Ezetimibe restores biliary cholesterol excretion in mice expressing Niemann-Pick C1-Like 1 only in liver

Niemann-Pick C1-Like 1 (NPC1L1) is highly expressed in the small intestine across mammalian species and is the target of ezetimibe, a potent cholesterol absorption inhibitor. In humans, NPC1L1 is also expressed in the liver. We found that transgenic overexpression of NPC1L1 in the wild-type mouse liver inhibits biliary cholesterol secretion and raises blood cholesterol, which can be reversed by ezetimibe treatment. Unfortunately, the high expression of endogenous NPC1L1 in the intestine hampered a definitive establishment of the role of hepatic NPC1L1 in cholesterol metabolism and ezetimibe action in the liver because intestinal NPC1L1 dramatically influences cholesterol homeostasis and is a target of ezetimibe. To circumvent this obstacle, we crossed liver-specific NPC1L1 transgenic mice to NPC1L1 knockout (L1-KO) mice and created a mouse line expressing no endogenous NPC1L1, but human NPC1L1 in liver only (L1(LivOnly) mice). Compared to L1-KO mice, L1(LivOnly) mice on a 0.2% cholesterol diet showed significantly increased hepatic and plasma cholesterol, and despite a 90% reduction in biliary cholesterol excretion, their fecal cholesterol excretion remained completely unaltered. Remarkably, 4days of ezetimibe treatment significantly restored biliary cholesterol secretion in L1(LivOnly) mice. These findings demonstrated a direct role of hepatic NPC1L1 in regulating biliary cholesterol excretion and hepatic/blood cholesterol levels, and unequivocally established hepatic NPC1L1 as a target of ezetimibe.     

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.     

Intracellular retention of caveolin 1 in presenilin-deficient cells

Mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early onset familial Alzheimer's disease. PS, a critical component of gamma-secretase, is responsible for the intramembranous cleavage of amyloid precursor protein and Notch. Other physiological functions have been assigned to PS without any clear identification of the mechanisms underlying these multiple biological roles. The early embryonic lethality of PS1 and PS2 double knock-out (PS1/2 null) mice prevents the evaluation of physiological roles of PS. To investigate new functions for presenilins, we performed a proteomic approach by using cells derived from PS1/2 null blastocysts and wild type controls. We identified a presenilin-dependent cell-surface binding of albumin. Binding of albumin depends on intact caveolae on the cellular surface. Abnormal caveolin 1 localization in PS1/2 null cells was associated with a loss of caveolae and an absence of caveolin 1 expression within lipid rafts. Expressing PS1 or PS2 but not the intracellular form of Notch1 in PS1/2 null cells restored normal caveolin 1 localization, demonstrating that presenilins are required for the subcellular trafficking of caveolin 1 independently from Notch activity. Despite an expression of both caveolin 1 and PS1 within lipid raft-enriched fractions after sucrose density centrifugation in wild type cells, no direct interaction between these two proteins was detected, implying that presenilins affect caveolin 1 trafficking in an indirect manner. We conclude that presenilins are required for caveolae formation by controlling transport of intracellular caveolin 1 to the plasma membrane.     

Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation

Intestinal NPC1L1 transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. NPC1L1 is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared with the small intestine. This study was undertaken to examine whether DNA methylation was responsible for segment-specific expression of NPC1L1. Treatment of mice with 5-azacytidine (i.p.) resulted in a significant dose-dependent increase in NPC1L1 mRNA expression in the colon. The lack of expression of NPC1L1 in the normal colon was associated with high levels of methylation in the area flanking the 3-kb fragment upstream of the initiation site of the mouse NPC1L1 gene in mouse colon as analyzed by EpiTYPER® MassARRAY®. The high level of methylation in the colon was observed in specific CpG dinucleotides and was significantly decreased in response to 5-azacytidine. Similar to mouse NPC1L1, 5-azacytidine treatment also increased the level of human NPC1L1 mRNA expression in the intestinal HuTu-80 cell line in a dose- and time-dependent manner. Silencing the expression of DNA methyltransferase DNMT1, -2, -3A, and -3B alone by siRNA did not affect NPC1L1 expression in HuTu-80 cells. However, the simultaneous attenuation of DNMT1 and -3B expression caused a significant increase in NPC1L1 mRNA expression as compared with control. Also, in vitro methylation of the human NPC1L1 promoter significantly decreased NPC1L1 promoter activity in human intestinal Caco2 cells. In conclusion, our data demonstrated for the first time that DNA methylation in the promoter region of the NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the length of the gastrointestinal tract.     

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.     

Caveolin-1 is not required for murine intestinal cholesterol transport

Caveolin-1 (CAV1) is the structural protein of the filamentous coat that decorates the cytoplasmic surface of each caveola. Cell culture studies have implicated CAV1 in playing an important role in intracellular cholesterol trafficking. In addition, it has been reported that CAV1 forms a detergent-resistant protein complex with Annexin-2 in enterocytes that can be disrupted by the cholesterol absorption inhibitor ezetimibe, suggesting a possible role for CAV1 in cholesterol absorption. In this report, we have evaluated cholesterol homeostasis in Cav1 knock-out mice. Deletion of CAV1 does not result in either a compensatory increase of CAV2 or CAV3 in intestine. In addition, Cav1 knock-out mice display normal mRNA and protein levels of Annexin-2 or the putative cholesterol transport protein Niemann-Pick C1-like 1 (NPC1L1) in proximal intestinal mucosa. Fractional cholesterol absorption and fecal neutral sterol excretion are statistically similar in Cav1 knock-out mice and their wild-type littermates. Moreover, oral administration of ezetimibe is equally effective in decreasing cholesterol absorption in Cav1 null mice and wild-type controls. The mRNA expression levels of genes sensitive to intracellular cholesterol concentration (ATP-binding cassette transporters ABCA1 and ABCG5, hydroxymethylglutaryl-CoA synthase and the LDL receptor) are similarly altered in the proximal intestinal mucosa of Cav1 null and wild-type mice following ezetimibe treatment. These results demonstrate that CAV1 is not required for cholesterol absorption or ezetimibe sensitivity in the mouse.     

NP c 1L l 在高脂血症和动脉粥样硬化中的表达分析

目的：研究NPC1L1（Niemann-Pick C1 Like 1）mRNA在单纯高脂血症大鼠和动脉粥样硬化大鼠小肠组织中的表达与差异,探讨其与脂质代谢和动脉粥样硬化之间的关系。方法：通过半定量RT-PCR方法分别检测正常普食组、单纯高脂饲养组和动脉粥样大鼠组小肠组织中NPC1L1 mRNA的表达差异。结果：三个组别大鼠小肠组织中均存在NPC1L2 mRNA,单纯高脂饮食和动脉粥样大鼠小肠组织中NPC1L1 mRNA表达明显高于正常对照大鼠（P〈0．01）;单纯高脂饮食和动脉粥样大鼠小肠组织中NPC1L1 mRNA表达之间无明显差异（P〉0．05）。结论：血脂代谢紊乱与小肠组织中NPC1L1的高表达有关,NPC1L1可能参与了血脂紊乱的病理生理过程;NPC1L1与促成动脉粥样硬化的发生无明显相关性。     

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.     

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.     

Cutting edge: impaired glycosphingolipid trafficking and NKT cell development in mice lacking Niemann-Pick type C1 protein

Niemann-Pick type C1 (NPC1) is a late endosomal/lysosomal transmembrane protein involved in the cellular transport of glycosphingolipids and cholesterol that is mutated in a majority of patients with Niemann-Pick C neurodegenerative disease. We found that NPC1-deficient mice lacked Valpha14-Jalpha18 NKT cells, a major population of CD1d-restricted T cells that is conserved in humans. NPC1-deficient mice also exhibited marked defects in the presentation of Sphingomonas cell wall Ags to NKT cells and in bacterial clearance in vivo. A synthetic fluorescent alpha-glycosylceramide analog of the Sphingomonas Ag trafficked to the lysosome of wild-type cells but accumulated in the late endosome of NPC1-deficient cells. These findings reveal a blockade of lipid trafficking between endosome and lysosome as a consequence of NPC1 deficiency and suggest a common mechanism for the defects in lipid presentation and development of Valpha14-Jalpha18 NKT cells.     

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.     

斑马鱼leg1直系同源基因mu-leg1在小鼠中的表达谱分析

基因leg1(liver-enriched gene 1)首先在斑马鱼中作为肝脏富集表达基因被鉴定。进一步的研究揭示leg1编码的Leg1蛋白代表一类新型外分泌蛋白, 它在斑马鱼胚期肝脏生长发育过程中起关键作用。小鼠leg1(mu-leg1)是斑马鱼leg1(zb-leg1)的直系同源基因, 二者编码的蛋白氨基酸序列相似性为31%。文章通过巢式PCR从成年小鼠肝脏中成功克隆了mu-leg1的cDNA序列, 并对该基因在成年小鼠不同组织中的表达特征进行分析和鉴定。Northern印迹杂交和半定量RT-PCR分析结果显示, mu-leg1在成年小鼠小肠中而非肝脏中富集表达。此外, 用制备的mu-Leg1多克隆抗体进行Western印迹杂交, 结果显示mu-Leg1也是一个分泌蛋白。同时, 还建立了mu-leg1基因条件性剔除杂合子小鼠。这些材料为今后深入研究和探讨mu-Leg1蛋白的生化功能奠定了基础。     

Raft association and lipid droplet targeting of flotillins are independent of caveolin

Lipid rafts are liquid ordered platforms that dynamically compartmentalize membranes. Caveolins and flotillins constitute a group of proteins that are enriched in these domains. Caveolin-1 has been shown to be an essential component of caveolae. Flotillins were also discovered as an integral component of caveolae and have since been suggested to interact with caveolins. However, flotillins are also expressed in non-caveolae-containing cells such as lymphocytes and neuronal cells. Hence, a discrepancy exists in the literature regarding the caveolin dependence of flotillin expression and their subcellular localization. To address this controversy, we used mouse embryonic fibroblasts (MEFs) from caveolin-1 knockout (Cav-1(-/-)) and wild-type mice to study flotillin expression and localization. Here we show that both membrane association and lipid raft partitioning of flotillins are not perturbed in Cav-1(-/-) MEFs, whereas membrane targeting and raft partitioning of caveolin-2, another caveolin family protein, is severely impaired. Moreover, we demonstrate that flotillin-1, but not flotillin-2, associates with lipid droplets upon oleic acid treatment and that this association is completely independent of caveolin. Taken together, our results show that flotillins are localized in lipid rafts independent of caveolin-1 and that translocation of flotillin-1 to lipid droplets is a caveolin-independent process.     

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse

Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.     

Mitochondrial uncoupling protein 1 expression in thymocytes

Using an antibody specific and selective to mitochondrial uncoupling protein 1 (UCP1) peptide, this study confirms the observation that UCP 1 is present in thymocytes isolated from UCP 1 wild-type, but not UCP 1 knock-out mice. UCP 1 is also shown to be present in thymocytes isolated from rat. It was also demonstrated that an antibody raised to the full-length UCP 1 protein appears to be non-specific for UCP 1, as it detects protein in UCP 1 wild-type and UCP 1 knock-out mice, protein in mitochondria isolated from brown adipose tissue of both UCP 1 wild-type and UCP 1 knock-out mice, as well as detecting protein in mitochondria isolated from rat spleen, kidney, skeletal muscle and liver, tissues that do not express UCP 1. We were also able to show that CIDEA, a soluble protein with a suggested role in regulating UCP 1 function, is equally abundant in thymocytes from UCP 1 wild-type and UCP 1 knock-out mice. Taken together our data demonstrate that (a) UCP 1 is present in rat and mouse thymocytes, (b) that the antibody to full-length UCP 1 is not specific for UCP 1 and (c) that the absence of UCP 1 does not affect native expression of CIDEA in thymocytes.     

Genetic inactivation of NPC1L1 protects against sitosterolemia in mice lacking ABCG5/ABCG8

Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (NPC1L1(-/-)mice) exhibit a defect in intestinal absorption of cholesterol and phytosterols. However, wild-type (WT) mice do not efficiently absorb and accumulate phytosterols either. Cell-based studies show that NPC1L1 is a much weaker transporter for phytosterols than cholesterol. In this study, we examined the role of NPC1L1 in phytosterol and cholesterol trafficking in mice lacking ATP-binding cassette (ABC) transporters G5 and G8 (G5/G8(-/-) mice). G5/G8(-/-) mice develop sitosterolemia, a genetic disorder characterized by the accumulation of phytosterols in blood and tissues. We found that mice lacking ABCG5/G8 and NPC1L1 [triple knockout (TKO) mice] did not accumulate phytosterols in plasma and the liver. TKO mice, like G5/G8(-/-) mice, still had a defect in hepatobiliary cholesterol secretion, which was consistent with TKO versus NPC1L1(-/-) mice exhibiting a 52% reduction in fecal cholesterol excretion. Because fractional cholesterol absorption was reduced similarly in NPC1L1(-/-) and TKO mice, by subtracting fecal cholesterol excretion in TKO mice from NPC1L1(-/-) mice, we estimated that a 25g NPC1L1(-/-) mouse may secrete about 4 mumol of cholesterol daily via the G5/G8 pathway. In conclusion, NPC1L1 is essential for phytosterols to enter the body in mice.     

Liver X receptor agonist T0901317 reduces athero-sclerotic lesions in apoE-/- mice by up-regulating NPC1 expression

In this study, we studied the effect of liver X receptor (LXR) agonist T0901317 on Niemann-Pick C1 protein (NPC1) expression in apoE-/- mice. Male apoE-/- mice were randomized into 4 groups, baseline group (n=10), control group (n=14), treatment group (n=14) and prevention group (n=14). All of the mice were fed with a high-fat/high-cholesterol (HFHC) diet containing 15% fat and 0.25% cholesterol. The baseline group treated with vehicle was sacrificed after 8 weeks of the diet. The control group and the prevention group were treated with either vehicle or T0901317 daily by oral gavage for 14 weeks. The treatment group was treated with vehicle for 8 weeks, and then was treated with the agonist T0901317 for additional 6 weeks. Gene and protein expression was analyzed by real-time quantitative PCR, immunohistochemistry and Western blotting, respectively. Plasma lipid concentrations were measured by commercially enzymatic methods. We used RNA interference technology to silence NPC1 gene expression in THP-1 macrophage-derived foam cells and then detected the effect of LXR agonist T0901317 on cholesterol efflux. Plasma triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and apoA-I concentrations were markedly increased in T0901317-treated groups. T0901317 treatment reduced the aortic atherosclerotic lesion area by 64.2% in the prevention group and 58.3% in the treatment group. LXR agonist treatment increased NPC1 mRNA expression and protein levels in the small intestine, liver and aorta of apoE-/- mice. Compared with the normal cells, cholesterol efflux of siRNA THP-1 macrophage-derived foam cells was significantly decreased, whereas cholesterol efflux of LXR agonist T0901317-treated THP-1 macrophage-derived foam cells was significantly increased. Our results suggest that LXR agonist T0901317 inhibits atherosclerosis development in apoE-/- mice, which is related to up-regulating NPC1 expression.