Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [ 3 H]AS. Chimeric and mutational studies indicate that high-affinity binding of W hole-body cholesterol homeostasis is maintained through three major pathways: de novo synthesis, intestinal absorption, and biliary excretion. Absorption of dietary and biliary cholesterol occurs in the proximal jejunum of the small intestine (1), and this process is blocked by ezetimibe (EZE), a drug used for the treatment of hypercholesterolemia. EZE, a potent cholesterol and phytosterol uptake inhibitor, effectively lowers circulating plasma cholesterol in humans by 15-20% (2), and its coadministration with 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitors (statins), inhibitors of cholesterol synthesis, leads to further reductions in cholesterol plasma levels (3).By searching expressed sequence tag databases for the presence of a sterol-sensing domain (SSD), a plasma membrane secretion signal, and enriched expression in intestinal enterocytes, the Niemann-Pick C1-Like 1 (NPC1L1) protein was identified in 2004 as a potential candidate gene for the EZE-sensitive pathway of cholesterol absorption (4). Mice deficient in NPC1L1 were found to have Ϸ70% reduction in sterol absorption, with the residual component being insensitive to EZE (4), suggesting that NPC1L1 is a critical component of cholesterol uptake in enterocytes (4). The use of enterocyte brush border membranes (BBMs) from several species, including NPC1L1 KO mice (5), or membranes derived from cells expressing recombinant NPC1L1, has provided strong evidence for NPC1L1 being the target to which EZE binds (5).More recently, in vitro studies have demonstrated EZE-sensitive cholesterol transport into McArdles RH7777 hepatoma (6), CaCo-2 (7), and MDCKII cells (8) overexpressing NPC1L1. However, although EZE-sensitive cholesterol transport correlates well with the amount of NPC1L1 expression (7, 8), it is not possible to determine whether NPC1L1 functions alone or as part of a multiprotein complex [SR-B1 (9-15), CD36 (14), CD13 (9), caveolin-1/annexin-2 (16)], facilitating the transfer of cholesterol from outside the cell to internal cholesterol pools (9, 15). Furthermore, it has been speculated that EZE may inhibit cholesterol transfer by binding to some of these other targets, in addition to its inhibition of NPC1L1 (9).In the present study, we attempted to determine whether EZE binds to NPC1L1 directly by purifying an EZE-NPC1L1 complex and analyzing its constituents by mass spectrometry. Quantitative analysis unambiguously demonstrated that NPC1L1 is the only prote...

Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [ 3 H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 ϫ 10 5 [ 3 H]AS sites/ cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC 50 blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.

Functional capacity in exercise stress testing is an independent predictor of cardiac events. Routine use of nuclear perfusion imaging increases radiation burden and cost. Our goal was to assess the clinical utility of exercise functional capacity with stress electrocardiogram (ECG) as an adjunct in predicting the presence of high-risk obstructive coronary artery disease (CAD) on diagnostic coronary angiography. We performed a retrospective study of patients who underwent exercise stress testing for the evaluation of chest pain and underwent diagnostic coronary angiography within the subsequent 3 months. High-risk CAD was defined as coronary artery diameter stenosis of ≥70% in the proximal left anterior descending artery, ≥70% diameter stenosis in 3 major epicardial arteries, or ≥50% diameter stenosis in the left main artery. Univariable and multivariable analyses were performed to identify predictors of high-risk CAD. Of the 412 patients, 105 (25%) had high-risk CAD on coronary angiography. On multivariate logistic regression, we found that positive stress ECG, abnormal stress imaging, left ventricular ejection fraction, and male gender were independent predictors of high-risk CAD. The strongest predictor was positive stress ECG (hazard ratio 3.16, 95% confidence interval 1.90 to 5.27, p <0.001). Functional capacity measures alone were not independent predictors of high-risk CAD. Achieving ≥10 METs with a negative stress ECG resulted in 94% sensitivity and 97% negative predictive value in identifying high-risk CAD. This supports the strategy for provisional use of myocardial perfusion imaging in patients with low functional capacity and/or abnormal stress ECG to minimize cost and radiation exposure.

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