title: Exogenous expression of glucagon-like peptide 1 receptor and human insulin in AtT-20 corticotrophs confers cAMP-mediated gene transcription and insulin secretion.
abstract: The insulinotrophic effects of glucagon-like peptide 1 (GLP-1) are mediated by its seven-transmembrane receptor (GLP-1R) in pancreatic beta-cells. We have transiently transfected the GLP-1R and a proopiomelanocortin (POMC) promoter-driven human preproinsulin gene vector (pIRES) into the AtT-20 pituitary corticotrophic cell line, to investigate the possibility of creating a regulated, insulin-expressing cell line. Receptor expression was confirmed by RT-PCR and functionality was demonstrated by measuring changes in cAMP levels in response to GLP-1. Rapid (5 min) stimulation of cAMP production was observed with 100 nM GLP-1, 24 h after transfection of 2 microg GLP-1R DNA. AtT-20 cells co-transfected with GLP-1R and human glycoprotein hormone alpha-subunit or rat POMC promoters revealed GLP-1-stimulated cAMP activation of transcription. Co-transfection of the pIRES vector with the GLP-1R resulted in GLP-1-stimulated activation of POMC promoter-driven preproinsulin gene transcription but insulin secretion was not detected. However, using an adenoviral expression system to infect AtT-20 cells with GLP-1R and the preproinsulin gene (including 120 bp of its own promoter) resulted in a 6.4 +/- 0.6-fold increase in cAMP and a 4.9 +/- 0.8-fold increase in insulin secretion in response to 100 nM GLP-1. These results demonstrate, for the first time, functional GLP-1R-mediated preproinsulin gene transcription and secretion in a transplantable cell line.
authors.affiliation: Department of Endocrinology, Barts and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK. k.k.sidhu@qmul.ac.uk
authors.forename: K K
authors.initials: KK
authors.lastname: Sidhu
authors.forename: R C
authors.initials: RC
authors.lastname: Fowkes
authors.forename: R H
authors.initials: RH
authors.lastname: Skelly
authors.forename: J M
authors.initials: JM
authors.lastname: Burrin
mesh_terms: D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D052216:Glucagon-Like Peptide 1 / Q000032:analysis*; D000067757:Glucagon-Like Peptide-1 Receptor; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D008156:Luciferases; D010902:Pituitary Gland / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000235:genetics; D011384:Proinsulin / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D018027:Receptors, Glucagon / Q000032:analysis*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: C000593833:GLP1R protein, human; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D007328:Insulin; D011498:Protein Precursors; D018027:Receptors, Glucagon; C013737:preproinsulin; D011333:Pro-Opiomelanocortin; D052216:Glucagon-Like Peptide 1; D011384:Proinsulin; D000242:Cyclic AMP; D008156:Luciferases