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A Role for Glycogen Synthase Kinase 3 beta in the Regulation of Glucagon Gene Transcription by Insulin

dc.contributor.advisorHardeland, Rüdiger Prof.
dc.contributor.authorDimopoulos, Nikolaosde
dc.description.abstractInsulin reguliert über seine Bindung an den Insulin Rezeptor in a-Zellen des endokrinen Pankreas eine Signalkaskade, die in einer Hemmung der Glucagongen-Transkription resulitert. In der vorliegenden Promotionsarbeit wurde dieser bis jetzt nicht vollständig aufgeklärte Mechanismus in Hinblick auf die Rolle von GSK3 untersucht. Es konnte gezeigt werden, dass die Überexpression von GSK3 die Glucagongen Trankription verstärkte.GSK3 Inhibitoren unterdrückten die Trankripiton des Glucagongens und zeigten somit den selben Effekt wie Insulin. Untersuchungen an Deletions- und Mutationsanalysen des Glucagonpromotors gaben Hinweise auf eine mögliche Wirkung von GSK3 über den Transkriptionsfaktor Pax6. Die transkriptionsaktivierende Wirkung von Pax6 und CBP wurde durch den GSK3 Inhibitor SB-216763 aufgehoben. Dies wurde in einem GAL4-System gezeigt. Rekombinantes GSK3b phosphorilierte die Transaktivierungsdomäne von Pax6 und ein CBP-Fragment, das die Aminosäuren 2040-2305 enthielt. Dies konnte durch in vitro Kinase assays gezeigt werden. Die Ergebnisse zeigen, das GSK3-b in die Regulation des Glucagongens involviert
dc.titleA Role for Glycogen Synthase Kinase 3 beta in the Regulation of Glucagon Gene Transcription by Insulinde
dc.title.translatedRolle der Glycogen Synthase Kinase 3 beta in der Regulation der Glucagongen-Transkription durch Insulinde
dc.contributor.refereePieler, Tomas Prof.
dc.subject.dnb570 Biowissenschaften, Biologiede
dc.description.abstractengInsulin binding to its receptor on the a-cells in endocrine pancreas initiates a signalling cascade that results in inhibition of glucagon gene transcription. Evidence suggests that the PI(3)K/PKB pathway might confer insulin-mediated inhibition, but the mechanism is still not completely understood. The present study characterized the role of GSK3b, a well known substrate of PKB, in the regulation of glucagon gene transcription.enhancement of glucagon gene transcription. The effect was due to its kinase activity, because a GSK3b kinase-dead mutant had no effect on transcription. Next, endogenous pools of GSK3 were assessed by using various recently developed selective GSK3 inhibitors. All of them inhibited transcription, thereby mimicking the effect of insulin. They could also abolish the effect of GSK3b overexpression on the stimulation of glucagon gene transcription. To validate the mode of action of the inhibitors, we could show that they modulated putative GSK3 substrates in vivo, e.g. they induced stabilization and accumulation of b-catenin levels.GSK3 might act through Pax6 and its potential coactivator CBP to regulate glucagon gene expression. Deletion and internal mutation analysis of the glucagon promoter indicated that Pax6 might play an important role to confer inhibition by the GSK3 inhibitor, SB-216763. Transcriptional activity of Pax6 and CBP was inhibited in response to SB-216763 within the glucagon promoter context, as revealed by employing the Gal4 system. Fusing full length CBP, as well as N- and C-terminal parts of CBP, with the Gal4 domain conferred transcriptional activity as well as responsiveness to GSK3b overexpression. The effect of GSK3b overexpression on the activity of the C-terminal part of CBP was further mapped.Finally, based on the above findings an in vitro kinase assay was performed. Recombinant GSK3b phosphorylated, even in the absence of priming phosphorylation by another kinase, the transactivation domain of Pax6 and a fragment of CBP consisting of amino acids 2040-2305 when fused to GST. However, the significance of this phosphorylation in vivo and in the context of the glucagon promoter remains to be shown.Taken together the results of the present study suggest the involvement of GSK3b in the regulation of glucagon gene transcription by insulin. Through inhibition of GSK3, insulin might repress gene transcription by disruption of a glucagon promoter-specific protein complex, whose critical components seem to be Pax6 and its coactivator CBP. Since hyperglucagonaemia is associated with type 2 diabetes mellitus, understanding the molecular mechanism of insulin-mediated inhibition of glucagon gene expression can prove a valuable tool to develop drugs to treat some of the aspects of the
dc.contributor.coRefereeGatz, Christiane Prof.
dc.contributor.thirdRefereeFicner, Ralf Prof.
dc.subject.topicMathematics and Computer Sciencede
dc.affiliation.instituteBiologische Fakultät inkl. Psychologiede

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