Retinyl esters are hydrolyzed in early endosomes of J774 macrophages

The aim of the current study was to identify the subcellular compartment(s) responsible for the hydrolysis of chylomicron remnant-retinyl esters, in J774.1 cells. The cells were incubated with medium containing chylomicron remnant [H-3]retinyl ester. Subcellular fractionation was used to separate early endosomes from late endosomes and lysosomes. About 26% and 80% of the total [H-3]retinyl esters taken up by the J774 cells were hydrolyzed after 10 min and 60 min of chase, respectively. In the early endosomes, there was a 4-fold increase of radioactivity (nearly all radioactivity associated with retinyl esters) during the first 10 min of chase. The radioactivity in early endosomes was reduced by 43% from 10 min to 60 min and remained stable from 60 to 180 min of chase. From 10 to 60 min the amount of retinol in early endosomes increased from 44% to 82%, indicating an efficient hydrolysis of retinyl esters, Less than 10% and 5% of the total cell-associated radioactivity was found in the late endosomes and lysosomes during the entire chase period. In the chase medium, 84% of the total amount of retinoid released during 180 min was present already after 10 min. The percentage of retinol in the medium increased from 25% to 82% during incubation from 10 to 180 min. These data suggest that retinyl esters are endocytosed together with the chylomicron remnant particle and hydrolyzed in the early endosomes in this cell model.