Glucose-dependent regulation of DNA synthesis in bovine retinal endothelial cells

Abstract

PURPOSE. Clinical trials have demonstrated that the onset and progression of diabetic retinopathy is influenced by the glucose control of the patient. The disease is characterised by the coexistence of impaired cell growth and excessive cell proliferation, and we wished to determine the effect that glucose has upon these parameters. METHODS. Bovine retinal endothelial cells were exposed to a range of glucose concentrations from 0–25 mmol/l. The level of DNA synthesis and cell number was then determined using pulse labelling with tritiated thymidine and a Coomassie blue dye-based assay, respectively. RESULTS. The level of DNA synthesis declined significantly as the concentration of glucose increased. DNA synthesis was further decreased by the presence of an inhibitor of PI3 kinase (Wortmannin). The decline in DNA synthesis was abrogated by the presence of a protein kinase C (PKC) inhibitor or by incubating the cells with antibodies specific for the GLUT-1 and GLUT-3 specific isoforms of glucose transporter proteins. TGF-ß antibody significantly increased the level of DNA synthesis in cells exposed to high concentrations of glucose. The changes that are observed in the level of DNA synthesis was not coincident with any significant changes in cell number as measured by the Coomassie blue assay. CONCLUSIONS. This demonstrated that the decline in DNA synthesis is dependent upon the entry of glucose into the cells and that this is mediated via a PKC dependent pathway.