Insulin Stimulation of Retinal Outer Segment Uptake by Cultured Human Retinal Pigment Epithelial Cells Determined by a Flow Cytometric Method

This study investigates the mechanism by which insulin stimulates phagocytosis in cultured human retinal pigment epithelium, using a flow cytometric assay of retinal outer segment uptake. RPE cells were isolated from adult human donors and grown in culture. Retinal outer segments were isolated from fresh bovine eyes and covalently labeled with the fluorescent dye carboxy-SNAFL-2. Retinal outer segment binding and uptake was quantified by direct visualization and the increase in cellular fluorescence measured using a flow cytometer and the methods compared. Uptake measured by flow cytometry was further characterized by concentration, time, temperature and serum stimulation, and shown to be comparable to other published methods. Uptake of retinal outer segments was acutely stimulated by insulin in the absence or presence of serum with half maximal stimulation occurring between 0·1 and 1·0 μg ml -1. Theophylline, forskolin and cholera toxin all reduced retinal outer segment uptake by RPE cells, but had no effect upon insulin stimulation. Measurements of cAMP showed that insulin did not change intracellular cAMP concentration compared to controls in the absence or presence of added retinal outer segments. One hundred nanomolar okadaic acid also inhibited retinal outer segment uptake but not insulin-stimulated uptake, nor did 100 μM genistein have an effect upon insulin-stimulated uptake. Preincubation of RPE cells with 25 μM ZnCl 2 overnight stimulated retinal outer segment uptake and appeared to inhibit the insulin stimulation. Preincubation of the cells in 25 mM glucose overnight also increased the uptake of retinal outer segments over control and reduced the effect of insulin. We conclude that insulin stimulates retinal outer segment phagocytosis by an as yet unknown process which may involve specific tyrosine phosphatases.