Abstract
It has been reported that endothelial cells are extremely sensitive to cytotoxic effect of active oxygen radicals (O2-, H2O, OH·). Oxygen radicals can be scavenged by superoxide dismutase, catalase or glutathione redox (GR) cycle. Interestingly, a previous paper suggest a significant role of GR cycle for maintaining endothelial cell integrity by scavenging hydrogen peroxide. It is clear that GR cycle activity is dependent on NADPH supply as a result of activation of hexose monophosphate shunt (HMS) pathway. Therefore, high medium glucose concentration can modulate intracellular GR cycke activity. In the present study, we studied a possible impairment of GR cycle in the cells cultured in the presence of high glucose media due to an insufficient NADPH supply as a result of impaired HMS activation in the presence of hydrogen peroxide in cultured human umbilical vein endothelial cells.
Glutathione redox cycle-dependent H2O2 degradation in endothelial cells cultured in the presence of 5.5 mM (NG), 11 mM, 22 mM, 33 mM (HG) or 66 mM D-glucose for 4-5 days decreased as a dosedependent manner. In contrast, neither 27.5 mM D-raffinose (HR) or L-glucose (HLG) in the media had no effect on the degradation. In contrast, there was no impairment of catalase-dependent H202-degradation in the cells cultured in both glucose concentrations.
There was no difference in basal NADPH level between HG and NG groups. However, after an exposure of 500 μM H2O2, NADPH level in HG group was 41% (P<0.01) and 36% (P<0.05) less than that in NG and HR groups, respectively. The glucose effect on NADPH concentration in the cells exposed by H2O2 was D-glucose concentrationdependent. Similarly, there was no difference in basal HMS activity which was calculated by [1-14C] Glut+[2-14C] Glu-2×[6-14C] Glu between HG and NG groups. However, after an exposure of H2O2, HMS activity in HG group was significantly (P<0.01) less than the values of NG group and HR group, respectively.
51Cr-release from cells in HG group utilized as a marker of cell injury which was measured in the presence of 20-500 μM H2O2 was significantly (P<0.01) greater than that of NG group. The high glucose-induced enhancement of H2O2-induced 51Cr-release was completely normalized by preincubating the cells in the presence of eigher 100 μM deferoxamine or 10 mM dimethylthiourea. Lipid peroxide content in plasma membrane fraction obtained from cells cultured in HG condition was 36% higher (P<0.05) than that of NG group.
These results indicate that glutathione redox cycle in cells cultured in high glucose media is impaired as D-glucose specific and medium glucose concentration-dependent manner. These abnormal radical scavenger functions in the cells cultured in high glucose media are impaired via an insufficient NADPH supply as a result of an impairment of HMS activity. The impairment of H2O2-degrading activity in high glucose condition may associate with a potentiation of oxygen radicals-induced endothelial cell injury.
