OBJECTIVE-␤-Cell proliferation is an important mechanism underlying ␤-cell mass adaptation to metabolic demands. We have examined effects, in particular those mediated through intracellular cAMP signaling, of the incretin hormone analog exendin-4 on cell cycle regulation in ␤-cells.RESEARCH DESIGN AND METHODS-Changes in islet protein levels of cyclins and of two critical cell cycle regulators cyclin kinase inhibitor p27 and S-phase kinase-associated protein 2 (Skp2) were assessed in mice treated with exendin-4 and in a mouse model with specific upregulation of nuclear cAMP signaling exhibiting increased ␤-cell proliferation (CBP-S436A mouse). Because cyclin A2 was stimulated by cAMP, we assessed the role of cylcin A2 in cell cycle progression in Min6 and isolated islet ␤-cells.RESULTS-Mice treated with exendin-4 showed increased ␤-cell proliferation, elevated islet protein levels of cyclin A2 with unchanged D-type cyclins, elevated PDX-1 and Skp2 levels, and reduced p27 levels. Exendin-4 stimulated cyclin A2 promoter activity via the cAMP-cAMP response element binding protein pathway. CBP-S436A islets exhibited elevated cyclin A2, reduced p27, and no changes in D-type cyclins, PDX-1, or Skp2. In cultured islets, exendin-4 increased cyclin A2 and Skp2 and reduced p27. Cyclin A2 overexpression in primary islets increased proliferation and reduced p27. In Min6 cells, cyclin A2 knockdown prevented exendin-4 -stimulated proliferation. PDX-1 knockdown reduced exendin-4 -stimulated cAMP synthesis and cyclin A2 transcription.CONCLUSIONS-Cyclin A2 is required for ␤-cell proliferation, exendin-4 stimulates cyclin A2 expression via the cAMP pathway, and exendin-4 stimulation of cAMP requires PDX-1. Diabetes 57:2371-2381, 2008 P ancreatic ␤-cell mass is dynamic and responds to variations in metabolic demand on insulin production. The inability of the endocrine pancreas to adapt to changing insulin demand (inadequate ␤-cell mass) is found both in type 1 and type 2 diabetes. Increasing ␤-cell mass by regeneration may ameliorate or correct both type 1 and 2 diabetes (1). Within the pancreas, ␤-cells regenerate predominantly by ␤-cell replication (2,3). In this context, insight into the mechanisms underlying ␤-cell proliferation and cell cycle regulation, may provide potential targets for therapy in situations of inadequate ␤-cell mass.The incretin hormone glucagon-like peptide-1 (GLP-1) and its long-acting peptide analog exendin-4 stimulate ␤-cell proliferation in vitro and in vivo (4), leading to increased ␤-cell mass in rodents and amelioration of glucose metabolism in diabetic animal models and human diabetic subjects (4). Downstream effectors of the GLP-1 signaling to the cell nucleus include 1) the epidermal growth factor receptor-phosphoinositol 3-kinase (PI 3-kinase)-protein kinase B (PKB/Akt)-forkhead box transcription factor O1 (FoxO1) pathway (5) and 2) the cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway (4). Activation of the PI 3-kinase pathway results in phosphorylation and nuclear...

The cyclic AMP (cAMP) signaling pathway is central in ␤-cell gene expression and function. In the nucleus, protein kinase A (PKA) phosphorylates CREB, resulting in recruitment of the transcriptional coactivators p300 and CREB binding protein (CBP). CBP, but not p300, is phosphorylated at serine 436 in response to insulin action. CBP phosphorylation disrupts CREB-CBP interaction and thus reduces nuclear cAMP action. To elucidate the importance of the cAMP-PKA-CREB-CBP pathway in pancreatic ␤ cells specifically at the nuclear level, we have examined mutant mice lacking the insulin-dependent phosphorylation site of CBP. In these mice, the CREB-CBP interaction is enhanced in both the absence and presence of cAMP stimulation. We found that islet and ␤-cell masses were increased twofold, while pancreas weights were not different from the weights of wild-type littermates. ␤-Cell proliferation was increased both in vivo and in vitro in isolated islet cultures. Surprisingly, glucose-stimulated insulin secretion from perfused, isolated mutant islets was reduced. However, ␤-cell depolarization with KCl induced similar levels of insulin release from mutant and wild-type islets, indicating normal insulin synthesis and storage. In addition, transcripts of pgc1a, which disrupts glucose-stimulated insulin secretion, were also markedly elevated. In conclusion, sustained activation of CBP-responsive genes results in increased ␤-cell proliferation. In these ␤ cells, however, glucose-stimulated insulin secretion was diminished, resulting from concomitant CREB-CBP-mediated pgc1a gene activation.Under circumstances of increased metabolic demand, such as pregnancy (2, 4), or in insulin-resistant states (3, 30), islet and ␤-cell masses increase to meet metabolic requirements. The failure of ␤ cells to adapt to metabolic requirements results in relative insulin deficiency and diabetes mellitus. Within the pancreas, three conceptual mechanisms may lead to an increase in ␤-cell mass: (i) neogenesis from nonendocrine pancreatic tissue, such as pancreatic-duct epithelium (2, 3, 27, 28), pancreatic acinar cells (␤ cells derived from non-␤ cells) (3, 38, 39), or undifferentiated cells within the islet (1, 9, 15, 44); (ii) proliferation of ␤ cells (␤ cells derived from existing ␤ cells) (6,11,38); and (iii) reduced ␤-cell apoptosis in the context of normal ␤-cell turnover (5, 26).Cyclic AMP (cAMP) signaling is critical in the physiologic function of ␤ cells. This is exemplified by the effects of the incretin hormone glucagon-like peptide-1 (GLP-1), which improves glucose-stimulated insulin secretion from pancreatic ␤ cells, in part by raising intracellular cAMP levels (13,18,28). In pharmacologic studies, GLP-1 also stimulated PDX-1 gene expression in pancreatic-duct epithelial cells and stimulates proliferation of ␤ cells (5,7,22,26,38). Binding of GLP-1 to its ␤-cell receptor elevates intracellular cAMP levels; cAMP in turn binds to the regulatory subunit of protein kinase A (PKA) and releases the PKA catalytic subunit. Elevation of cAMP als...

The CD31 Ag is a surface glycoprotein of 130 kDa with a broad cellular distribution. We show that among peripheral human blood cells, it is expressed on monocytes, granulocytes, platelets, and a subpopulation of lymphocytes. Activation of granulocytes leads to down-regulation of CD31 molecule expression. Sequence analysis and quantitative measurements of the relatedness of the CD31 molecule to other known proteins demonstrate that it consists of six Ig constant domains and that each domain bears substantial similarity to Fc gamma R domains. We find, however, that the CD31 molecule does not bind Ig Fc domains. On human monocytes we demonstrate that CD31 mAb recognizing certain epitopes of the CD31 molecule induce the generation of reactive oxygen metabolites. No such effect was seen with human granulocytes. By using two CD31 mAb, termed 1B5 and 7E4, we analyzed the requirements for activation of the monocyte respiratory burst via CD31 Ag in more detail. We show that signal transduction occurs via formation of a CD31 Ag-mAb-Fc gamma R complex involving either Fc gamma RI (CD64) or Fc gamma RII (CDw32) molecules.