Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptors (LXRalpha and LXRbeta) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARalpha and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARalpha ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis.     

Luteolin and Quercetin Affect the Cholesterol Absorption Mediated by Epithelial Cholesterol Transporter Niemann–Pick C1-Like 1 in Caco-2 Cells and Rats

Niemann–Pick C1-Like 1 (NPC1L1) mediates cholesterol absorption, and ezetimibe is a potent NPC1L1 inhibitor applicable for medication of hypercholesterolemia. Epidemiological studies demonstrated that consumption of polyphenols correlates with a decreased risk for atherosclerosis due to their antioxidant effect. This activity can hardly be attributable to the antioxidant activity only, and we hypothesized that polyphenols inhibit intestinal transport of cholesterol. We elucidated the kinetic parameters of intestinal cholesterol absorption, screened several polyphenols for their ability to specifically inhibit intestinal cholesterol absorption, and determined the inhibitory effects of selected flavonoids in vitro and in vivo. The concentration-dependent uptake of cholesterol by Caco-2 cells obeyed a monophasic saturation process. This indicates the involvement of an active-passive transport, i.e., NPC1L1. Parameters of cholesterol uptake by Caco-2 cells were as follows: J _max, K _t, and K _d were 6.89±2.96 19.03±11.58 µM, and 0.11±0.02 pmol/min/mg protein, respectively. Luteolin and quercetin inhibited cholesterol absorption by Caco-2 cells and human embryonic kidney 293T cells expressing NPC1L1. When preincubated Caco-2 cells with luteolin and quercetin before the assay, cholesterol uptake significantly decreased. The inhibitory effects of these flavonoids were maintained for up to 120 min. The level of inhibition and irreversible effects were similar to that of ezetimibe. Serum cholesterol levels significantly decreased more in rats fed both cholesterol and luteolin (or quercetin), than in those observed in the cholesterol feeding group. As quercetin induced a significant decrease in the levels of NPC1L1 mRNA in Caco-2 cells, the in vivo inhibitory effect may be due to the expression of NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels by specifically inhibiting intestinal cholesterol absorption mediated by NPC1L1.     

Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells

Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.     

Modulation of human Niemann-Pick C1-like 1 gene expression by sterol: Role of sterol regulatory element binding protein 2

Niemann-Pick C1-like 1 (NPC1L1) is an essential intestinal component of cholesterol absorption. However, little is known about the molecular regulation of intestinal NPC1L1 expression and promoter activity. We demonstrated that human NPC1L1 mRNA expression was significantly decreased by 25-hydroxycholesterol but increased in response to cellular cholesterol depletion achieved by incubation with Mevinolin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) in human intestinal Caco-2 cells. We also showed that a -1741/+56 fragment of the NPC1L1 gene demonstrated high promoter activity in Caco-2 cells that was reduced by 25-hydroxycholesterol and stimulated by cholesterol depletion. Interestingly, we showed that the NPC1L1 promoter is remarkably transactivated by the overexpression of sterol regulatory element (SRE) binding protein (SREBP)-2, suggesting its involvement in the sterol-induced alteration in NPC1L1 promoter activity. Finally, we identified two putative SREs in the human NPC1L1 promoter and established their essential roles in mediating the effects of cholesterol on promoter activity. Our study demonstrated the modulation of human NPC1L1 expression and promoter activity by cholesterol in a SREBP-2-dependent mechanism.     

小肠胆固醇吸收的研究进展

胆固醇是生命活动必不可少的脂类物质,但当体内胆固醇水平过高时,就会引起高胆固醇血症,进而导致动脉粥样硬化、脑中风和冠心病。人体内胆固醇有两种来源:以乙酰辅酶A为原料从头合成,或者通过小肠从食物中吸收。现今,过量的胆固醇摄取是引起高胆固醇血症的重要原因。胆固醇在小肠中的吸收是一个复杂的、由多个步骤组成的连续的分解、转运以及重新酯化的过程。其中由Niemann-Pick C1 Like 1(NPC1L1)蛋白介导肠道中胆固醇进入吸收细胞,是胆固醇吸收的限速步骤。本文重点总结了小肠胆固醇吸收的分子途径、调控机制、医药研发现状及与low-density lipoprotein receptor(LDLR)内吞过程的比较。     

Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid

To address the effect of the n-3 fatty acid, docosahexaenoic acid (22:6), on proteins that play a role in cholesterol absorption, CaCo-2 cells were incubated with taurocholate micelles alone or micelles containing 22:6 or oleic acid (18:1). Compared with controls or 18:1, 22:6 did not interfere with the cellular uptake of micellar cholesterol. Apical cholesterol efflux was enhanced in cells incubated with 22:6. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was decreased by 22:6. 22:6 decreased Niemann-Pick C1-Like 1 (NPC1L1) protein and mRNA levels without altering gene or protein expression of ACAT2, annexin-2, caveolin-1, or ABCG8. Peroxisome proliferator-activated receptor delta (PPARdelta) activation decreased NPC1L1 mRNA levels and cholesterol trafficking to the endoplasmic reticulum, suggesting that 22:6 may act through PPARdelta. Compared with hamsters fed a control diet or olive oil (enriched 18:1), NPC1L1 mRNA levels were decreased in duodenum and jejunum of hamsters ingesting fish oil (enriched 22:6). In an intestinal cell, independent of changes in ABCG8 expression, 22:6 increases the apical efflux of cholesterol. 22:6 interferes with cholesterol trafficking to the endoplasmic reticulum by the suppression of NPC1L1, perhaps through the activation of PPARdelta. Moreover, a diet enriched in n-3 fatty acids decreases the gene expression of NPC1L1 in duodenum and jejunum of hamster.     

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.     

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.     

Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia

NPC1L1, a recently identified relative of Niemann-Pick C1, was characterized to determine its subcellular location and potential function(s). NPC1L1 was highly expressed in HepG2 cells and localized in a subcellular vesicular compartment rich in the small GTPase Rab5. mRNA expression profiling revealed significant differences between mouse and man with highest expression found in human liver and significant expression in the small intestine. In contrast, liver expression in mouse was extremely low with mouse small intestine exhibiting the highest NPC1L1 expression. A mouse knock-out model of NPC1L1 was generated and revealed that mice lacking a functional NPC1L1 have multiple lipid transport defects. Surprisingly, lack of NPC1L1 exerts a protective effect against diet-induced hyperlipidemia. Further characterization of cell lines generated from wild-type and knock-out mice revealed that in contrast to wild-type cells, NPC1L1 cells exhibit aberrant plasma membrane uptake and subsequent transport of various lipids, including cholesterol and sphingolipids. Furthermore, lack of NPC1L1 activity causes a deregulation of caveolin transport and localization, suggesting that the observed lipid transport defects may be the indirect result of an inability of NPC1L1 null cells to properly target and/or regulate caveolin expression.     

Polyunsaturated fatty acids down-regulate in vitro expression of the key intestinal cholesterol absorption protein NPC1L1: no effect of monounsaturated nor saturated fatty acids

Several transporter proteins regulate intestinal cholesterol absorption. Of these proteins, NPC1L1 is a major contributor to this process. Fatty acids (FAs) modulate cholesterol absorption by a mechanism that remains unknown. We evaluate the effect of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) on the expression of NPC1L1 and others proteins associated with cholesterol absorption (SR-BI, ABCG5, ABCG8, ABCA1, CAV-1, ANX-2) in human enterocytes in vitro. The role of SREBPs, PPARs, LXR and RXR in this process was also investigated. Caco-2/TC-7 enterocytes were incubated for 24 h with a wide range of concentrations of FA–bovine serum albumin (50–300 μM). Gene expression was analyzed by quantitative real-time PCR. The NPC1L1 protein present in enterocyte membranes was analyzed using Western blot. NPC1L1 mRNA levels were reduced 35–58% by the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (P<.05). Linoleic acid (n-6), palmitic acid and oleic acid did not affect NPC1L1 mRNA expression. ABCA1 mRNA levels were reduced 44–70% by n-6 arachidonic acid and 43–55% by n-3 EPA (P<.05). LXR and LXR+RXR agonists decreased NPC1L1 mRNA expression by 28% and 57%, respectively (P<.05). A concentration of 200 μM of EPA and DHA decreased NPC1L1 protein expression in enterocyte membranes by 58% and 59%, respectively. We have demonstrated that the PUFAs n-3 EPA and DHA down-regulate NPC1L1 mRNA expression. In addition, PUFAs also down-regulate NPC1L1 protein expression in enterocyte membranes. LXR and RXR activation induced a similar repression effect. The lipid-lowering effect of n-3 PUFAs could be mediated in part by their action at the NPC1L1 gene level.     

Origins of intestinal ABCA1-mediated HDL-cholesterol

The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2. Influx of apical membrane cholesterol increases ABCA1 mRNA and mass, resulting in enhanced efflux of HDL-cholesterol. Luminal (micellar) cholesterol and newly synthesized cholesterol are not transported directly to ABCA1 but reach the ABCA1 pool after incorporation into the apical membrane. Depleting the apical or the basolateral membrane of cholesterol by cyclodextrin attenuates the amount of cholesterol transported by ABCA1 without altering ABCA1 expression. Filipin added to the apical side but not the basal side attenuates ABCA1-mediated cholesterol efflux, suggesting that apical membrane "microdomains," or rafts, supply cholesterol for HDL. Preventing cholesterol esterification increases the amount of cholesterol available for HDL. Ezetimibe, a Niemann-Pick C1-like 1 protein inhibitor, does not alter ABCA1-mediated cholesterol efflux. U18666A and imipramine, agents that mimic cholesterol trafficking defects of Neimann-Pick type C disease, attenuate cholesterol efflux without altering ABCA1 expression; thus, intestinal NPC1 may facilitate cholesterol movement to ABCA1. ABCA1-mediated cholesterol efflux is independent of cholesterol synthesis. The results suggest that following incorporation into plasma membrane and rafts of the apical membrane, dietary/biliary and newly synthesized cholesterol contribute to the ABCA1 pool and HDL-cholesterol. NPC1 may have a role in this process.     

Up-regulation of Cholesterol Absorption Is a Mechanism for Cholecystokinin-induced Hypercholesterolemia

Excessive absorption of intestinal cholesterol is a risk factor for atherosclerosis. This report examines the effect of cholecystokinin (CCK) on plasma cholesterol level and intestinal cholesterol absorption using the in vivo models of C57BL/6 wild-type and low density lipoprotein receptor knock-out (LDLR^−/−) mice. These data were supported by in vitro studies involving mouse primary intestinal epithelial cells and human Caco-2 cells; both express CCK receptor 1 and 2 (CCK1R and CCK2R). We found that intravenous injection of [Thr²⁸,Nle³¹]CCK increased plasma cholesterol levels and intestinal cholesterol absorption in both wild-type and LDLR^−/− mice. Treatment of mouse primary intestinal epithelial cells with [Thr²⁸,Nle³¹]CCK increased cholesterol absorption, whereas selective inhibition of CCK1R and CCK2R with antagonists attenuated CCK-induced cholesterol absorption. In Caco-2 cells, CCK enhanced CCK1R/CCK2R heterodimerization. Knockdown of both CCK1R and CCK2 or either one of them diminished CCK-induced cholesterol absorption to the same extent. CCK also increased cell surface-associated NPC1L1 (Niemann-Pick C1-like 1) transporters but did not alter their total protein expression. Inhibition or knockdown of NPC1L1 attenuated CCK-induced cholesterol absorption. CCK enhanced phosphatidylinositide 3-kinase (PI3K) and Akt phosphorylation and augmented the interaction between NPC1L1 and Rab11a (Rab-GTPase-11a), whereas knockdown of CCK receptors or inhibition of G protein βγ dimer (Gβγ) diminished CCK-induced PI3K and Akt phosphorylation. Inhibition of PI3K and Akt or knockdown of PI3K diminished CCK-induced NPC1L1-Rab11a interaction and cholesterol absorption. Knockdown of Rab11a suppressed CCK-induced NPC1L1 translocation and cholesterol absorption. These data imply that CCK enhances cholesterol absorption by activation of a pathway involving CCK1R/CCK2R, Gβγ, PI3K, Akt, Rab11a, and NPC1L.     

NPC1L1 (Niemann-Pick C1-like 1) mediates sterol-specific unidirectional transport of non-esterified cholesterol in McArdle-RH7777 hepatoma cells

Recent evidence suggests that NPC1L1 (Niemann-Pick C1-like 1) is critical for intestinal sterol absorption in mice, yet mechanisms by which NPC1L1 regulates cellular sterol transport are lacking. In the study we used a McArdle-RH7777 rat hepatoma cell line stably expressing NPC1L1 to examine the sterol-specificity and directionality of NPC1L1-mediated sterol transport. As previously described, cholesterol-depletion-driven recycling of NPC1L1 to the cell surface facilitates cellular uptake of non-esterified (free) cholesterol. However, it has no impact on the uptake of esterified cholesterol, indicating free sterol specificity. Interestingly, the endocytic recycling of NPC1L1 was also without effect on beta-sitosterol uptake, indicating that NPC1L1 can differentiate between free sterols of animal and plant origin in hepatoma cells. Furthermore, NPC1L1-driven free cholesterol transport was unidirectional, since cellular cholesterol efflux to apolipoprotein A-I, high-density lipoprotein or serum was unaffected by NPC1L1 expression or localization. Additionally, NPC1L1 facilitates mass non-esterified-cholesterol uptake only when it is located on the cell surface and not when it resides intracellularly. Finally, NPC1L1-dependent cholesterol uptake required adequate intracellular K(+), yet did not rely on intracellular Ca(2+), the cytoskeleton or signalling downstream of protein kinase A, protein kinase C or pertussis-toxin-sensitive G-protein-coupled receptors. Collectively, these findings support the notion that NPC1L1 can selectively recognize non-esterified cholesterol and promote its unidirectional transport into hepatoma cells.     

Intestinal SR-BI is upregulated in insulin-resistant states and is associated with overproduction of intestinal apoB48-containing lipoproteins

Intestinal lipid dysregulation is a common feature of insulin-resistant states. The present study investigated alterations in gene expression of key proteins involved in the active absorption of dietary fat and cholesterol in response to development of insulin resistance. Studies were conducted in two diet-induced animal models of insulin resistance: fructose-fed hamster and high-fat-fed mouse. Changes in the mRNA abundance of lipid transporters, adenosine triphosphate cassette (ABC) G5, ABCG8, FA-CoA ligase fatty acid translocase P4, Niemann-Pick C1-Like1 (NPC1L1), fatty acid transport protein 4 (FATP4), and Scavenger Receptor Class B Type I (SR-BI), were assessed in intestinal fragments (duodenum, jejunum, and ileum) using quantitative real-time PCR. Of all the transporters evaluated, SR-B1 showed the most significant changes in both animal models examined. A marked stimulation of SR-B1 expression was observed in all intestinal segments examined in both insulin-resistant animal models. The link between SR-BI expression and intestinal lipoprotein production was then examined in the Caco-2 cell model. SR-B1 overexpression in Caco-2 cells increased apolipoprotein B (apoB) 100 and apoB48 secretion, whereas RNAi knock down of SR-B1 decreased secretion of both apoB100 and apoB48. We also observed changes in subcellular distribution of SR-B1 in response to exogenous lipid and insulin. Confocal microscopy revealed marked changes in SR-BI subcellular distribution in response to both exogenous lipids (oleate) and insulin. In summary, marked stimulation of intestinal SR-BI occurs in vivo in animal models of diet-induced insulin resistance, and modulation of SR-BI in vitro regulates production of apoB-containing lipoprotein particles. We postulate that apical and/or basolateral SR-BI may play an important role in intestinal chylomicron production and may contribute to chylomicron overproduction normally observed in insulin-resistant states.     

Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia

Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.