NPC1L1-dependent transport of 27-alkyne cholesterol in intestinal epithelial cells

Abstract

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol is not fully understood due to the lack of a suitable NPC1L1-substrate that can be traced by fluorescence imaging and biochemical methods. 27-alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labelled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ~75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a non-radioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

Factors Influencing Gallstone Formation: A Review of the Literature

“…Biosynthetic and dietary cholesterol are absorbed by Niemann-Pick C1-Like Protein1 (NPC1L1) on the membranes of intestinal epithelium and further esterified by ACAT (the alternative name is acyl-coenzymeA: cholesterol acyltransferase), which then enters the bloodstream through the lymphatic system and is eventually absorbed by the liver as chylomicrons [15,16]. The proportion of total cholesterol from diet also depends mainly on the efficiency of absorption of cholesterol from the intestinal tract and the amount of cholesterol consumed daily.…”

Characterization of interactions of dietary cholesterol with the murine and human gut microbiome

“…To gain a better understanding of how dietary cholesterol interacts with the gut microbiome, we exposed mice to a modified form of cholesterol (cholesterol alkyne (Chol Alk )) that has been readily used to investigate cholesterol metabolism in established systems 24 , 25 and can be traced through diet–microbiome interactions using the BOSSS workflow 21 , 24 (Extended Data Fig. 1 ).…”

Effects of phytosterol supplementation on lipid profiles in patients with hypercholesterolemia: a systematic review and meta-analysis of randomized controlled trials

“…ABCA1 is a key mediator for the efflorescence of intracellular cholesterol to apolipoprotein A-I (apoA-I) to produce HDL. 104 Phytosterols enter the digestive tract with food and bind to the transporter Niemann-Pick C1-Like 1 (NPC1L1) in the lumen and near the small intestine, 105 where they may compete with cholesterol. Increased expression of these proteins encourages small intestinal cells to secrete absorbed Food & Function Review cholesterol into the lumen, thereby reducing the amount of cholesterol entering the bloodstream.…”