title: Regulation of forkhead box O1 (FOXO1) by protein kinase B and glucocorticoids: different mechanisms of induction of beta cell death in vitro.
abstract: OBJECTIVE
  In steroid diabetes insulin secretion does not adequately compensate for enhanced hepatic gluconeogenesis and peripheral insulin resistance. Previous studies suggest that activation of the transcription factor forkhead box O1 (FOXO1) contributes to glucocorticoid-induced beta cell death. This study examines the role and regulation of FOXO1 in insulin-secreting cells.
  
  
  METHODS
  INS-1E cells and mouse islet cells were cultured in the presence of dexamethasone. Signalling pathways were modified pharmacologically or by small interfering (si)RNA-mediated inhibition of protein synthesis. Changes in protein abundance and phosphorylation were analysed by western blotting, and subcellular localisation was assessed using confocal microscopy. Transcript levels were examined by RT-PCR.
  
  
  RESULTS
  Surprisingly, downregulation of FOXO1 by siRNA did not affect dexamethasone-induced apoptosis or Bim expression, but it prevented the effects of the pan protein kinase B (AKT) inhibitor (Akti-1/2). Indeed, dexamethasone and Akti-1/2 synergistically increased beta cell death and Bim expression. Akti-1/2 triggered dephosphorylation and nuclear translocation of FOXO1. Glucocorticoid-receptor activation stimulated Foxo1 transcription, but FOXO1 phosphorylation was unchanged and the cytosolic concentration of FOXO1 remained high in relation to its nuclear concentration. However, subcellular fractionation revealed a significant increase in both cytosolic and nuclear FOXO1 compared with untreated cells. Dexamethasone diminished Pdx1 mRNA level, an effect which was not reversed by siRNA against Foxo1. Downregulation of AKT isoforms and serum/glucocorticoid-regulated kinase 1 (SGK1) suggests that only sustained suppression of all three AKT isoforms caused dephosphorylation and nuclear accumulation of FOXO1.
  
  
  CONCLUSIONS
  This study reveals that FOXO1 is not the main mediator of glucocorticoid-receptor-induced beta cell apoptosis, but rather that it escalates beta cell death when AKT activity is inhibited by distinct pathways.
authors: Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical Chemistry, Department of Internal Medicine IV, University of Tübingen, Otfried-Müller-Strasse 10, 72076, Tübingen, Germany.
authors: G
authors: G
authors: Kaiser
authors: F
authors: F
authors: Gerst
authors: D
authors: D
authors: Michael
authors: S
authors: S
authors: Berchtold
authors: B
authors: B
authors: Friedrich
authors: N
authors: N
authors: Strutz-Seebohm
authors: F
authors: F
authors: Lang
authors: H-U
authors: HU
authors: Häring
authors: S
authors: S
authors: Ullrich
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D005938:Glucocorticoids / Q000494:pharmacology*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D005938:Glucocorticoids; D034741:RNA, Small Interfering; D003907:Dexamethasone; D051057:Proto-Oncogene Proteins c-akt