To maintain proper cellular function, the amount and distribution of cholesterol residing within cellular membranes must be regulated. The principal disorder affecting transport of cholesterol through the late endosomal/lysosomal system and intracellular cholesterol homeostasis is Niemann-Pick type C (NPC) disease. The genes responsible for NPC disease have been identified, and the encoded Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (HE1/NPC2) proteins are currently the subject of intense investigation. This review provides a detailed examination of NPC1 and HE1/NPC2 in regulating the transport of cholesterol through the late endosomal/lysosomal system to other cellular compartments responsible for maintaining intracellular cholesterol homeostasis, and how defective function of these proteins may be responsible for the pathophysiology associated with NPC disease.     

The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1

Niemann-Pick C1-like 1 (NPC1L1) is a polytopic transmembrane protein that plays a critical role in cholesterol absorption. Ezetimibe, a hypocholesterolemic drug, has been reported to bind NPC1L1 and block cholesterol absorption. However, the molecular mechanism of NPC1L1-mediated cholesterol uptake and how ezetimibe inhibits this process are poorly defined. Here we find that cholesterol specifically promotes the internalization of NPC1L1 and that this process requires microfilaments and the clathrin/AP2 complex. Blocking NPC1L1 endocytosis dramatically decreases cholesterol internalization, indicating that NPC1L1 mediates cholesterol uptake via its vesicular endocytosis. Ezetimibe prevents NPC1L1 from incorporating into clathrin-coated vesicles and thus inhibits cholesterol uptake. Together, our data suggest a model wherein cholesterol is internalized into cells with NPC1L1 through clathrin/AP2-mediated endocytosis and ezetimibe inhibits cholesterol absorption by blocking the internalization of NPC1L1.     

Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport

The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann-Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC.     

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 small GTPase Cdc42 interacts with Niemann-Pick C1-like 1 (NPC1L1) and controls its movement from endocytic recycling compartment to plasma membrane in a cholesterol-dependent manner

Niemann-Pick C1-like 1 (NPC1L1) is a multi-transmembrane protein that mediates the absorption of dietary and biliary cholesterol through vesicular endocytosis. The subcellular localization of NPC1L1 is regulated by cholesterol. Cholesterol depletion induces the transport of NPC1L1 to plasma membrane (PM) from endocytic recycling compartment that requires MyoVb·Rab11a·Rab11-FIP2 triple complex, and cholesterol-replenishment renders the internalization of NPC1L1 together with cholesterol. Here, we find that GTP-bound Cdc42 interacts with NPC1L1. Cholesterol depletion regulates the activation of Cdc42 and enhances NPC1L1-Cdc42 interaction. Overexpression of constitutive GTP-bound Cdc42 mutant form or knockdown of Cdc42 inhibits the transport of NPC1L1 to the PM and disturbs the cholesterol-regulated binding of NPC1L1 to Rab11a, MyoVb, and actin. Knockdown of Cdc42 downstream effectors N-WASP or Arp3 also leads to the similar results. In liver-specific Cdc42 knock-out (Cdc42 LKO) mice, NPC1L1 fails to localize to bile canaliculi, and the biliary cholesterol cannot be efficiently reabsorbed. These results indicate that Cdc42 controls the cholesterol-regulated transport and localization of NPC1L1, and plays a role in cholesterol absorption.     

Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

Recent studies have documented the importance of Niemann-Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1-/- mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1-/- mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1-/- mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1.     

Molecular mechanisms of cholesterol absorption and transport in the intestine

Many enzymes and transport proteins participate in cholesterol absorption. This review summarizes recent results on several proteins that are important for each step of the cholesterol absorption pathway, including the important roles of: (i) pancreatic triglyceride lipase (PTL), carboxyl ester lipase (CEL), and ileal bile acid transporter in determining the rate of cholesterol absorption; (ii) ATP binding cassette (ABC) transporters and the Niemann-Pick C-1 like-1 (NPC1L1) protein as intestinal membrane gatekeepers for cholesterol efflux and influx; and (iii) intracellular membrane vesicles and transport proteins in lipid trafficking through intracellular compartments prior to lipoprotein assembly and secretion to plasma circulation.