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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.

The proinflammatory cytokines interleukin-1 (IL-1 ), interferon-(IFN-) and tumour necrosis factor-(TNF-) are toxic to pancreatic -cells and are implicated in the pathogenesis of type 1 diabetes. We have previously found that GH and prolactin (PRL) stimulate both proliferation and insulin production in pancreatic -cells and rat insulin-producing INS-1 cells. Here we report that human (h) GH can prevent the apoptotic effects of IL-1 , IFN-and TNF-in INS-1 and INS-1E cells. Using adenovirus-mediated gene transfer, we found that the anti-apoptotic effect of hGH is abrogated by expression of a dominant negative signal transducer and activator of transcription (STAT5) mutant in INS-1E cells. hGH and the cytotoxic cytokines was found to additively increase suppressor of cytokine signalling-3 mRNA expression after 4 h of exposure. In order to identify possible targets for the STAT5-mediated protection of INS-1E cells, we studied the effect of hGH on activation of the transcription factors STAT1 and nuclear factor-B (NF-B) by IFNand IL-1 +TNF-respectively. Gel retardation experiments showed that hGH affects neither IFN-+ TNF--induced STAT1 DNA binding nor IL-1 and IFN-+TNF--induced NF B DNA binding. The lack of influence of hGH on cytokine-mediated activation of STAT1 and NF B is in accordance with the finding that hGH had only a minor effect on cytokine-induced inducible nitric oxide synthase (iNOS) gene expression and in fact augmented the IL-1 -stimulated nitric oxide production. As the anti-apoptotic Bcl-x L gene has been shown to harbour a STAT5-binding element we measured the expression of Bcl-x L as well as the pro-apoptotic Bax. We found that hGH increased the Bcl-x L /Bax ratio both in the absence and in the presence of cytotoxic cytokines. In conclusion, these results suggested that GH and PRL protect -cells against cytotoxic cytokines via STAT5-dependent mechanisms distal to iNOS activation possibly at the level of Bcl-x L .

Pancreatic ␤-cell growth and survival and insulin production are stimulated by growth hormone and prolactin through activation of the transcription factor signal transducer and activator of transcription (STAT)5. To assess the role of STAT5 activity in ␤-cells in vivo, we generated transgenic mice that expressed a dominant-negative mutant of STAT5a (DNSTAT5) or constitutive active mutant of STAT5b (CASTAT5) under control of the rat insulin 1 promoter (RIP). When subjected to a high-fat diet, RIP-DNSTAT5 mice showed higher body weight, increased plasma glucose levels, and impairment of glucose tolerance, whereas RIP-CASTAT5 mice were more glucose tolerant and less hyperleptinemic than wild-type mice. Although the pancreatic insulin content and relative ␤-cell area were increased in high-fat diet-fed RIP-DNSTAT5 mice compared with wild-type or RIP-CASTAT5 mice, RIP-DNSTAT5 mice showed reduced ␤-cell proliferation at 6 months of age. The inhibitory effect of high-fat diet or leptin on insulin secretion was diminished in isolated islets from RIP-DNSTAT5 mice compared with wild-type islets. Upon multiple low-dose streptozotocin treatment, RIP-DNSTAT5 mice exhibited higher plasma glucose levels, lower plasma insulin levels, and lower pancreatic insulin content than wild-type mice, whereas RIP-CASTAT5 mice maintained higher levels of plasma insulin. In conclusion, our results indicate that STAT5 activity in ␤-cells influences the susceptibility to experimentally induced type 1 and type 2 diabetes.

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