Piro Salvatore, Spampinato Daniela, Spadaro Luisa, Oliveri Conchita Emanuela, Purrello Francesco, Rabuazzo Agata Maria
Clinica di Medicina Interna, Laboratorio di Medicina Molecolare, Dipartimento di Medicina Interna e Medicina Specialistica, Università degli Studi di Catania, Ospedale Garibaldi "Nesima", via Palermo 636, 95122, Catania, Italy.
Nutr Metab Cardiovasc Dis. 2008 Feb;18(2):96-104. doi: 10.1016/j.numecd.2007.01.009. Epub 2007 Jun 8.
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.
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.
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.
内皮细胞损伤是糖尿病相关动脉粥样硬化及血管并发症发病机制中的关键事件。细胞凋亡增加可能导致内皮完整性丧失并引发心血管疾病。本研究旨在阐明高水平游离脂肪酸（FFA）是否会导致细胞凋亡，若如此，胰岛素信号改变在决定这一效应中可能发挥何种作用。
用高水平FFA培养人脐静脉内皮细胞（HUVECs）72小时后，通过Annexin V-FITC和PI检测发现凋亡细胞增多。随后，通过RT-PCR和蛋白质印迹法检测发现Bcl-2/Bax比值降低（促凋亡状态）。由于Akt通路参与胰岛素信号传导和细胞凋亡，我们研究了Akt是否介导FFA的凋亡效应。与对照组相比，暴露于FFA的HUVECs中总Akt蛋白含量相等。在HUVECs中，FFA导致磷酸化活性Akt显著降低。此外，磷酸化eNOS（活性形式）降低，而裂解的caspase-9（活性形式）增加。当在含FFA的培养基中加入10⁻⁸M胰岛素时，这些改变被阻止。胰岛素的抗凋亡作用被PI3K/Akt抑制剂Ly29400阻断。
高水平FFA通过抑制Akt导致HUVECs凋亡；胰岛素可预防这些效应。FFA不适当升高可能通过激活凋亡损害细胞存活，从而影响血管内皮，进而导致2型糖尿病患者心血管疾病的发生。
