Reactive oxygen species downregulate glucose transport system in retinal 3 endothelial cells 4 5

26 Retinal endothelial cells are believed to play an important role in the pathogenesis of 27 diabetic retinopathy. In previous studies, we and others demonstrated that glucose 28 transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased 29 oxidative stress is likely to be the event whereby hyperglycemia is transduced into 30 endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 31 regulation are not clearly established. The objective of this study is to evaluate the 32 effect of increased oxidative stress on glucose transport and on GLUT1 subcellular 33 distribution in a retinal endothelial cell line and to elucidate the signaling pathways 34 associated with such regulation. Conditionally immortalized rat retinal endothelial cells 35 (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular 36 hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed 37 that oxidative stress did not alter the total levels of GLUT1 protein, although the levels 38 of mRNA were decreased, and there was a subcellular redistribution of GLUT1, 39 decreasing its content at the plasma membrane. Consistently, the halflife of the protein 40 at plasma membrane markedly decreased under oxidative stress. The proteasome 41 appears to be involved in GLUT1 regulation in response to oxidative stress, as 42 revealed by an increase in stabilization of the protein present at the plasma membrane 43 and normalization of glucose transport following proteasome inhibition. Indeed, levels 44 of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. 45 Furthermore, data indicates that Akt activation is involved in the stabilization of GLUT1 46 at the plasma membrane. Thus, subcellular redistribution of GLUT1 under conditions of 47 oxidative stress is likely to contribute to the disruption of glucose homeostasis in 48 diabetes. 49