UNIPROT:P01275 - FACTA Search

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Query: UNIPROT:P01275 (glucagon)
26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glucagon-like peptide 1 (GLP-1), a hormonal activator of adenyl cyclase, stimulates insulin gene transcription, an effect mediated by the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1). Here we demonstrate that the signaling mechanism underlying stimulatory effects of GLP-1 on insulin gene transcription results from protein kinase A (PKA)-independent activation of the RIP1 CRE. Although GLP-1 stimulates cAMP production in rat INS-1 insulinoma cells, we find accompanying activation of a -410-bp RIP1 luciferase construct (-410RIP1-LUC) to exist independently of this second messenger. GLP-1 produced a dose-dependent stimulation of -410RIP1-LUC (EC50 0.43 nmol/l), an effect reproduced by the GLP-1 receptor agonist exendin-4 and abolished by the antagonist exendin(9-39). Activation of RIP1 by GLP-1 was not affected by cotransfection with dominant-negative Gs alpha, was not blocked by cAMP antagonist Rp-cAMPS, and was insensitive to PKA antagonist H-89. Truncation of -410RIP1-LUC to generate -307-, -206-, and -166-bp constructs revealed 2 segments of RIP1 targeted by GLP-1. The first segment, not regulated by forskolin, was located between -410 and -307 bp of the promoter. The second segment, regulated by both GLP-1 and forskolin, included the CRE and was located between -206 and -166 bp. Consistent with these observations, stimulatory effects of GLP-1 at RIP1 were reduced after introduction of delta-182 and delta-183/180 inactivating deletions at the CRE. The action of GLP-1 at -410RIP1-LUC was also reduced by cotransfection with A-CREB, a genetically engineered isoform of the CRE binding protein CREB, which dimerizes with and prevents binding of basic-region-leucine-zipper (bZIP) transcription factors to the CRE. In contrast, the action of GLP-1 at the CRE was not blocked by cotransfection with M1-CREB, an isoform that lacks a consensus serine residue serving as substrate for PKA-mediated phosphorylation. On the basis of these studies, it is proposed that PKA-independent stimulatory actions of GLP-1 at RIP1 are mediated by bZIP transcription factors related in structure but not identical to CREB.
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PMID:Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element. 1090 73

AR42J is an exocrine pancreatic cell line that has been reported to differentiate towards an endocrine phenotype when stimulated with various growth factors, such as activin A, hepatocyte growth factor (HGF), betacellulin or glucagon-like peptide 1. In our experiments, AR42J-B13 cells differentiated morphologically in response to the growth factor treatment as reported previously. However, they failed to express the insulin gene. We found that the cells did not express several transcription factors known to be found in the beta-cell, including Nkx6.1, isl-1, Pax4 and Pax6. In addition, the mRNA level for pdx-1 and Nkx2.2 were very low in comparison to the insulinoma cell lines INS-1 and RINm5F. However, some transcription factors typically found in beta-cells and neuroendocrine cells were expressed also in the AR42J-B13 cells. These included BETA2/NeuroD, HNF1alpha, C/EBPbeta and IA-1. Unlike the insulinoma cells, AR42J cells expressed the exocrine transcription factor p48. In order to induce endocrine differentiation, we transfected the AR42J-B13 cells with the full length cDNAs of isl-1, Nkx6.1, Nkx2.2 and pdx-1 under the control of the CMV promoter, both separately and in combinations. The expression of Nkx2.2 led consistently to the appearance of pancreatic polypeptide but not insulin, glucagon or somatostatin mRNA. The PP mRNA expression in Nkx2.2 cDNA transfected cells was independent of the growth factor treatment used for differentiating AR42J cells. In conclusion, the AR42J-B13 line possesses some features of a pancreatic neuroendocrine cell. However, we were unable to confirm the capacity of these cells to differentiate into insulin-producing cells. Our results indicate that Nkx2.2 plays a role in the transcriptional regulation of PP expression.
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PMID:Transcription factor expression and hormone production in pancreatic AR42J cells. 1094 Apr 82

The regulation of glucose-dependent insulin secretion in pancreatic beta-cells is linked to the expression and function of the ATP-sensitive potassium channel (K(ATP)), which is composed of a sulfonylurea receptor (SUR1) and an inwardly rectifying potassium channel (Kir6.2). Previous animal and human genetic studies have demonstrated that disruption or defective expression of K(ATP) subunit genes has a profound impact on the regulation of insulin secretion. Little is known about how SUR1 and Kir6.2 gene expression is regulated. Here we show that high glucose concentrations lead to a marked decrease (approximately 70%) in Kir6.2 messenger RNA (mRNA) levels in isolated rat pancreatic islets as well as in the INS-1 beta-cell line. This effect is reversible, because exposure to low glucose reinduces Kir6.2 transcript levels. The cognate K(ATP) channel subunit SUR1 showed similar down-regulation at high glucose concentration. The K(ATP) channel activity of INS-1 cells cultivated at high glucose was reduced by 33-51%. In contrast, glucagon-like peptide-1 (GLP-1) induced Kir6.2 mRNA steady state levels and was able to prevent glucose-dependent inhibition of Kir6.2 mRNA and K(ATP) channel activity. To provide further insight into the mechanisms by which glucose and GLP-1 regulate beta-cell K(ATP) channel genes, we have cloned and initiated the characterization of the Kir6.2 gene transcriptional regulatory regions contained within the entire 4.5 kb flanked by the SUR1 and Kir6.2 genes. Transient transfection experiments with five deletion constructs in a pancreatic beta-cell line (INS-1) showed that the proximal 988 bp of the Kir6.2 promoter sequence contributes only 25-30% to the total basal promoter activity. The minimal promoter region -67/+140, also encompassing parts of the 5'-untranslated region, confers sensitivity to GLP-1, which stimulates transcriptional activity of the Kir6.2 minigene by about 2-fold. We propose that glucose- and GLP-1-dependent regulation of K(ATP) subunit genes may be important in the adaptation of beta-cells to changes in secretory demands in physiological and diseased states.
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PMID:Regulated expression of adenosine triphosphate-sensitive potassium channel subunits in pancreatic beta-cells. 1114 75

The insulin gene promoter contains many transcriptional response elements that predispose the gene to a wide range of regulatory signals. Glucagon-like peptide 1 (GLP-1) stimulates insulin gene transcription by intracellular second messenger cascades leading to direct transcription factor activation or to the up-regulation of insulin promoter specific transcription factors. In these studies, we have identified a novel regulatory signaling mechanism acting on the rat insulin 1 promoter (rINS1) in the INS-1 beta-cell line. In the presence of stimulatory concentrations of GLP-1 (0.1--100 nM) on rINS1 activity, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) using SB 203580 resulted in a marked increase in promoter activity (maximum 3-fold) over GLP-1 alone, as determined by rINS1 promoter-luciferase reporter gene expression. This effect was revealed to be mediated via the cAMP response element (CRE) of rINS1, because site directed mutagenesis of the CRE motif in rINS1 abolished the increased response to SB 203580. Furthermore, inhibition of p38 MAPK uncovered a similar, more pronounced, response in the expression of a generic CRE promoter driven reporter gene. Time course dose-response studies indicate that the p38 MAPK induced inhibitory response may involve expression of immediate early genes (IEGs); maximum repression of rINS1 activity occurred after 4 h of treatment, comparable with regulatory responses by IEGs. In conclusion, these results demonstrate a novel signaling mechanism whereby p38 MAPK represses rINS1 promoter activity in response to GLP-1, suggesting the involvement of a robust regulatory control by p38 MAPK in insulin gene expression. The relevance of this mechanism may be most apparent during periods of cellular stress in which p38 MAPK activity is stimulated. In this regard, reduced insulin expression levels caused by chronic hyperglycemia (glucotoxicity) and/or hyperlipidemia (lipotoxicity) may be a direct consequence of this mechanism.
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PMID:Insulinotropic hormone glucagon-like peptide 1 (GLP-1) activation of insulin gene promoter inhibited by p38 mitogen-activated protein kinase. 1118 33

Protein kinase B/Akt (PKB/Akt) is activated by phosphatidylinositol 3-kinase (PI 3-K) and is a central mediator of cellular proliferation and protection against apoptosis. Insulin, insulin-like growth factor (IGF-1), and glucagon-like peptide-1 (GLP-1) act as glucose-dependent growth factors for pancreatic beta-cells. We assessed signaling pathways and stimulation patterns of PKB/Akt activation by these ligands in the beta-cell line INS-1. Insulin, IGF-1, and GLP-1 induced distinctive time dependent, dose dependent, and glucose dependent phosphorylation of PKB/Akt. Insulin and IGF-1 stimulated PI 3-K activity was mainly associated with insulin receptor substrate (IRS) isoforms IRS-1 and IRS-2 and less so with the IRS-isoform Grb-2 associated binder-1 (Gab-1). In contrast, GLP-1 induced PI 3-K activity mainly in Gab-1 and also in IRS-2 immunoprecipitates, although in an attenuated kinetic. Thus, activation pathways of PKB/Akt by insulin, IGF-1, and GLP-1 converge at the level of IRS-isoforms and PI 3-K inducing differential activation of PKB/Akt. These data indicate an essential role of PKB/Akt in regulation of beta-cell proliferation.
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PMID:Integrative mitogenic role of protein kinase B/Akt in beta-cells. 1119 29

In the pancreas, ligands of receptor tyrosine kinases (RTKs) are thought to be implicated in the development and function of the islets of Langerhans, which represent the endocrine part of the pancreas. In a previous study, we randomly screened by reverse transcriptase-polymerase chain reaction for RTKs expressed in the embryonic pancreas. One cDNA fragment that was cloned during this screen corresponded to the KIT receptor. The objective of the present study was to analyze the pattern of Kit expression in the pancreas. We demonstrated that Kit is expressed and functional in terms of signal transduction in the insulin-producing cell line INS-1. Indeed, upon treatment with the KIT ligand (KITL), the extracellular signal-regulated protein kinase was phosphorylated, and the expression of early responsive genes was induced. We also demonstrated that Kit mRNAs are present in fetal and adult rat islets. We next used mice that had integrated the lacZ reporter gene into the Kit locus. In these mice, beta-galactosidase (beta-gal) served as a convenient marker for expression of the endogenous Kit gene. Kit was found to be specifically transcribed in beta-cells (insulin-expressing cells), whereas no expression was found in other endocrine cell types or in the exocrine tissue. Interestingly, not all mature beta-cells expressed Kit, indicating that Kit is a marker of a subpopulation of beta-cells. Finally, by following beta-gal expression in the pancreas during fetal life, we found that at E14.5, Kit is expressed in both insulin- and glucagon-expressing cells present at that stage, and also in a specific cell population present in the epithelium that stained negative for endocrine markers. These data suggest that these Kit-positive/endocrine-negative cells could represent a subpopulation of endocrine cell precursors.
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PMID:Expression of the receptor tyrosine kinase KIT in mature beta-cells and in the pancreas in development. 1152 67

The winged helix transcription factors, hepatocyte nuclear factors 3alpha, -beta, and -gamma (HNF-3, encoded by the Foxa1, -a2, and -a3 genes, respectively), are expressed early in embryonic endoderm and play important roles in the regulation of gene expression in liver and pancreas. Foxa1 has been shown to be required for glucagon secretion in the pancreas, whereas Foxa2 is critical for the regulation of insulin secretion in pancreatic beta-cells. Here we address the role of Foxa3 in the maintenance of glucose homeostasis. Mice homozygous for a null mutation in Foxa3 appear normal under fed conditions. However, when fasted, Foxa3(-/-) mice have a significantly lower blood glucose compared with control mice. The fasting hypoglycemia in Foxa3(-/-) mice could not be attributed to defects in pancreatic hormone secretion, ketone production, or hepatic glycogen breakdown. Surprisingly, mRNA levels for several gluconeogenic enzymes were up-regulated appropriately in fasted Foxa3(-/-) mice, despite the fact that the corresponding genes had been shown to be activated by FOXA proteins in vitro. However, the mRNA for the plasma membrane glucose transporter GLUT2 was decreased by 64% in the fasted and 93% in the fed state, suggesting that efflux of newly synthesized glucose is limiting in Foxa3(-/-) hepatocytes. Thus, Foxa3 is the dominating transcriptional regulator of GLUT2 expression in hepatocytes in vivo. In addition, we investigated the hepatic transcription factor network in Foxa3(-/-) mice and found that the normal activation of HNF-4alpha, HNF-1alpha, and PGC-1 induced by fasting is attenuated in mice lacking Foxa3.
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PMID:Foxa3 (hepatocyte nuclear factor 3gamma ) is required for the regulation of hepatic GLUT2 expression and the maintenance of glucose homeostasis during a prolonged fast. 1154 10

To develop transplantable beta-cell lines for the treatment of diabetes mellitus, we have taken advantage of the property of INS-1 cells to synthesize and secrete not only insulin, but also small quantities of the insulinotropic hormone glucagon-like peptide-1 (GLP-1). In INS-1 cells over-expressing the beta-cell GLP-1 receptor (GLP-1-R), we have shown, by radioimmune assay and bioassay of conditioned medium, that an autocrine signaling mechanism of hormone action exists whereby self-secreted GLP-1 acts as a competence factor in support of insulin gene transcription. INS-1 cells also exhibit insulin gene promoter activity, as assayed in cells transfected with a rat insulin gene I promoter-luciferase construct (RIP1-Luc). The GLP-1-R agonist exendin-4 stimulates RIP1-Luc activity in a glucose-dependent manner, an effect mediated by endogenous GLP-1-Rs, and is blocked by the serine/threonine protein kinase inhibitor Ro 31-8220. Over-expression of GLP-1-R in transfected INS-1 cells reduces the threshold for exendin-4 agonist action, whereas basal RIP1-Luc activity increases 2.5-fold in the absence of added agonist. The increase of basal RIP1-Luc activity is a consequence of autocrine stimulation by self-secreted GLP-1 and is blocked by introduction of (1) an inactivating W39A mutation in the N-terminus ligand-binding domain of GLP-1-R or (2) mutations in the third cytoplasmic loop that prevent G protein coupling. No evidence for constitutive ligand-independent signaling properties of the GLP-1-R has been obtained. Over-expression of GLP-1-R increases the potency and efficacy of D-glucose as a stimulator of RIP1-Luc. Thus, INS-1 cells over-expressing the GLP-1-R recapitulate the incretin hormone effect of circulating GLP-1, thereby providing a possible strategy by which beta-cell lines may be engineered for efficient glucose-dependent insulin biosynthesis and secretion.
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PMID:Over-expression of the glucagon-like peptide-1 receptor on INS-1 cells confers autocrine stimulation of insulin gene promoter activity: a strategy for production of pancreatic beta-cell lines for use in transplantation. 1184 26

Currently there is intense interest to define the mechanism of action of glucagon-like peptide-1 (GLP-1) in regulating beta-cell function, including insulin gene transcription. In this study, GLP-1 (100 nmol/l), in the presence of glucose (11 mmol/l), induced a similar71-fold increase in insulin gene promoter activity in INS-1 pancreatic beta-cells, an effect that was an order of magnitude larger than with either stimulant alone. The response to GLP-1 was mimicked by forskolin and largely inhibited by the protein kinase A (PKA) inhibitors, H89 and myristoylated PKI(14--22) amide, indicating partial mediation via a cAMP/PKA pathway. Significantly, the actions of both GLP-1 and forskolin were abolished by the selective Ca(2+)/calmodulin-dependent phosphatase 2B (calcineurin) inhibitor, FK506, as well as by the chelation of intracellular Ca(2+) by BAPTA (bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate). Glucose and GLP-1 also synergistically activated NFAT (nuclear factor of activated T-cells)-mediated transcription from a minimal promoter construct containing tandem NFAT consensus sequences. Furthermore, two-point base pair mutations in any of the three identified NFAT sites within the rat insulin I promoter resulted in a significant reduction in the combined effect of glucose and GLP-1. These data suggest that the synergistic action of glucose and GLP-1 to promote insulin gene transcription is mediated through NFAT via PKA- and calcineurin-dependent pathways in pancreatic beta-cells.
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PMID:NFAT regulates insulin gene promoter activity in response to synergistic pathways induced by glucose and glucagon-like peptide-1. 1187 68

The transcription factor Foxa2 is implicated in blood glucose homeostasis. Conditional expression of Foxa2 or its dominant-negative mutant DN-Foxa2 in INS-1 cells reveals that Foxa2 regulates the expression of genes important for glucose sensing in pancreatic beta-cells. Overexpression of Foxa2 results in blunted glucose-stimulated insulin secretion, whereas induction of DN-Foxa2 causes a left shift of glucose-induced insulin release. The mRNA levels of GLUT2 and glucokinase are drastically decreased after induction of Foxa2. In contrast, loss of Foxa2 function leads to up-regulation of hexokinase (HK) I and II and glucokinase (HK-IV) mRNA expression. The glucokinase and the low K(m) hexokinase activities as well as glycolysis are increased proportionally. In addition, induction of DN-Foxa2 also reduces the expression of beta-cell K(ATP) channel subunits Sur1 and Kir6.2 by 70%. Furthermore, in contrast to previous reports, induction of Foxa2 causes pronounced decreases in the HNF4alpha and HNF1alpha mRNA levels. Foxa2 fails to regulate the expression of Pdx1 transcripts. The expression of insulin and islet amyloid polypeptide is markedly suppressed after induction of Foxa2, while the glucagon mRNA levels are significantly increased. Conversely, Foxa2 is required for glucagon expression in these INS-1-derived cells. These results suggest that Foxa2 is a vital transcription factor evolved to control the expression of genes essential for maintaining beta-cell glucose sensing and glucose homeostasis.
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PMID:Foxa2 (HNF3beta ) controls multiple genes implicated in metabolism-secretion coupling of glucose-induced insulin release. 1187 61

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