Advanced glycosylation end products increase diacylglycerol levels in cultured human umbilical vein endothelial cells

Objective To study whether the diacylglycerol (Dia) signaling pathway is stimulated by advanced glycosylation end products (AGEP) and to test the effect of vitamin E and aminoguanidine (AG) on the elevation of Dia induced by AGEP in cultured human umbilical vein endothelial cells (HUVECs). Methods The effects of AGEP on Dia levels in cultured HUVEC were studied with radio-enzymatic assay. Quantitative measurements of (32)P phosphatidic acid were achieved by thin-layer chromatography and autoradiography. Results The Dia levels in HUVECs were increased by AGEP modified bovine serum albumin (AGEP-BSA) in a dose-dependent, biphasic manner. The early phase was rapid and transient, peaking at 15 s; the late phase reached the maximal level at 10 min and then decayed slowly. Dia levels in HUVEC exposed to different concentrations (50, 100 and 200 mg/L) of AGEP-BSA (341±14, 678±16, and 873±18 pmol/L, respectively vs control 225±10 pmol/L) and AGEP-BSA samples with various glycosylation times (4, 8 and 12 weeks) were significantly increased (270±12, 394±16, and 556±19 pmol/L) as compared with the controls. 50 and 100 mmol/L of vitamin E can reduce AGEP-BSA-induced Dia levels from 873±18 pmol/L to 764±29 and 441±21 pmol/L in HUVEC, respectively. In AG-treated (100 mmol/L) groups, the same concentration (100 and 200 mg/L) of AGEP-BSA-induced elevation of Dia was decreased to 312±8 and 351±13 pmol/L, respectively. Glycosylated low-density lipoprotein (LDL) did not affect Dia levels. Conclusion AGEP causes a robust stimulation of the Dia/protein kinase C pathway in HUVEC. Vitamin E can attenuate the AGEP-BSA-induced elevation of Dia levels. AG can suppress the ability of AGEP-BSA to increase Dia levels in HUVEC.