Abstract
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.
Highlights
The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2
To address whether cholesterol influx from the apical membrane of intestinal cells contributes to ABCA1-mediated HDL formation, cells that were cultured on membranes separating an upper and lower well were labeled with cholesterol overnight to uniformly label all cholesterol pools
The results of this study show that in intestinal cells, the influx of endogenous apical membrane cholesterol enhances gene and protein expression of ABCA1, resulting in increased efflux of cellular cholesterol to apolipoprotein A-I (apoA-I) and HDL production
Summary
The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2. In cells incubated in the absence of the LXR agonist (open bars), the amount of labeled cholesterol recovered in the basolateral medium increased in a concentration-dependent manner in response to the influx of micellar cholesterol (Fig. 1B).
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