Protein kinase B activity is sufficient to mimic the effect of insulin on glucagon gene transcription.
Insulin inhibits glucagon gene transcription, and insulin deficiency is associated with hyperglucagonemia that contributes to hyperglycemia in diabetes mellitus. However, the insulin signaling pathway to the glucagon gene is unknown. Protein kinase B (PKB) is a key regulator of insulin signaling and glucose homeostasis. Impaired PKB function leads to insulin resistance and diabetes mellitus. Therefore, the role of PKB in the regulation of glucagon gene transcription was investigated. After transient transfections of glucagon promoter-reporter genes into a glucagon-producing islet cell line, the use of kinase inhibitors indicated that the inhibition of glucagon gene transcription by insulin depends on phosphatidylinositol (PI) 3-kinase. Furthermore, insulin caused a PI 3-kinase-dependent phosphorylation and activation of PKB in this cell line as revealed by phospho-immunoblotting and kinase assays. Overexpression of constitutively active PKB mimicked the effect of insulin on glucagon gene transcription. Both insulin and PKB responsiveness of the glucagon promoter were abolished when the binding sites for the transcription factor Pax6 within the G1 and G3 promoter elements were mutated. Recruitment of Pax6 or its potential coactivator, the CREB-binding protein (CBP), to G1 and G3 by using the GAL4 system restored both insulin and PKB responsiveness. These data suggest that insulin inhibits glucagon gene transcription by signaling via PI 3-kinase and PKB, with the transcription factor Pax6 and its potential coactivator CBP being critical components of the targeted promoter-specific nucleoprotein complex. The present data emphasize the importance of PKB in insulin signaling and glucose homeostasis by defining the glucagon gene as a novel target gene for PKB.
Schinner
Sven
S
Department of Molecular Pharmacology, University of Göttingen, 37099 Göttingen, Germany.
Barthel
Andreas
A
Dellas
Claudia
C
Grzeskowiak
Rafal
R
Sharma
Sanjeev K
SK
Oetjen
Elke
E
Blume
Roland
R
Knepel
Willhart
W
D000818:Animals; D050882:CREB-Binding Protein; D002460:Cell Line; D005136:Eye Proteins; D005934:Glucagon / Q000235:genetics*; D018398:Homeodomain Proteins / Q000502:physiology; D007328:Insulin / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D018716:Molecular Mimicry; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000502:physiology; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D012097:Repressor Proteins; D015398:Signal Transduction; D015534:Trans-Activators / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D050882:CREB-Binding Protein; D005136:Eye Proteins; D005934:Glucagon; D018398:Homeodomain Proteins; D007328:Insulin; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012097:Repressor Proteins; D015534:Trans-Activators; C000605732:Pax6 protein, mouse; C000605733:Pax6 protein, rat; C494364:Crebbp protein, mouse; C494365:Crebbp protein, rat