PURPOSE: To investigate the effect of chronic hyperglycemia on the levels of the glucose transporter GLUT1 in retina and its ubiquitinylation. METHODS: Two diabetic animal models (Goto Kakizaki rats and alloxan-induced diabetic rabbits) and retinal endothelial cells in culture were used. GLUT1 content was determined by western blotting. Glut1 mRNA was determined by RT-PCR and northern blotting. Ubiquitin conjugates were evaluated by western blot analysis. In vitro ubiquitin conjugation activity was evaluated in supernatants using radiolabeled ubiquitin. Evidence for GLUT1 ubiquitinylation was further investigated by transfecting HEK293 cells with a hemagglutinin (HA)-tagged ubiquitin cDNA followed by immunoprecipitation of the cell lysates. RESULTS: Chronic hyperglycemia resulted in a significant decrease on the amount of GLUT1 protein without significant changes on the GLUT1 mRNA in the retinas of diabetic GK rats and alloxan treated rabbits, and in high glucose treated retinal endothelial cells, compared to controls. The content of high molecular weight ubiquitin conjugates was higher both in the membrane fractions of diabetic retinas and in endothelial cells incubated with high glucose concentrations. GLUT1 immunoprecipitated from diabetic retinas crossreacted with antibodies directed against ubiquitin suggesting that GLUT1 is posttranslationally modified by monoubiquitinylation. Cells transfected with HA-tagged ubiquitin revealed crossreactivity with anti-GLUT1 antibodies on the HA immunoprecipitates. CONCLUSIONS: The data indicate that retinal GLUT1 abundance decreases in experimental diabetes and with exposure of retinal endothelial cells to elevated glucose concentrations. Results further suggest that decreased abundance of GLUT1 may be associated with its increased degradation by a ubiquitin dependent mechanism. Ubiquitinylation of GLUT1 may be the mechanism targeting GLUT1 for degradation in diabetes.
PURPOSE: To investigate the effect of chronic hyperglycemia on the levels of the glucose transporter GLUT1 in retina and its ubiquitinylation. METHODS: Two diabetic animal models (Goto Kakizaki rats and alloxan-induced diabetic rabbits) and retinal endothelial cells in culture were used. GLUT1 content was determined by western blotting. Glut1 mRNA was determined by RT-PCR and northern blotting. Ubiquitin conjugates were evaluated by western blot analysis. In vitro ubiquitin conjugation activity was evaluated in supernatants using radiolabeled ubiquitin. Evidence for GLUT1 ubiquitinylation was further investigated by transfecting HEK293 cells with a hemagglutinin (HA)-tagged ubiquitin cDNA followed by immunoprecipitation of the cell lysates. RESULTS: Chronic hyperglycemia resulted in a significant decrease on the amount of GLUT1 protein without significant changes on the GLUT1 mRNA in the retinas of diabetic GK rats and alloxan treated rabbits, and in high glucose treated retinal endothelial cells, compared to controls. The content of high molecular weight ubiquitin conjugates was higher both in the membrane fractions of diabetic retinas and in endothelial cells incubated with high glucose concentrations. GLUT1 immunoprecipitated from diabetic retinas crossreacted with antibodies directed against ubiquitin suggesting that GLUT1 is posttranslationally modified by monoubiquitinylation. Cells transfected with HA-tagged ubiquitin revealed crossreactivity with anti-GLUT1 antibodies on the HA immunoprecipitates. CONCLUSIONS: The data indicate that retinal GLUT1 abundance decreases in experimental diabetes and with exposure of retinal endothelial cells to elevated glucose concentrations. Results further suggest that decreased abundance of GLUT1 may be associated with its increased degradation by a ubiquitin dependent mechanism. Ubiquitinylation of GLUT1 may be the mechanism targeting GLUT1 for degradation in diabetes.
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