Retinol transport in cultured fat-storing cells of rat liver. Quantitative analysis by anchored cell analysis and sorting system

Abstract

It is difficult to study the mechanism of specific transport of vitamin A in fat-storing cells (FSC) in vivo. In this study, transport of vitamin A added to the medium was quantitatively analyzed in cultured FSCs by means of the spontaneous fluorescence emitted by vitamin A. By density-gradient centrifugation with 38% Percoll, an FSC-rich fraction was separated from normal rat liver cells. The FSCs were observed to retain cytoplasmic fat droplets even on days 3 and 4 of culture. The FSCs containing fat droplets were selected for this experiment by checking their emission of vitamin A fluorescence. To analyze the vitamin A content of isolated cells, we employed a newly developed anchored cell analysis and sorting system (ACAS 470), which provides fluorescence analysis and sorting of adherent cells under the phase contrast microscope by utilizing a laser with its irradiation range narrowed to 1 micron. Vitamin A fluorescence was detectable by this system even in the cultured FSCs. After 24 hours of culture of FSCs in medium with 1 x 10(-6) M vitamin A added, the strength of fluorescence per FSC was 24.3 +/- 11.2 x 10(5)/cell for control, 61.5 +/- 17.6 x 10(5)/cell for retinyl acetate, 26.0 +/- 12.6 x 10(5)/cell for retinyl palmitate, and 59.0 +/- 15.1 x 10(5)/cell for retinol. Thus, retinol and retinyl acetate were transferred to FSCs in significant amounts without the participation of retinol-binding protein. Furthermore, an extended examination was made of the mechanism of the retinol transport observed in this study. Transport was never inhibited by the presence of vitamin E or azide. Retinol may be transferred by passive transport attributable to the concentration gradient rather than by active transport or through cell membrane damage by retinol itself. There was a tendency for inhibition of the transport of retinol into the cells in fetal calf serum. This inhibition may have occurred because the retinol-binding protein or other serum proteins had decreased the concentration of free retinol.