Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1).
Free fatty acids (FFA) have been reported to reduce pancreatic beta-cell mitogenesis and to increase apoptosis. Here we show that the FFA, oleic acid, increased apoptosis 16-fold in the pancreatic beta-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 mm glucose and/or in the added presence of insulin-like growth factor 1, FFA-induced beta-cell apoptosis was lessened compared with that at a basal 5 mm glucose. However, most strikingly, adenoviral mediated expression of a constitutively active PKB, but not a "kinase-dead" PKB variant, essentially prevented FFA-induced beta-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-3alpha/beta and the forkhead transcription factor, FoxO1, in protection of FFA-induced beta-cell apoptosis. In addition, down-regulation of the pro-apoptotic tumor suppressor protein, p53, via PKB-mediated phosphorylation of MDM2 might also play a role in partially protecting beta-cells from FFA-induced apoptosis. Adenoviral mediated expression of wild type p53 potentiated FFA-induced beta-cell apoptosis, whereas expression of a dominant negative p53 partly inhibited beta-cell apoptosis by approximately 50%. Hence, these data demonstrate that PKB activation plays an important role in promoting pancreatic beta-cell survival in part via inhibition of the pro-apoptotic proteins glycogen synthase kinase-3alpha/beta, FoxO1, and p53. This, in turn, provides novel insight into the mechanisms involved in FFA-induced beta-cell apoptosis.
Wrede
Christian E
CE
Pacific Northwest Research Institute and Department of Pharmacology, University of Washington, Seattle, WA 98122, USA.
Dickson
Lorna M
LM
Lingohr
Melissa K
MK
Briaud
Isabelle
I
Rhodes
Christopher J
CJ
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation; D005227:Fatty Acids / Q000494:pharmacology*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007515:Islets of Langerhans / Q000473:pathology*; D009336:Necrosis; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D005227:Fatty Acids; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D007334:Insulin-Like Growth Factor I; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; C506434:Casp9 protein, mouse; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D005947:Glucose