Endothelial dysfunction plays an important role in atherogenesis in type 2 diabetes. Previous works have shown that incubation of the endothelial cells (E-cells) in high glucose media results in production of reactive oxygen species (ROS) and lipid peroxidation. We have previously shown that T-lymphocytes (T-cells), which in their native state have no insulin receptors, upon incubation in high glucose become activated and exhibit de novo growth factor receptors for insulin, IGF1, IL-2, and GLUT-4 and become insulin sensitive. Thus both T-cells and E-cells show certain similarities in glucose-induced production of free radicals. We hypothesized that E-cells in high glucose media may behave similar to T-cells, induce insulin receptors and metabolize glucose since this has not been previously reported.
Methods E-cells were incubated in 5, 15, and 30 mM glucose, and 25 mM mannitol for 6, 12, 24, and 48 hours at 37°C. We measured 125I insulin binding as well as uptake of 14C deoxyglucose in response to 50 μU/mL of insulin and analyzed expression of insulin receptor (IR) and glucose transporters GLUT1 and GLUT4 by Western blotting.
Results Binding of 125I insulin to cells incubated in 30 mM glucose (but no 5 mM) was time- and temperature-dependent and demonstrated 100% dissociation with 100 ng of insulin per mL. Uptake of 14C deoxyglucose was also time- and glucose concentration-dependent with greatest uptake at 48 hours, but none at 6 hours. Uptake of 14C deoxyglucose was highest at 30 mM, intermediate with 15 mM, and none at 5 mM glucose. These effects of high glucose were not attributed to hyperosmotic action of sugar, since 25 mM mannitol had no effect 14C deoxyglucose uptake. Demonstration of time-dependent increase in insulin sensitivity was further confirmed by increased IR expression in E-cells by the use of Western blotting analysis after incubation of E-cells with 30 mM glucose, but not with 5 mM glucose. Immunoblotting experiments also showed that incubation of E-cells in 30 mM glucose was accompanied by 2-4-fold elevation in expression of GLUT1 and GLUT4 as compared with 5 mM glucose values. In conclusion, we have established certain similarities between E-cells and T-cells in which both tissues are originally insulin insensitive but become insulin sensitive and develop insulin receptors in high concentration of glucose exhibiting production of ROS and lipid peroxidation. The use of T-cells as an easily available tissue may be an attractive alternative as a surrogate biomarker to predict early atherogenic changes.