Protein hydrolysates stimulate proglucagon gene transcription in intestinal endocrine cells via two elements related to cyclic AMP response element

Abstract

Aims/hypothesis. Protein hydrolysates (peptones) increase not only glucagon-like peptide-1 (GLP-1) secretion but also transcription of the proglucagon (PG) gene in the intestine. The critical physiological roles of gut-derived GLPs raised hope for their therapeutic use in several disorders, especially GLP-1 in diabetes. We aimed to investigate the molecular mechanisms involved in this nutrient-PG gene interaction. Methods. Wild-type and mutated PG promoter fragments fused to the luciferase reporter gene were trans-fected into enteroendocrine STC-1 cells, which were then either treated or not with peptones. Co-transfection with expression vectors of dominant-negative forms of cAMP response element binding protein (CREB) and protein kinase A (PKA) proteins were performed, as well as electrophoresis mobility shift assays. Results. Deletion analysis showed that the promoter region spanning between -350 and -292 bp was crucial for the transcriptional stimulation induced by peptones. Site-directed mutagenesis of the canonical cAMP response element (CRE PG) and of the adjacent putative CRE site (CRE-like1) led to a dramatic inhibition of the promoter responsiveness to peptones. Over expression of a dominant-negative mutant of CREB or of PKA produced a comparable and selective inhibitory effect on the activity of transfected promoter fragment containing the -350/-292 sequence. EMSA showed that CREB and fra2 transcription factors bound to CREPG and CRE-like1 elements respectively, independently of peptone treatment. Conclusions/interpretation. Our report identified cis-and trans-regulatory elements implicated in the transcriptional control of PG gene by nutrients in enteroendocrine cells. It highlights the role of a previously unsuspected CRE-like1 element, and emphasises the importance of CRE-related sequences in the regulation of PG gene transcription in the intestine.