In the mouse, insulin is produced from two similar but nonallelic genes that encode proinsulins I and II. We have investigated expression of these two genes during mouse embryonic development, using a PCR to detect the two gene transcripts and immunocytochemistry to visualize the two corresponding proteins. At appearance of the dorsal pancreatic anlage at day 9.5 of gestation, both mRNAs could be detected in the embryos, and both proteins were present together in the same cells of the developing pancreas. At days 9.5 and 10.5, when the ventral anlage appears, there were fewer proinsulin H mRNAs than proinsulin I mRNAs. At day 12.5 this ratio was reversed. Proinsulin II mRNA, but not proinsulin I mRNA, could be detected at day 8.5 in the prepancreatic embryo. Proinsulin II mRNA, but not proinsulin I mRNA, was also found in the heads of embryos at day 9.5 and at all later stages studied. These results indicate that the two proinsulin genes are regulated independently, at least in part. They also suggest that insulin might play a role as a growth factor in the developing mouse brain.Insulin, a key hormone in metabolic homeostasis, is synthesized, stored, and secreted by beta cells of the pancreatic islets. The protein is synthetized in the form of a precursor, preproinsulin, which is highly conserved among animal species. Unlike most mammals, mice and rats express two nonallelic genes that encode proinsulins I and II. In the mouse these two proteins differ by two amino acids in the B chain and three amino acids in the C peptide. Mouse C peptide I also lacks the Gly-Ala residues present in positions 17 and 18 of C peptide 11(1, 2). The corresponding genes in mouse and rat are highly homologous, and their organization is similar, except that the preproinsulin I gene possesses only the first of two introns present in the preproinsulin II gene (1, 3).On previous studies concerning the regulation of these two nonallelic genes, mRNAs were reported to be present in nearly equal quantities in adult pancreas of mouse (1, 4), as well as of rat (5) Here we have investigated regulation of the expression of the two proinsulin genes during mouse development. In the mouse, the pancreas is derived from the duodenum as two evaginations evolving at days 9.5 (dorsal) and 10.5 (ventral) of the gestation. The evaginations coalesce at day 11, and insulin has been first detected in previous studies at day 11.5 (9, 10). We have used a reverse-transcriptase-PCR (RT-PCR) assay, which allows identification and estimation of the relative amounts of mRNA transcribed from each of the two proinsulin genes. This RT-PCR, using a single pair ofprimers and a single probe for the two transcripts, allowed comparison of the relative amounts of proinsulin I and II mRNAs in the samples (11). Immunocytochemistry with antibodies specific for each of the two proinsulins allowed us to distinguish the products of the two genes in embryo sections. We have, thus, been able to detect insulin mRNAs and proteins much earlier, to distinguish the two forms, ...

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.

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Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371,[606][607][608][609]. In adults, IPF1 expression is restricted to the a-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of 8-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet a-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta2/NeuroD, which is expressed in both a-and P-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-8 islet cells, which suggests an important role of IPF1 in a-cell maturation.Insulin promoter factor 1 (IPF1) is expressed in precursor cells during pancreas ontogeny (1, 2), and expression is required for pancreas formation (3,4). During ontogeny, IPF1 expression becomes restricted to the nuclei of the insulin-producing pancreatic islet (3-cells, suggesting that maintenance of IPF1 expression is necessary for the differentiation islet p3-cells from an IPF1-positive precursor common to all islet cells (2, 5). This restricted expression profile within the islets is reflected in the transplantable rat pancreatic insulinoma (IN) and glucagonoma (AN), which show substantial similarity to the mature islet f3-and a-cells, respectively (6-8). Thus, the AN is lacking IPF1 expression, as is the normal a-cell, and was recently found to be similar to normal a-cells in its expression of glucokinase as well as of the glucose-regulated insulinotropic peptide and glucagon-like peptide 1 receptors (9, 10). In vitro IPF1 binds to multiple sites in the insulin promoter and activates insulin gene reporter constructs when cotransfected into cell lines (1,5,11,12). This activity is dependent on cooperation between IPF1 and insulin enhancer factor-1 (IEF-1; refs. 5 and 12), a heterodimer composed of Beta2/ NeuroD, which is present in both a-and (3-cells, and ubiquitous class A helix-loop-helix proteins, such as Betal/rat E-box binding protein (REB; ref. 13) and products of the E2A gene (E47, E12, and ITF-1; refs. 14-19). In addition to IPF1 and IEF-1 binding sites, transcriptional regulation of the insulin gene requires a number of other cis-elements to which factors not yet cloned are binding (20)(21)(22)(23). To address whether IPF1 could activate transcription of the otherwise silent insulin genes in islet cells lacking IPF1 but expressing at least a subset of the other insulin gene transcription factors, a cDNA encoding rat IPF1 (24) under transcriptional control of the cytomegalovirus promoter was stably tra...