Effect of rosiglitazone and PPARγ on insulin gene transcription and mapping of PPARγ domains involved in insulin/glucagon gene transcriptional regulation

Abstract

Thiazolidindiones are a new class of oral antidiabetic drugs. They reduce hepatic glucose output, insulin resistance as well as plasma glucose, insulin and triglyceride levels. The thiazolidindione rosiglitazone is a ligand of peroxysome proliferator activated receptor γ (PPARγ) and was shown to inhibit glucagon gene transcription by abolishing the transcription activational function of the transcription factor Pax6. In this study we addressed the question whether rosiglitazone through PPARγ also act on human insulin gene transcription and which domains of PPARγ are mediating this effect in comparison to glucagon gene transcriptional inhibition. To study insulin and glucagon gene promoter activities, luciferase reporter gene constructs carrying fragments of the human insulin (–336hInsLuc) or rat glucagon promoter (–350rGluLuc) were used in transient transfections in the tumor cell lines HIT and InR1G9, respectively. PPARγ or deletional mutants of PPARγ lacking the DNA binding domain (aa 175–475) or DNA binding domain and hinge region (aa 249–475) were cotransfected and cells were treated with 30µM of rosiglitazone for 24 hours before harvesting. Basal activity of –336hInsLuc was inhibited by approximately 60% after cotransfection of PPARγ and rosiglitazone treatment. Cotransfection of PPARγ (175–475) and PPARγ (249–475) did not show any effect on insulin promoter activity after treatment with rosiglitazone. In contrast, -350rGluLuc was inhibited by approximately 80% after cotransfection of PPARγ and rosiglitazone treatment, whereas the cotransfection of PPARγ (175–475) and PPARγ (249–475) caused an inhibition by about 35% after rosiglitazone treatment. These results show that rosiglitazone/PPARγ inhibit insulin gene transcription. Furthermore these results suggest that different domains of PPARγ may be involved in the inhibition of glucagon and insulin gene implying that PPARγ may be involved in different inhibitory regulatory pathways.