INSULIN STIMULATES ADRENOMEDULLIN (AM) SYNTHESIS IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS (hRPE).

Abstract

Abstract: : Purpose: Several years after the onset, diabetic retinopathy (DR) can occur in patients with diabetes mellitus (DM). Since insulin is the most common therapy in advanced type I and II DM and elevated levels of AM have been reported in the vitreous fluid of DR patients, we investigated the effect of insulin on adrenomedullin synthesis in hRPE cells. Methods: Primary hRPE cell cultures were established from human eyes. Cells were treated with insulin and tyrphostin AG490 (AG490), an inhibitor of STAT signaling pathway. Cell proliferation was monitored by 3H–thymidine (3H–thy) incorporation and the trypan blue exclusion methods. 14C–methionine labeled intracellular AM (14C–AM) synthesis was determined by immunoprecipitation using AM specific antibody. AM specific antibody was also used for immunohistochemistry. Data were analyzed by Student 't' test. Results: Insulin increased hRPE cell proliferation in a dose dependent manner. The addition of AG490 inhibited insulin stimulated hRPE cell proliferation as monitored by 3H–thy incorporation (1433.5±112.2 vs. 3521.0±224.4, p<=0.05, mean±SEM,n=4). Insulin also increased 14C–AM synthesis in a dose dependent manner. The addition of AG490 to insulin exposed hRPE cells inhibited corresponding 14C–AM synthesis (3157.2±1734.0 vs.4512.2±2452.5, p<=0.05, mean±SEM,n=4). Immunohistochemical studies showed increased positive immunoreactivity of AM in hRPE cells exposed to insulin. The addition of AG490 to insulin exposed to hRPE cells inhibited corresponding AM protein immunoreactivity. Conclusion: Insulin is a mitogen for hRPE cells and it stimulates AM synthesis. AG490 inhibits insulin stimulated AM synthesis as well as hRPE cell proliferation. The data suggest that AM produced locally in hRPE cells may be involved in the pathogenesis of diabetic retinopathy.

Keywords: diabetic retinopathy • growth factors/growth factor receptors • diabetes