Role of Beta-Adrenergic Receptor Signaling in Regulation of Apoptosis in Differing Glucose Environments

Abstract

Purpose: : The goal of this study was to investigate the cellular signaling involved in beta-adrenergic receptor regulation of apoptosis in serum-starved human retinal endothelial cells (HREC) cultured in high and low glucose conditions

Methods: : Cells were grown in high and low glucose until 80% confluency was reached. Cells cultured in high (25mM) and low glucose (5mM) conditions were serum starved for 18-24 hours, followed by treatment with a beta-1-adrenergic receptor agonist, xamoterol (10uM), for 15, 30, and 45 minutes. Non-treated controls were also collected. In addition, high glucose or low glucose serum-starved cells were treated with Fas inhibitor, followed by stimulation with xamoterol at the 30-minute time point. Immunoblotting was performed for Fas ligand, Fas, and Fas-Associated Death Domain (FADD) for all not treated samples compared to the collected time points in high and low glucose. In addition, ELISA analyses for active caspase-8 and -3 were completed. TUNEL slides were prepared for all to visualize apoptosis.

Results: : Treatment of HREC in high glucose media with xamoterol significantly decreased levels of pro-apoptotic Fas-ligand, while Fas-ligand levels were increased in low glucose cells. Fas receptor levels were significantly decreased in high glucose samples treated with xamoterol, in contrast to low glucose levels, where the protein levels were elevated. Xamoterol significantly increased the phosphorylation of FADD in HREC cultured in both high and low glucose conditions. Cleaved caspase-8 levels decreased significantly in high glucose following xamoterol treatment and increased in low glucose. Caspase-3 levels decreased significantly in high glucose cells treated with xamoterol, but was increased significantly in low glucose treated cells. High glucose caspase-3 levels showed no change in cells treated with Fas inhibitor and xamoterol, indicating that the effects of xamoterol on apoptosis occur through Fas signaling.

Keywords: apoptosis/cell death • signal transduction • diabetic retinopathy