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J Zhou, BK Deo MS, T Terasaki PhD, K Hosoya PhD, AK Kumagai MD; Identification and Initial Characterization of Differentially Expressed Genes in Rat Retinal Endothelial Cells With Glucose Transporter 1 (GLUT1) Overexpression . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1344.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Hyperglycemia from poorly controlled diabetes has been associated with an increased risk of diabetic retinopathy. In order to understand the effects of increased glucose transport per se on retinal endothelial cell gene expression, we used a rat retinal endothelial cell culture model with stable GLUT1 overexpression to identify genes that may be involved in glucose transport and/or glucose metabolism in retinal endothelial cells. Methods:A rat retinal endothelial cell line (TRiBRB) was transfected with vector containing full-length human GLUT1 cDNA. Clones overexpressing GLUT1 were propagated through G418 selection. Expression of GLUT1 was determined by Western Blot. Changes in Vmax and glucose transport were assessed by [3H]-2-deoxyglucose (2DG) uptake. To identify differentially expressed genes, we isolated mRNA from cells with GLUT1 overexpression and control cells and performed suppression subtractive hybridization using a PCR-select cDNA Subtraction kit (CLONTECH). Differentially expressed genes were confirmed by Northern blot and QT RT-PCR. Results:There was approximately a four-fold increase in GLUT1 expression in stable transfected TRiBRB cells as compared to control cells. The increased GLUT1 protein expression resulted in approximately 30-60% increases in Vmax and glucose transport as assessed by 2DG uptake. By using subtractive hybridization, we isolated and partially sequenced four genes that were either up or down regulated upon increased GLUT1 expression. The extent of up or down regulation at mRNA level was up to 50% as determined by Northern blot and QT RT-PCR. The tissue distribution of those genes were also examined by Northern blot and QT RT-PCR. Conclusion:This study has identified differentially expressed genes using rat retinal endothelial cells with GLUT1 overexpression. The altered expression pattern of those genes suggests they may play a role in regulating glucose transport and/or glucose metabolism upon increased flux of glucose in rat retinal endothelial cells. Further characterization of those genes and gene products will help to elucidate their roles in the formation of diabetic retinopathy.
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