Yoshihide Yamanashi

Niemann-Pick C1-Like 1 Mediates α-Tocopherol Transport

Dietary lipids and fat-soluble micronutrients are solubilized in mixed micelles and absorbed in the small intestine. Based on an assumption that cholesterol and other fat-soluble molecules share a number of transport mechanisms and the fact that Niemann-Pick C1-like 1 (NPC1L1) is critical for intestinal cholesterol absorption, we hypothesized that some fat-soluble molecules may be transported by NPC1L1. To investigate this hypothesis, we compared the cellular uptake and inhibitory effects of ezetimibe, the molecular target of which is NPC1L1, between cholesterol and some fat-soluble molecules using rat NPC1L1-overexpressing Caco-2 cells. The in vitro analysis suggested that NPC1L1 mediates the uptake of α-tocopherol (vitamin E) in an ezetimibe-sensitive manner as well as the uptake of cholesterol but does not mediate the uptake of retinol (vitamin A) or cyclosporin A. To confirm the ezetimibe-sensitive uptake of α-tocopherol in vivo, we performed an in vivo absorption study using rats and the results suggested a physiologically significant role of NPC1L1-mediated α-tocopherol absorption. Furthermore, using human NPC1L1 overexpression system, we demonstrated that both cholesterol and α-tocopherol uptake was also significantly increased by the overexpression of human NPC1L1 and ezetimibe inhibited their uptake. Mutual inhibition studies of cholesterol and α-tocopherol in human NPC1L1-mediated uptake revealed the inhibitory effect of cholesterol and the stimulatory effect of α-tocopherol on the NPC1L1-mediated transport of both substrates. The present data suggest, for the first time, that NPC1L1 has the ability to transport α-tocopherol and that ezetimibe is able to inhibit the intestinal absorption of α-tocopherol.

Niemann-Pick C1-Like 1 Overexpression Facilitates Ezetimibe-Sensitive Cholesterol and β-Sitosterol Uptake in CaCo-2 Cells

Previous in vivo studies including those with knockout mice suggested that Niemann-Pick C1-like 1 (NPC1L1) plays an essential role in the intestinal absorption of cholesterol. To characterize the mechanism of cholesterol uptake mediated by NPC1L1, an in vitro system reflecting the function of this transporter needs to be established. In the present study, we constructed NPC1L1 overexpressing CaCo-2 cells as an in vitro model and characterized the transport properties of NPC1L1. Immunohistochemical staining revealed that CaCo-2 cells express NPC1L1 on the apical membrane. It was also demonstrated that the uptakes of both cholesterol and β-sitosterol are increased by NPC1L1 overexpression. In addition, the uptake of cholesterol was increased in a dose-dependent manner by an increase in the content of taurocholate in micelles, whereas micellar phosphatidylcholine showed a negative correlation with cholesterol uptake. Furthermore, it was confirmed that sterol uptake increased by NPC1L1 overexpression was inhibited by ezetimibe. We could thus establish an in vitro intestinal model to study the mechanism of NPC1L1-dependent sterol uptake and to screen drug candidates whose target is NPC1L1.

NPC1L1 over-expression facilitates ezetimibe-sensitive cholesterol and β-sitosterol uptake in CaCo-2 cells

Previous in vivo studies including those with knockout mice suggested that Niemann-Pick C1-like 1 (NPC1L1) plays an essential role in the intestinal absorption of cholesterol. To characterize the mechanism of cholesterol uptake mediated by NPC1L1, an in vitro system reflecting the function of this transporter needs to be established. In the present study, we constructed NPC1L1 overexpressing CaCo-2 cells as an in vitro model and characterized the transport properties of NPC1L1. Immunohistochemical staining revealed that CaCo-2 cells express NPC1L1 on the apical membrane. It was also demonstrated that the uptakes of both cholesterol and β-sitosterol are increased by NPC1L1 overexpression. In addition, the uptake of cholesterol was increased in a dose-dependent manner by an increase in the content of taurocholate in micelles, whereas micellar phosphatidylcholine showed a negative correlation with cholesterol uptake. Furthermore, it was confirmed that sterol uptake increased by NPC1L1 overexpression was inhibited by ezetimibe. We could thus establish an in vitro intestinal model to study the mechanism of NPC1L1-dependent sterol uptake and to screen drug candidates whose target is NPC1L1.

NPC2 Regulates Biliary Cholesterol Secretion via Stimulation of ABCG5/G8-Mediated Cholesterol Transport

BACKGROUND & AIMS Biliary cholesterol secretion helps maintain cholesterol homeostasis; it is regulated by the cholesterol exporter adenosine triphosphate-binding cassettes G5 and G8 (ABCG5/G8) and the cholesterol importer Niemann-Pick C1-like 1 (NPC1L1). We studied another putative regulator of cholesterol secretion into bile, Niemann-Pick C2 (NPC2)--a cholesterol-binding protein secreted by the biliary system--and determined its effects on transporter-mediated biliary secretion of cholesterol. METHODS Mice with hepatic knockdown of Npc2 or that overexpressed NPC2 were created using adenovirus-mediated gene transfer; biliary lipids were characterized. The effects of secreted NPC2 on cholesterol transporter activity were examined in vitro using cells that overexpressed ABCG5/G8 or NPC1L1. RESULTS Studies of mice with altered hepatic expression of NPC2 revealed that this expression positively regulates the biliary secretion of cholesterol, supported by the correlation between levels of NPC2 protein and cholesterol in human bile. In vitro analysis showed that secreted NPC2 stimulated ABCG5/G8-mediated cholesterol efflux but not NPC1L1-mediated cholesterol uptake. Consistent with these observations, no significant changes in biliary cholesterol secretion were observed on hepatic overexpression of NPC2 in ABCG5/G8-null mice, indicating that NPC2 requires ABCG5/G8 to stimulate cholesterol secretion. Analyses of NPC2 mutants showed that the stimulatory effect of biliary NPC2 was independent of the function of lysosomal NPC2 as a regulator of intracellular cholesterol trafficking. CONCLUSIONS NPC2 is a positive regulator of biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport.

ABCG2 Dysfunction Increases Serum Uric Acid by Decreased Intestinal Urate Excretion

ATP-binding cassette transporter G2 (ABCG2), also known as breast cancer resistance protein (BCRP), is identified as a high-capacity urate exporter and its dysfunction has an association with serum uric acid (SUA) levels and gout/hyperuricemia risk. However, pathophysiologically important pathway(s) responsible for the ABCG2-mediated urate excretion were unknown. In this study, we investigated how ABCG2 dysfunction affected the urate excretion pathways. First, we revealed that mouse Abcg2 mediates urate transport using the membrane vesicle system. The export process by mouse Abcg2 was ATP-dependent and not saturable under the physiological concentration of urate. Then, we characterized the excretion of urate into urine, bile, and intestinal lumen using in vivo mouse model. SUA of Abcg2-knockout mice was significantly higher than that of control mice. Under this condition, the renal urate excretion was increased in Abcg2-knockout mice, whereas the urate excretion from the intestine was decreased to less than a half. Biliary urate excretion showed no significant difference regardless of Abcg2 genotype. From these results, we estimated the relative contribution of each pathway to total urate excretion; in wild-type mice, the renal excretion pathway contributes approximately two-thirds, the intestinal excretion pathway contributes one-third of the total urate excretion, and the urate excretion into bile is minor. Decreased intestinal excretion could account for the increased SUA of Abcg2-knockout mice. Thus, ABCG2 is suggested to have an important role in extra-renal urate excretion, especially in intestinal excretion. Accordingly, increased SUA in patients with ABCG2 dysfunction could be explained by the decreased excretion of urate from the intestine.

In-vitro characterization of the six clustered variants of NPC1L1 observed in cholesterol low absorbers.

Objectives Niemann-Pick C1-like 1 (NPC1L1) has been shown to be involved in cholesterol transport. Among nonsynonymous variants found from cholesterol low absorbers, six variants were located within only 39 amino acids in the predicted extracellular loop of NPC1L1 protein, suggesting the importance of the region with regard to the function of NPC1L1. In this study, we performed in-vitro analysis to determine the protein expression, cellular localization, and intrinsic activity of these variants. As &agr;-tocopherol is also transported by NPC1L1, we compared the transport activity of NPC1L1 variants between cholesterol and &agr;-tocopherol. Methods and Results Expression vectors for the variants or wild type of NPC1L1 were constructed and transiently transfected into Caco-2 cells, which revealed that four kinds of variants (D398G, T413M, R417W, and G434R) are associated with the reduced expression level and altered subcellular localization of NPC1L1 protein. As four variants (A395V, G402S, R417W, and G434R) are expressed to some extent on the apical membrane, we constructed Caco-2 cells stably overexpressing these variants. All of these variants showed significantly lower transport activity of cholesterol and &agr;-tocopherol than the wild-type NPC1L1, although the transport was ezetimibe-sensitive. Discussion These results account for the reduced intestinal cholesterol absorption in subjects with these six kinds of variants and suggest the possibility of reduced &agr;-tocopherol absorption in carriers of the six variants, due to their decreased expression level, altered subcellular localization or lower intrinsic activity compared with wild-type NPC1L1.

NPC1L1 is a key regulator of intestinal vitamin K absorption and a modulator of warfarin therapy

NPC1L1, a cholesterol importer and a molecular target of ezetimibe, mediates the physiological absorption of vitamin K and therefore modulates the anticoagulant effect of warfarin. A tug of war: Two drugs modulate vitamin K function Kale was the superfood of 2014, with even soda-laden convenience stores like 7-Eleven adding kale juices to their inventories. But patients on warfarin—a commonly prescribed drug to prevent blood clotting—should avoid eating these healthy, leafy greens. Greens such as kale and spinach harbor loads of vitamin K (VK), which can interfere with the drug’s activity. The mechanism linking VK to warfarin action remains unclear; however, in a new study in rodent models, Takada et al. reveal that Niemann-Pick C1-like 1 (NPC1L1) protein, a cholesterol transporter, plays a key role in vitamin transport, too. The consequence of this interaction between VK and NPC1L1? Animals taking lipid-lowering drugs like ezetimibe, which act on NPC1L1, demonstrate heightened activity of warfarin, presumably because VK is not absorbed properly. When the animals were given oral VK supplementation, warfarin activity returned to normal, even if the animals were taking both drugs. Takada and colleagues retrospectively evaluated the medical records of patients taking warfarin alone or warfarin with ezetimibe and confirmed that the anticoagulant activity of warfarin was increased in more than 85% of patients also taking ezetimibe. This drug-drug interaction will be an important consideration when prescribing patients both warfarin and ezetimibe (or similar anticoagulant and cholesterol drugs), but lucky for those patients, this study further suggests that the interaction can be countered in part by a kale salad or, for those averse to this trendy roughage, a daily supplement of VK. Vitamin K (VK) is a micronutrient that facilitates blood coagulation. VK antagonists, such as warfarin, are used in the clinic to prevent thromboembolism. Because VK is not synthesized in the body, its intestinal absorption is crucial for maintaining whole-body VK levels. However, the molecular mechanism of this absorption is unclear. We demonstrate that Niemann-Pick C1-like 1 (NPC1L1) protein, a cholesterol transporter, plays a central role in intestinal VK uptake and modulates the anticoagulant effect of warfarin. In vitro studies using NPC1L1-overexpressing intestinal cells and in vivo studies with Npc1l1-knockout mice revealed that intestinal VK absorption is NPC1L1-dependent and inhibited by ezetimibe, an NPC1L1-selective inhibitor clinically used for dyslipidemia. In addition, in vivo pharmacological studies demonstrated that the coadministration of ezetimibe and warfarin caused a reduction in hepatic VK levels and enhanced the pharmacological effect of warfarin. Adverse events caused by the coadministration of ezetimibe and warfarin were rescued by oral VK supplementation, suggesting that the drug-drug interaction effects observed were the consequence of ezetimibe-mediated VK malabsorption. This mechanism was supported by a retrospective evaluation of clinical data showing that, in more than 85% of warfarin-treated patients, the anticoagulant activity was enhanced by cotreatment with ezetimibe. Our findings provide insight into the molecular mechanism of VK absorption. This new drug-drug interaction mechanism between ezetimibe (a cholesterol transport inhibitor) and warfarin (a VK antagonist and anticoagulant) could inform clinical care of patients on these medications, such as by altering the kinetics of essential, fat-soluble vitamins.

Human NPC1L1 Expression is Positively Regulated by PPARα

ABSTRACTPurposeNiemann-Pick C1-like 1 (NPC1L1), a pharmacological target of ezetimibe, is responsible for cholesterol absorption in enterocytes and hepatocytes. In the present study, the involvement of peroxisome proliferator-activated receptor α (PPARα) and its cofactor, PPARγ coactivator 1α (PGC1α) in the transcriptional regulation of human NPC1L1 was analyzed.MethodsReporter gene assays and electrophoretic mobility shift assays (EMSAs) were performed with the 5′-flanking region of the human NPC1L1 gene and the effect of siPPARα was examined.ResultsPPARα-mediated transactivation was observed with human NPC1L1 promoter constructs. Detailed analyses using deletion- and mutated-promoter constructs revealed the presence of a functional PPARα-response element (PPRE) upstream of the human NPC1L1 gene (−846/−834), a direct binding of PPARα and RXRα to which was confirmed by EMSAs. Moreover, PPARα-specific knockdown resulted in a significant decrease in the endogenous expression of NPC1L1 mRNA and protein in human-derived HepG2 cells. Furthermore, cotransfection of PGC1α stimulated the SREBP2/HNF4α- and PPARα/RXRα-mediated activation of the human NPC1L1 promoter.ConclusionsWe found that PPARα positively regulates human NPC1L1 transcription via direct binding to a PPRE. Additionally, PGC1α stimulates the SREBP2/HNF4α- and PPARα/RXRα-mediated transactivation of human NPC1L1. These findings may provide new insights into the close relationship of glucose, fatty acids and cholesterol homeostasis.

Sindbis Viral Vectors Transiently Deliver Tumor-associated Antigens to Lymph Nodes and Elicit Diversified Antitumor CD8+ T-cell Immunity

Tumors are theoretically capable of eliciting an antitumor immune response, but are often poorly immunogenic. Oncolytic viruses (OVs) have recently emerged as a promising strategy for the immunogenic delivery of tumor-associated antigens (TAAs) to cancer patients. However, safe and effective OV/TAA therapies have not yet been established. We have previously demonstrated that vectors based on Sindbis virus (SV) can inhibit tumor growth and activate the innate immune system in mice. Here, we demonstrate that SV vectors carrying a TAA generate a dramatically enhanced therapeutic effect in mice bearing subcutaneous, intraperitoneal, and lung cancers. Notably, SV/TAA efficacy was not dependent on tumor cell targeting, but was characterized by the transient expression of TAAs in lymph nodes draining the injection site. Early T-cell activation at this site was followed by a robust influx of NKG2D expressing antigen-specific cytotoxic CD8+ T cells into the tumor site, subsequently leading to the generation of long-lasting memory T cells which conferred protection against rechallenge with TAA-positive as well as TAA-negative tumor cells. By combining in vivo imaging, flow cytometry, cytotoxicity/cytokine assays, and tetramer analysis, we investigated the relationship between these events and propose a model for CD8+ T-cell activation during SV/TAA therapy.

Novel function of Niemann‐Pick C1‐like 1 as a negative regulator of Niemann‐Pick C2 protein

The hepatic expression of Niemann‐Pick C1‐like 1 (NPC1L1), which is a key molecule in intestinal cholesterol absorption, is high in humans. In addition to NPC1L1, Niemann‐Pick C2 (NPC2), a secretory cholesterol‐binding protein involved in intracellular cholesterol trafficking and the stimulation of biliary cholesterol secretion, is also expressed in the liver. In this study, we examined the molecular interaction and functional association between NPC1L1 and NPC2. In vitro studies with adenovirus‐based or plasmid‐mediated gene transfer systems revealed that NPC1L1 negatively regulated the protein expression and secretion of NPC2 without affecting the level of NPC2 messenger RNA. Experiments with small interfering RNA against NPC1L1 confirmed the endogenous association of these proteins. In addition, endocytosed NPC2 could compensate for the reduction of NPC2 in NPC1L1‐overexpressing cells, and this demonstrated that the posttranscriptional regulation of NPC2 was dependent on a novel ability of NPC1L1 to inhibit the maturation of NPC2 and accelerate the degradation of NPC2 during its maturation. Furthermore, to confirm the physiological relevance of NPC1L1‐mediated regulation, we analyzed human liver specimens and found a negative correlation between the protein levels of hepatic NPC1L1 and hepatic NPC2. Conclusion: NPC1L1 down‐regulates the expression and secretion of NPC2 by inhibiting its maturation and accelerating its degradation. NPC2 functions as a regulator of intracellular cholesterol trafficking and biliary cholesterol secretion; therefore, in addition to its role in cholesterol re‐uptake from the bile by hepatocytes, hepatic NPC1L1 may control cholesterol homeostasis via the down‐regulation of NPC2. (HEPATOLOGY 2011)