The insulinotrophic effects of glucagon-like peptide 1 (GLP-1) are mediated by its seven-transmembrane receptor (GLP-1R) in pancreatic β-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 μg GLP-1R DNA. AtT-20 cells co-transfected with GLP-1R and human glycoprotein hormone α-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.

The aim of the present study was to examine whether triiodo-l-thyronine (T₃) or l-thyroxine (T₄) rapidly activated the mitogen-activated protein kinase (MAPK) intracellular signalling cascade in osteoblast-like cells and investigate whether this activation was initiated at the integrin α_Vβ₃ cell surface receptor. Using PCR and western blotting, the expression of integrin α_Vβ₃ mRNA and protein was demonstrated in the human osteoblast-like cell lines MG-63 and SaOS-2. The treatment of MG-63 cells with T₃ (10 nM) or T₄ (100 nM) for 10 min stimulated extracellular signal-regulated kinase activity (ERK, a component of the MAPK pathway) as determined by fluorescent immunocytochemistry and an immunocomplex activity assay (T₃ by 10.7-fold, P<0.01 and T₄ by 10.4-fold, P<0.01 compared with control). T₃ (10 nM) and T₄ (100 nM) also significantly stimulated thymidine incorporation into MG-63 cells by 2.3±0.7-fold (P<0.01) and 2.1±0.1-fold (P<0.05) respectively. To establish whether transient ERK activation via the integrin α_Vβ₃ cell surface receptor mediated these effects, MG-63 cells were pretreated for 30 min with the specific MAPK kinase inhibitor, U0126 (1 μM), or an anti-integrin α_Vβ₃-blocking antibody. Both pretreatments significantly inhibited T₃- and T₄-stimulated ERK activation and abolished T₃-stimulated thymidine incorporation (P<0.01). T₄-stimulated incorporation was significantly inhibited from 2.1- to 1.3-fold above control (P<0.05). Thus, our results suggest that T₃ and T₄ rapidly stimulate ERK activation in MG-63 cells via integrin α_Vβ₃ and that one functional effect of this ERK activation is increased DNA synthesis.