Induction of autophagy by different glucose levels in human retinal pigment epithelium

Purpose: Diabetic retinopathy(DR) is one of the leading causes of blindness in the world. Autophagy as an intracellular clearance mechanism gets activated under varying nutrient conditions.Our aim was to investigate the presence of autophagy in retinal pigment epithelial(RPE) cells under different glucose concentrations as in vitro model for DR.
Methods:ARPE-19 and primary human RPE cells were cultured in media containing three different glucose concentrations: F12-DMEM medium (3.18g/L), DMEM High glucose(4.5g/L) and DMEM Low glucose(1g/L). In addition, a daily alternating(low and high glucose) medium was used to mimic diabetic conditions. Both cell types were kept in culture for 6 days.Light microscopy was performed to determine morphological differences,while proliferation rate and cell viability were measured in parallel to detection of autophagy by Western blot (quantification of LC3II/I ratio, p62 expression)and fluorescence microscopy of GFP-LC3 transfected cells.
Results: Remarkable morphological differences were found in both cell types with respect to different glucose concentrations. Cells grown in alternating glucose medium assumed polygonal morphology and large size compared to those grown in high and low glucose-containing medium – the latter were more elongated and round,respectively. F-12-DMEM medium-grown cells were inhomogenous in shape and smallest in size. Viability of the cells under all growth conditions showed similar pattern, while proliferation rate was highest in alternating glucose medium, followed by high glucose, F-12-DMEM and low glucose-containing medium. Autophagy was induced under high and alternating glucose concentrations, as well as F12-DMEM medium.
Conclusions:Alternating glucose levels can lead to higher proliferation rate,bigger size and higher induction of autophagy in RPE cells in vitro. Exploring metabolic pathways under different glucose concentrations can be a good model for studying DR, as well as finding new pharmacological agents affecting this disease.