Free fatty acids (FFA) have been reported to reduce pancreatic β-cell mitogenesis and to increase apoptosis. Here we show that the FFA, oleic acid, increased apoptosis 16-fold in the pancreatic β-cell line, INS-1, over a 18-h period as assessed by Hoechst 33342/propidium iodide staining and caspase-3 and -9 activation, with negligible necrosis. A parallel analysis of the phosphorylation activation of protein kinase B (PKB) showed this was reduced in the presence of FFA that correlated with the incidence of apoptosis. At stimulatory 15 mmglucose and/or in the added presence of insulin-like growth factor 1, FFA-induced β-cell apoptosis was lessened compared with that at a basal 5 mmglucose. However, most strikingly, adenoviral mediated expression of a constitutively active PKB, but not a “kinase-dead” PKB variant, essentially prevented FFA-induced β-cell apoptosis under all glucose/insulin-like growth factor 1 conditions. Further analysis of pro-apoptotic downstream targets of PKB, implicated a role for PKB-mediated phosphorylation inhibition of glycogen synthase kinase-3α/β and the forkhead transcription factor, FoxO1, in protection of FFA-induced β-cell apoptosis. In addition, down-regulation of the pro-apoptotic tumor suppresser protein, p53, via PKB-mediated phosphorylation of MDM2 might also play a role in partially protecting β-cells from FFA-induced apoptosis. Adenoviral mediated expression of wild type p53 potentiated FFA-induced β-cell apoptosis, whereas expression of a dominant negative p53 partly inhibited β-cell apoptosis by ∼50%. Hence, these data demonstrate that PKB activation plays an important role in promoting pancreatic β-cell survival in part via inhibition of the pro-apoptotic proteins glycogen synthase kinase-3α/β, FoxO1, and p53. This, in turn, provides novel insight into the mechanisms involved in FFA-induced β-cell apoptosis.
Protein Kinase B/Akt Prevents Fatty Acid-induced Apoptosis in Pancreatic β-Cells (INS-1)
