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The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), have been suggested to act as -cell growth factors and may therefore be of critical importance for the maintenance of a proper -cell mass. We have investigated the molecular mechanism of incretin-induced -cell replication in primary monolayer cultures of newborn rat islet cells. GLP-1, GIP and the long-acting GLP-1 derivative, liraglutide, increased -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 (90% increase) and was additive (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 -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 -cell replication in humans which would have significant impact on long-term diabetes treatment.

No abstract

Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic beta-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the beta-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.

GH and PRL stimulate proliferation and insulin production of pancreatic beta-cells. Whereas GH- and PRL-regulated transcription of the insulin gene in insulinoma cells has been shown to depend on STAT5 (signal transducer and activator of transcription 5), the signaling pathways involved in GH/PRL-induced beta-cell replication are unknown. The roles of various signaling pathways in human GH (hGH)-induced DNA synthesis were studied by analysis of the effect of specific inhibitors in both the insulin-producing cell line, INS-1, and in primary beta-cells. The mitogen-activated protein kinase kinase (MEK)-inhibitor, PD98059, as well as the mitogen-activated protein kinase p38 (MAPKp38) inhibitor, SB203580, partially inhibited hGH- induced proliferation in INS-1 cells but had no significant effect in primary beta-cells. Staurosporine, a protein kinase C (PKC) and protein kinase A (PKA) inhibitor, blocked both basal and hGH-induced proliferation in INS-1 cells, but had no inhibitory effect in primary beta-cells. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited hGH-induced proliferation neither in INS-1 cells nor in primary beta-cells, whereas the tyrosine kinase inhibitor, genistein, completely inhibited hGH- induced proliferation in both primary beta-cells and INS-1 cells. To analyze the possible role of STAT5 in hGH-induced proliferation, a dominant negative STAT5 mutant, STAT5Delta749, was expressed in INS-1 cells under the control of a doxycycline- inducible promoter by stable transfection. Two clones were found to exhibit dose-dependent, doxycycline-inducible expression of STAT5Delta749 and suppression of hGH-stimulated transcriptional activation of a STAT5-regulated PRL receptor (PRLR) promoter-reporter construct. Furthermore, induction of STAT5Delta749 expression completely inhibited hGH-induced DNA synthesis. Analysis of endogenous gene expression revealed a doxycycline-dependent inhibition of hGH-stimulated PRLR and cyclin D2 mRNA levels. Our results suggest that GH/PRL-induced beta-cell proliferation is dependent on the Janus Kinase2 (JAK2)/STAT5 signaling pathway but not the MAPK, PI3K, and PKC signaling pathways. Furthermore, the cell cycle regulator cyclin D2 may be a crucial target gene for STAT5 in this process.