OBJECTIVE Endothelial cell injury is a key event in the pathogenesis of diabetes-associated atherosclerosis and vascular complications. Increased apoptosis may contribute to the loss of endothelial integrity and leads to cardiovascular disease. This study was designed to elucidate whether high levels of free fatty acids (FFA) cause apoptosis and if so what is the possible role of insulin signaling alteration(s) in determining this effect. RESULTS In human umbilical vein endothelial cells (HUVECs) cultured for 72h with high levels of FFA, apoptotic cells, detected by Annexin V-FITC and PI, were increased. Then we observed a decrease of Bcl-2/Bax ratio (pro-apoptotic condition), measured by RT-PCR and Western blot. As the Akt pathway is involved in insulin signaling and apoptosis, we investigated whether Akt mediated FFA apoptotic effects. HUVECs exposed to FFA showed an equal amount of total Akt protein content compared to controls. In HUVECs, FFA induced a significant decrease in phosphorylated active Akt. Furthermore, phosphorylated eNOS (active form) was decreased and cleaved caspase-9 (active form) was increased. These alterations were prevented when insulin at 10(-8)M was added in culture medium containing FFA. The insulin anti-apoptotic effect was prevented by Ly29400, a PI3K/Akt inhibitor. CONCLUSIONS High levels of FFA cause HUVECs apoptosis through Akt inhibition; insulin can prevent these effects. Inappropriate FFA elevation may affect vascular endothelium by impairing cell survival via activation of apoptosis, thus contributing to the development of cardiovascular disease in type 2 diabetic patients.