Glycated low-density lipoprotein attenuates shear stress-induced nitric oxide synthesis by inhibition of shear stress-activated L-arginine uptake in endothelial cells.
Little is known about the mechanism(s) of endothelial dysfunction in diabetes. In this study, the effect of nonenzymatic glycated LDL, a phenomenon induced by elevated D-glucose levels associated with diabetes, on porcine aortic endothelial cells was investigated. Two fractions of LDL from diabetic patients were separated by affinity column chromatography and are referred to herein as fraction alpha (nonglycated LDL) and fraction beta (glycated LDL). Incubation of endothelial cells for 24 h with total LDL isolated from diabetic subjects (dLDL) increased the release of superoxide anions (*O2-) by fivefold, while no effect of LDL isolated from healthy individuals (nLDL) was found. Fraction beta, but not fraction alpha, evoked the *O2- release. In vitro-glycated LDL mimicked the effect of dLDL/fraction beta on *O2- release that correlated with its degree of glycation (R2 = 0.96). Moreover, nitric oxide (NO) stability (measured with a porphyrinic-based electrode) and NO bioactivity (measured by its ability to elevate cellular cGMP levels) were reduced in cells treated with dLDL by 46 and 41%, respectively. dLDL (but not nLDL or fraction alpha) abolished shear stress-induced L-arginine uptake. The inhibitory effect of dLDL on shear stress-induced L-arginine uptake was mimicked by in vitro-glycated LDL. The efficiency of in vitro-glycated LDL to diminish shear stress-evoked L-arginine uptake correlated with the extent of glycation (R2 = 0.88). Moreover, dLDL, but not nLDL or fraction alpha, reduced shear stress-mediated cGMP formation and NOx production by 47 and 88%, respectively. This effect was also mimicked by in vitro-glycated LDL, correlating with its degree of glycation (R2 = 0.86). Under these experimental conditions, glycated LDL reduced shear stress-induced increase in NO synthesis by inhibition of shear stress-stimulated L-arginine uptake and NO bioactivity due to increased endothelial cell *O2- release. These properties may contribute to the reduced vasodilatory response and the vascular complications in diabetes.