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R. Fernandes, A.L. Carvalho, A.K. Kumagai, R. Seiça, K.–I. Hosoya, T. Terasaki, J. Murta, P. Pereira, C. Faro; Downregulation of retinal GLUT1 in diabetes by the ubiquitin proteasome pathway . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3255.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the effect of chronic hyperglycemia on regulation of the levels of the glucose transporter GLUT1 in retina by a ubiquitin–proteasome pathway mechanism. Methods: GLUT1 content was determined by Western Blotting. Glut1 mRNA was determined by RT–PCR and Northern Blotting. Ubiquitin conjugates were evaluated by Western blot analyses. Evidence for GLUT1 ubiquitinylation was determined by transfecting HEK293 cells with a 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 animals and in the high glucose–treated 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. GLUT1 is posttranslationally modified in response to proteasome inhibitors showing an extra band compatible with monoubiquitinylated GLUT1. Cells transfected with hemagglutinin–tagged ubiquitin revealed crossreactivity with anti–GLUT1 antibodies on the HA immunoprecipitates. Conclusions: Data shows 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 to its increased degradation by a ubiquitin–proteasome dependent mechanism. Ubiquitin proteasome pathway may perform a relevant role on GLUT1 degradation on diabetes.
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