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J. Zhou, T. Terasaki, K. Hosoya, A.K. Kumagai; Increased JNK-interacting Protein 1 (JIP-1) and Oxidative Stress in Response to GLUT1 Overexpression in Rat Retinal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3906.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Diabetic retinopathy (DR), one of the major microvascular complications of diabetes, is characterized by retinal capillary damage, including death of pericytes and endothelial cells and capillary closure. Exposure of human umbilical vein endothelial cells to high glucose causes increased oxidative stress and increased apoptosis through activation of JNK signaling. In this study, we investigated whether increased glucose transport per se causes changes in JNK signaling and oxidative stress in rat retinal endothelial cells. Methods: An established GLUT1-overexpressing rat retinal endothelial cell line was used in this study. Expression of GLUT1 was determined by western blot. Intracellular glucose was measured by GC-Mass spectrometry. Changes in JIP-1 gene expression were detected by cDNA microarray in replicated experiments. Expression of JIP-1 gene was confirmed by Northern blot. Western blot and immunoprecipitation were used to determine protein expression. Results: Clones overexpressing GLUT1 showed greater than six-fold increase in GLUT1 and 30-60% increases in Vmax and glucose transport, as assessed by 2DG uptake. In addition, the intracellular glucose was also increased approximately 44% in GLUT1-overexpressing cells. GLUT1-overexpressing cells had a 71% increase in DCF fluorescence, indicative of the presence of elevated levels of reactive oxygen species (ROS). Furthermore, western blot analysis demonstrated increased lipid peroxidation in GLUT1-overexpressing cells, as evidenced by increased malondialdehyde (MDA)-protein adducts. Immunoblotting also showed an increase in phospho-JNK (active form) and no change in total JNK in GLUT1-overexpressing cells. Analysis of differentially expressed genes in GLUT1-overexpressing cells by cDNA microarray showed increased expression of JIP-1, a scaffold protein required for JNK activation. There was an approximate 80% increase in JIP-1 mRNA by Northern blot. JIP-1 protein level was also increased. Conclusions: GLUT1 overexpression and increased glucose flux resulted in increased oxidative stress and JNK phosphorylation in rat retinal endothelial cells. In addition, the expression of JIP-1, a protein critical in the regulation of JNK activity, was also increased. Further study of JIP-1 and its role in regulating JNK activity in response to increased GLUT1 expression and increased glucose flux may help to elucidate the mechanisms of diabetic retinopathy.
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