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K K Sidhu Department of Endocrinology, Barts and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK
Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK

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R C Fowkes Department of Endocrinology, Barts and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK
Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK

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R H Skelly Department of Endocrinology, Barts and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK
Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK

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J M Burrin Department of Endocrinology, Barts and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK
Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK

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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.

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A Scarlett Bart's and the London School of Medicine and Dentistry, William Harvey Research Institute, Queen Mary, Centre for Endocrinology, University of London, Charterhouse Square, London EC1M 6BQ, UK

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M P Parsons Bart's and the London School of Medicine and Dentistry, William Harvey Research Institute, Queen Mary, Centre for Endocrinology, University of London, Charterhouse Square, London EC1M 6BQ, UK

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P L Hanson Bart's and the London School of Medicine and Dentistry, William Harvey Research Institute, Queen Mary, Centre for Endocrinology, University of London, Charterhouse Square, London EC1M 6BQ, UK

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K K Sidhu Bart's and the London School of Medicine and Dentistry, William Harvey Research Institute, Queen Mary, Centre for Endocrinology, University of London, Charterhouse Square, London EC1M 6BQ, UK

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T P Milligan Bart's and the London School of Medicine and Dentistry, William Harvey Research Institute, Queen Mary, Centre for Endocrinology, University of London, Charterhouse Square, London EC1M 6BQ, UK

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J M Burrin Bart's and the London School of Medicine and Dentistry, William Harvey Research Institute, Queen Mary, Centre for Endocrinology, University of London, Charterhouse Square, London EC1M 6BQ, UK

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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 αVβ3 cell surface receptor. Using PCR and western blotting, the expression of integrin αVβ3 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 αVβ3 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 αVβ3-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 αVβ3 and that one functional effect of this ERK activation is increased DNA synthesis.

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