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.

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.Cholesterol absorption of both dietary cholesterol and cholesterol cleared from the liver through biliary secretion contributes along with regulation of cholesterol biosynthesis to maintain a tight control of cholesterol homeostasis. The mechanism by which cholesterol moves from the intestinal lumen into the absorptive enterocytes lining the proximal small intestine is poorly understood. The identification of ezetimibe as a potent selective inhibitor of intestinal cholesterol uptake and absorption confirmed this mechanism as a key point of therapeutic intervention for lowering plasma cholesterol levels and indicated that this process is mediated by a specific transporter (1-4). Based on the properties of ezetimibe in animal models of cholesterol uptake, it was predicted that such a transporter would be expressed in jejunal enterocytes and localized to the brush border membrane, which forms the interface between the intestinal lumen and the intracellular compartments responsible for cholesterol esterification and packaging into chylomicrons.Through studies designed to understand the mechanism by which ezetimibe inhibits cholesterol absorption, we recently identified Niemann-Pick C1 Like 1 (NPC1L1) 1 as a critical protein for the intestinal absorption of dietary and biliary cholesterol (5). NPC1L1 was identified through a genomics-bioinformatics approach by sequencing an expression sequence tags library from rat jejunum, annotating the sequences, and searching databases for intestinal proteins with features of a cholesterol transporter (5). NPC1L1 was found to be highly expressed in the jejunum and localized on the surface of the absorptive enterocytes. Mice deficient in NPC1L1 exhibited a significant reduction in chol...

The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface receptor that can bind and is activated by BAs, but its physiological role is unclear. Using targeted deletion of the Gpbar1 gene in mice, we show that the gene plays a critical role in the maintenance of bile lipid homoeostasis. Mice lacking Gpbar1 expression were viable, developed normally and did not show significant difference in the levels of cholesterol, BAs or any other bile constituents. However, they did not form cholesterol gallstones when fed a cholic acid-containing high-fat diet, and liver-specific gene expression indicated that Gpbar1-deficient mice have altered feedback regulation of BA synthesis. These results suggest that Gpbar1 plays a critical role in the formation of gallstones, possibly via a regulatory mechanism involving the cholesterol 7alpha-hydroxylase pathway.

1 Previous studies described the metabolism-based discovery of a potent, selective inhibitor of intestinal absorption of cholesterol, SCH58235 (Ezetimibe). Here we demonstrate that the phenolic glucuronide (SCH60663) of SCH58235, was more potent at inhibiting cholesterol absorption in rats than SCH58235, when administered by the intraduodenal route. 2 To understand the increased potency of the glucuronide, the metabolism and distribution of SCH58235 and SCH60663 were studied in bile duct-cannulated rats. 3 One minute after intraduodenal delivery of SCH58235, signi®cant levels of compound were detected in portal plasma; 495% was glucuronidated, indicating that the intestine was metabolizing SCH58235 to its glucuronide. When intraduodenally delivered as SCH58235, the compound was glucuronidated, moved through the intestinal wall, into portal plasma, through the liver, and into bile. However, when delivered as SCH60663, 495% of the compound remained in the intestinal lumen and wall, which may explain its increased potency. Signi®cant inhibition of cholesterol absorption and glucuronidation of SCH58235 occurred when SCH58235 was intravenously injected into bile duct-cannulated rats. Autoradiographic analysis demonstrated that drug related material was located throughout the intestinal villi, but concentrated in the villus tip. 4 These data indicate that (a) SCH58235 is rapidly metabolized in the intestine to its glucuronide; (b) once glucuronidated, the dose is excreted in the bile, thereby delivering drug related material back to the site of action and (c) the glucuronide is more potent than the parent possibly because it localizes to the intestine. Taken together, these data may explain the potency of SCH58235 in the rat (ID 50 =0.0015 mg kg 71) and rhesus monkey (ID 50 =0.0005 mg kg 71 ).

Abstract-Ezetimibe (SCH58235) is a potent, selective, cholesterol absorption inhibitor. The objective of this study was to determine whether ezetimibe reduces plasma cholesterol and inhibits atherogenesis in apolipoprotein E knockout (apoEϪ/Ϫ) mice. Cholesterol absorption was inhibited by Ͼ90% at doses of ezetimibe Ͼ3 mg/kg in apoEϪ/Ϫ mice. Atherosclerosis and lipoprotein changes were determined in apoEϪ/Ϫ mice fed a high-fat (0.15% cholesterol) "western" diet, a low-fat (0.15% cholesterol) diet, or a semisynthetic cholesterol-free diet with or without ezetimibe (5 mg/kg per day) for 6 months. Ezetimibe reduced plasma cholesterol levels from 964 to 374 mg/dL, from 726 to 231 mg/dL, and from 516 to 178 mg/dL in the western, low-fat, and cholesterol-free diet groups, respectively. The reductions occurred in the very low density and low density lipoprotein fractions, whereas high density lipoprotein cholesterol levels were increased by ezetimibe treatment. Ezetimibe reduced aortic atherosclerotic lesion surface area from 20.2% to 4.1% in the western diet group and from 24.1% to 7.0% in the low-fat cholesterol diet group. Ezetimibe reduced carotid artery atherosclerotic lesion cross-sectional area by 97% in the western and low-fat cholesterol groups and by 91% in the cholesterol-free group. Ezetimibe inhibits cholesterol absorption, reduces plasma cholesterol, increases high density lipoprotein levels, and inhibits the progression of atherosclerosis under western, low-fat, and cholesterol-free dietary conditions in apoEϪ/Ϫ mice. Although apoEϪ/Ϫ mice are more hypercholesterolemic than are humans and low density lipoprotein reductions with ezetimibe are not as pronounced clinically, ezetimibe may inhibit atherogenesis in individuals consuming restricted-fat or western diets. Key Words: cholesterol absorption Ⅲ ezetimibe Ⅲ atherosclerosis Ⅲ hypercholesterolemia T he abnormal metabolism and elevation of plasma cholesterol and lipoproteins are well-documented risk factors for the development of atherosclerosis. Evidence from clinical trials indicates that reducing plasma cholesterol by dietary and/or pharmacological means leads to reductions in the incidence of death from cardiovascular disease. 1-4 Present pharmacological interventions include the inhibition of cholesterol biosynthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase (statins) either alone or in combination with other agents. Ezetimibe (SCH58235) is the first of a new class of selective cholesterol absorption inhibitors that may represent a new option for the pharmacological treatment of hypercholesterolemia. 5,6 Ezetimibe selectively inhibits the intestinal uptake and absorption of dietary and biliary cholesterol at the brush border of small intestinal enterocytes, confining cholesterol to the intestinal lumen for subsequent excretion. 6,7 Results from preclinical studies in various animal models have demonstrated the lipid-lowering properties of ezetimibe as a single agent [5][6][7][8][9] and the synergistic cholesterol-lowering...

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1 Ezetimibe potently inhibits the transport of cholesterol across the intestinal wall, thereby reducing plasma cholesterol in preclinical animal models of hypercholesterolemia. The e ect of ezetimibe on known absorptive processes was determined in the present studies. 2 Experiments were conducted in the hamster and/or rat to determine whether ezetimibe would a ect the absorption of molecules other than free cholesterol, namely cholesteryl ester, triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid. In addition, to determine whether exocrine pancreatic function is involved in the mechanism of action of ezetimibe, a biliary anastomosis model, which eliminates exocrine pancreatic function from the intestine while maintaining bile¯ow, was established in the rat. 3 Ezetimibe reduced plasma cholesterol and hepatic cholesterol accumulation in cholesterol-fed hamsters with an ED 50 of 0.04 mg kg 71 . Utilizing cholesteryl esters labelled on either the cholesterol or the fatty acid moiety, we demonstrated that ezetimibe did not a ect cholesteryl ester hydrolysis and the absorption of fatty acid thus generated in both hamsters and rats. The free cholesterol from this hydrolysis, however, was not absorbed (92 ± 96% inhibition) in the presence of ezetimibe. Eliminating pancreatic function in rats abolished hydrolysis of cholesteryl esters, but did not a ect the ability of ezetimibe to block absorption of free cholesterol (794%). Ezetimibe did not a ect the absorption of triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid in rats. 4 Ezetimibe is a potent inhibitor of intestinal free cholesterol absorption that does not require exocrine pancreatic function for activity. Ezetimibe does not a ect the absorption of triglyceride as a pancreatic lipase inhibitor (Orlistat) would, nor does it a ect the absorption of vitamin A, D or taurocholate, as a bile acid sequestrant (cholestyramine) would.

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