title: Stimulation of pancreatic beta-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways.
abstract: The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), have been suggested to act as beta-cell growth factors and may therefore be of critical importance for the maintenance of a proper beta-cell mass. We have investigated the molecular mechanism of incretin-induced beta-cell replication in primary monolayer cultures of newborn rat islet cells. GLP-1, GIP and the long-acting GLP-1 derivative, liraglutide, increased beta-cell replication 50-80% at 10-100 nM upon a 24 h stimulus, whereas glucagon at a similar concentration had no significant effect. The stimulatory effect of GLP-1 and GIP was efficiently mimicked by the adenylate cyclase activator, forskolin, at 10 nM (approximately 90% increase) and was additive (approximately 170-250% increase) with the growth response to human growth hormone (hGH), indicating the use of distinct intracellular signalling pathways leading to mitosis by incretins and cytokines, respectively. The response to both GLP-1 and GIP was completely blocked by the protein kinase A (PKA) inhibitor, H89. In addition, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin and the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, both inhibited GLP-1- and GIP-stimulated proliferation. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, had no inhibitory effect on either GLP-1 or GIP stimulated proliferation. Cyclin Ds act as molecular switches for the G0/G1-S phase transition in many cell types and we have previously demonstrated hGH-induced cyclin D2 expression in the insulinoma cell line, INS-1. GLP-1 time-dependently induced the cyclin D1 mRNA and protein levels in INS-1E, whereas the cyclin D2 levels were unaffected. However, minor effect of GLP-1 stimulation was observed on the cyclin D3 mRNA levels. Transient transfection of a cyclin D1 promoter-luciferase reporter construct into islet monolayer cells or INS-1 cells revealed approximately a 2-3 fold increase of transcriptional activity in response to GLP-1 and GIP, and a 4-7 fold increase in response to forskolin. However, treatment of either cell type with hGH had no effect on cyclin D1 promoter activity. The stimulation of the cyclin D1 promoter by GLP-1 was inhibited by H89, wortmannin, and PD98059. We conclude that incretin-induced beta-cell replication is dependent on cAMP/PKA, p42 MAPK and PI3K activities, which may involve transcriptional induction of cyclin D1. GLP-1, GIP and liraglutide may have the potential to increase beta-cell replication in humans which would have significant impact on long-term diabetes treatment.
authors.affiliation: Novo Nordisk A/S, Department of Islet Discovery Research, Krogshoejvej 31, 9R2.28, 2880 Bagsvaerd, Denmark.
authors.forename: Birgitte N
authors.initials: BN
authors.lastname: Friedrichsen
authors.forename: Nicole
authors.initials: N
authors.lastname: Neubauer
authors.forename: Ying C
authors.initials: YC
authors.lastname: Lee
authors.forename: Vivian K
authors.initials: VK
authors.lastname: Gram
authors.forename: Niels
authors.initials: N
authors.lastname: Blume
authors.forename: Jacob S
authors.initials: JS
authors.lastname: Petersen
authors.forename: Jens H
authors.initials: JH
authors.lastname: Nielsen
authors.forename: Annette
authors.initials: A
authors.lastname: Møldrup
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005419:Flavonoids / Q000494:pharmacology; D005749:Gastric Inhibitory Polypeptide / Q000494:pharmacology*; D052216:Glucagon-Like Peptide 1 / Q000031:analogs & derivatives / Q000494:pharmacology*; D019382:Human Growth Hormone / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D000069450:Liraglutide; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D013268:Stimulation, Chemical; D013449:Sulfonamides / Q000494:pharmacology; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D005419:Flavonoids; D007093:Imidazoles; D007546:Isoquinolines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011725:Pyridines; D013449:Sulfonamides; D019382:Human Growth Hormone; D019938:Cyclin D1; D005576:Colforsin; D005749:Gastric Inhibitory Polypeptide; D000069450:Liraglutide; D052216:Glucagon-Like Peptide 1; D017868:Cyclic AMP-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D000262:Adenylyl Cyclases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin