Exendin-4, as an analogue of glucagon-like peptide-1, is currently used for the treatment of type 2 diabetes by enhancement of glucose-induced insulin secretion. Preliminary studies showed that secretion of glucagon is inhibited by exendin-4 in vivo and in vitro. However, the detailed mechanism in exendin-4 regulated glucagon expression is not clear.In this study, we firstly proved that exendin-4 decreased the mRNA and protein expression of glucagon in a dose-dependent manner. Following, the promoter activity of glucagon was significantly decreased by exendin-4 and inhibition of Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) signaling pathway by its specific inhibitor, STO-609 rescued this effect. Since glucagon secretion is regulated by AMP-activiated protein kinase (AMPK) and AMPK is able to be activated by CaMKK, we checked the role of AMPK in this process and found exendin-4 activated AMPK at Thr172 site. Additionally, inhibition of AMPK activity by its specific inhibitor or overexpression of domain-negative AMPK canceled the effect of exendin-4 on glucagon promoter activity. As activation of AMPK enhances FoxO transcriptional activity, to further study the transcription of glucagon, we used ChIP assay to demonstrate that transcription factor FoxO1 could bind to glucagon promoter region and exendin-4 decreased glucagon expression via down-regulation of FoxO1. Mutation of FoxO1-binding site in glucagon promoter or silence of FoxO1 canceled the effect of exendin-4 on glucagon expression.
In summary, our results proved that exendin-4 decreases the expression and transcription of glucagon via CaMKK/AMPK/FoxO1 pathway in pancreatic alpha cells, contributing to new knowledge about the regulation of glucagon gene by exendin-4.
J. Lyu: None. H. Imachi: None. K. Fukunaga: None. S. Sato: None. T. Dong: None. T. Ibata: None. T. Kobayashi: None. K. Murao: Research Support; Self; Novartis Pharma K.K..