Direct apoptotic effects of free fatty acids on human endothelial cells.
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.
Piro
Salvatore
S
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.
Spampinato
Daniela
D
Spadaro
Luisa
L
Oliveri
Conchita Emanuela
CE
Purrello
Francesco
F
Rabuazzo
Agata Maria
AM
D017209:Apoptosis / Q000187:drug effects; D053453:Caspase 9 / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D005230:Fatty Acids, Nonesterified / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494826:BAX protein, human; D005230:Fatty Acids, Nonesterified; D007328:Insulin; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D053453:Caspase 9