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Abstract: 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. [Copyright &y& Elsevier]

Abstract: Fluorescence-based reverse transcription real-time quantitative polymerase chain reaction (RT–QPCR) is a highly sensitive method for the detection and quantitation of mRNA. To control and correct for sample variability, some common housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and ubiquitin are often used as endogenous standards. Other internal calibrators such as 18S–ribosomal RNA (18S–RNA) have also been used, but further methodological concerns arise given that ribosomal RNA lacks the 3′ poly-A tail typically associated with messenger RNA. To take advantage of the constant expression levels of 18S–RNA and the precision of oligo-(dT) primed first-strand synthesis, we have developed a method that combines oligo-(dT) with an 18S–RNA-specific primer in the initial reverse transcription (RT) reaction. This strategy, termed coapplication reverse transcription (Co–RT), allows for the analysis of multiple target genes with the advantages of 18S–RNA normalization from a single RT reaction. In this article, we describe Co–RT and present tissue distribution and expression level analysis of several target genes using this method. Co–RT provides increased sensitivity and higher accuracy than do the standard random primed RT methods. [Copyright &y& Elsevier]

Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1 Like 1 (NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption. [ABSTRACT FROM AUTHOR]

The molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining candidate genes involved in these processes a fluorescence-activated cell sorter-based, retroviral-mediated expression cloning strategy has been devised. SCH354909, a fluorescent derivative of ezetimibe, a compound which blocks intestinal cholesterol absorption but whose mechanism of action is unknown, was synthesized and shown to block intestinal cholesterol absorption in rats. Pools of cDNAs prepared from rat intestinal cells enriched in enterocytes were introduced into BW5147 cells and screened for SCH354909 binding. Several independent clones were isolated and all found to encode the scavenger receptor class B, type I (SR-BI), a protein suggested by others to play a role in cholesterol absorption. SCH354909 bound to Chinese hamster ovary (CHO) cells expressing SR-BI in specific and saturable fashion and with high affinity (Kd∼18 nM). Overexpression of SR-BI in CHO cells resulted in increased cholesterol uptake that was blocked by micromolar concentrations of ezetimibe. Analysis of rat intestinal sections by in situ hybridization demonstrated that SR-BI expression was restricted to enterocytes. Cholesterol absorption was determined in SR-B1 knockout mice using both an acute, 2-h, assay and a more chronic fecal dual isotope ratio method. The level of intestinal cholesterol uptake and absorption was similar to that seen in wild-type mice. When assayed in the SR-B1 knockout mice, the dose of ezetimibe required to inhibit hepatic cholesterol accumulation induced by a cholesterol-containing ‘western’ diet was similar to wild-type mice. Thus, the binding of ezetimibe to cells expressing SR-B1 and the functional blockade of SR-B1-mediated cholesterol absorption in vitro suggest that SR-B1 plays a role in intestinal cholesterol metabolism and the inhibitory activity of ezetimibe. In contrast studies with SR-B1 knockout mice suggest that SR-B1 is not essential for intestinal cholesterol absorption or the activity of ezetimibe. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Niemann­Pick 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. [ABSTRACT FROM AUTHOR]

Fluorescent analogues of the cholesterol absorption inhibitor (CAI), Sch 58235, have been designed and synthesized as single enantiomers. Biological testing reveals that they are potent CAIs and are suitable tools for the investigation of the azetidinone CAI mechanism of action (MOA). [Copyright &y& Elsevier]