Purchase this article with an account.
AK Kumagai, JA Jamali, BK Deo, K Hosoya, T Terasaki; Overexpression of GLUT1 and Increased Glucose Transport Leads to Apoptosis in Retinal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1342.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Elevated glucose concentrations cause increased endothelial cell death in vitro and in vivo. To determine whether this phenomenon is due to increased glucose flux per se rather than interactions with cell surface receptors (e.g., RAGE or integrins), changes associated with apoptosis were assessed in TRiBRB, a rat retinal endothelial cell line, with stable overexpression of the GLUT1 glucose transporter and increased glucose transport. Methods: Untransfected TRiBRB (Naïve), TRiBRB with GLUT1 overexpression (GLUT1), and vector controls (Vector) were grown in 5 mM or 25 mM glucose for 5 days, and DNA fragmentation in single cells was assessed by a Comet assay. Morphological changes (nuclear condensation and fragmentation) were assessed in GLUT1 or Vector cells grown in 5 mM glucose by staining with bisbenzamide. The effects of inhibition of caspase cleavage on these changes were assessed in the presence and absence of ZVAD-fmk (20 uM), a generalized inhibitor of caspase activity. Results: Raising the glucose concentration from 5 to 25 mM produced approximately 2-fold and 4.5-fold increases in DNA fragmentation in Naïve and Vector control cells, respectively. In contrast, GLUT1 cells grown in 5 mM glucose had a 7-fold increase in DNA fragmentation in comparison to Vector controls in 5 mM glucose and demonstrated no additional increase in DNA fragmentation in 25 mM glucose. Quantitative analysis of bisbenzamide staining revealed a 6.4-fold increase in cells demonstrating apoptotic nuclear changes in the GLUT1 cells compared to Vector controls (22.6 ± 2.0% versus 3.5 ± 0.7%, p < 0.0001). Inhibition of caspase activity with ZVAD-fmk completely reversed both the increased DNA fragmentation and nuclear changes in GLUT1-overexpressing cells grown in 5 mM glucose. Conclusion: Increased retinal endothelial cell glucose transport, via overexpression of GLUT1, increases DNA fragmentation and morphological nuclear changes in a manner mimicking the effects of exposure to high glucose concentrations. Prevention of these changes by caspase inhibition suggests that they are due to programmed cell death. These studies suggest that increased glucose flux and intracellular glucose are responsible for apoptosis in endothelial cells exposed to high glucose.
This PDF is available to Subscribers Only