Abstract
Purpose: :
Increased oxidative stress is implicated in the pathogenesis of diabetic complications, including diabetic retinopathy. The objective of this study is to assess the effect of oxidative stress in regulating GLUT-1 dependent glucose transport in retinal endothelial cells.
Methods: :
Retinal endothelial cells in culture (TR-iBRB) were subjected to oxidative stress by incubation with glucose oxidase. Formation of protein carbonyls was used as a marker of oxidative damage to TR-iBRB. Level of GLUT1 mRNA was determined by real time RT-PCR. GLUT1 protein levels were determined by western blot. Where appropriate, the pool of GLUT-1 present at plasma membrane was assessed following biotinylation of the membrane proteins. The glucose transport into endothelial cells and activity of GLUT-1 were measured by 3H-deoxyglucose uptake. Subcellular distribution of GLUT-1 was assessed by fluorescence confocal microscopy.
Results: :
Incubation of endothelial cells with glucose oxidase leads to an accumulation of oxidized proteins. Oxidative stress also induces a decrease in the levels of both GLUT-1 mRNA and protein. Biotinylation assays and confocal microscopy further revealed that exposure to oxidative stress reduces the pool of GLUt-1 present at plasma membrane. Consistently, glucose transport, as revealed by 3H-deoxyglucose uptake, is decreased under oxidative stress. Incubation of TR-iBRB, previously exposed to oxidative stress, with the proteasome inhibitors MG-132 or lactacystine restores glucose transport to control levels.
Conclusions: :
Data suggested that exposure of retinal endothelial cells to oxidative stress leads to a downregulation of glucose transport, which presumably results both from a down-regulation of GLUT-1 production and a proteasome-dependent subcellular re-distribution of the glucose transporter. The reduction of GLUT-1 at plasma membrane, induced by increased oxidative stress, may contribute for the deregulation of glucose homeostasis in diabetes.
Keywords: diabetic retinopathy • oxidation/oxidative or free radical damage • retinal degenerations: cell biology