Insulin-mediated stimulation of protein kinase Akt - A potent survival signaling cascade for endothelial cells

Insulin exerts potent antiapoptotic effects in neuronal cells and has been suggested to promote angiogenesis. Therefore, we investigated whether insul in inhibits tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in hu man umbilical vein endothelial cells (HUVECs). Because insulin has been sho wn to stimulate the protein kinase Akt, we investigated whether activation of Akt contributes to the apoptosis-suppressive effect of insulin and chara cterized the downstream signaling pathway. Incubation with insulin dose-dep endently prevented apoptosis induced by TNF-alpha (50 ng/mL), The extent of apoptosis suppression by insulin was similar to the effect of vascular end othelial growth factor. Pharmacological inhibition of Akt activation or ove rexpression of a dominant-negative Akt mutant prevented the antiapoptotic e ffect of insulin. Furthermore, we investigated the effect of TNF-alpha on A kt phosphorylation by Western blot analysis with the use of a phosphospecif ic Akt antibody. Incubation of HUVECs with TNF-alpha induced a marked depho sphorylation of Akt. Insulin counteracted this TNF-alpha-induced dephosphor ylation of Akt. Furthermore, we investigated the downstream signaling event s, Akt has been shown to mediate its apoptosis-suppressive effects via phos phorylation of Bad or caspase-9. However, incubation with insulin did not l ead to enhanced phosphorylation of Bad at Ser 136 or Ser 112. In contrast, insulin inhibited caspase-9 activity and prevented caspase-9-induced apopto sis. Mutation of the Akt site within caspase-9 significantly reduced the ap optosis-suppressive effect of insulin. The present study demonstrates an im portant role for insulin-mediated Akt activation in the prevention of endot helial cell apoptosis, which may importantly contribute to cell homeostasis and the integrity of the endothelium. In endothelial cells, Akt seems to m ediate its antiapoptotic effect, at least in part, via phosphorylation of c aspase-9 rather than Bad.