Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P01275 (glucagon)
 26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesterol and cholesterol secretion into bile in the liver. Previously, we identified an SR-BI-associated protein, termed PDZK1, from rat liver membrane extracts. PDZK1 contains four PSD-95/Dlg/ZO-1 (PDZ) domains, the first of which in the N-terminal region is responsible for the association with SR-BI. PDZK1 controls hepatic SR-BI expression in a posttranscriptional fashion both in cell culture and in vivo. In this study, we demonstrated that the C-terminal region of PDZK1 is crucial for up-regulating SR-BI protein expression. Metabolic labeling experiments and phosphoamino acid analysis revealed that PDZK1 is phosphorylated at Ser residues within this region. Point-mutation analysis demonstrated that PDZK1 is phosphorylated at Ser-509. Interestingly, a mutant PDZK1, in which Ser-509 was replaced with Ala, lost the ability to up-regulate SR-BI protein. We identified Ser-509 of PDZK1 as the residue that is phosphorylated by the cAMP-dependent PKA in vitro as well as in cell culture. Ser-509 of PDZK1 in rat liver was also phosphorylated, as shown by an Ab that specifically detects phosphorylated Ser-509. Administration of glucagon to Wistar rats increased PDZK1 phosphorylation as well as hepatic SR-BI and PDZK1 expression while it decreased plasma HDL levels, indicating that PDZK1 phosphorylation is hormonally regulated. These findings suggest that phosphorylation of PDZK1 has an important role in the regulation of hepatic SR-BI expression and, thus, influences plasma HDL levels.
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PMID:Regulation of SR-BI protein levels by phosphorylation of its associated protein, PDZK1. 1617 36

Glucagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) that code for transcription factors implicated in cell proliferation, differentiation, and apoptosis. We recently observed that early growth response factor-1 (EGR-1), an IEG product, was required for transcriptional activation of Ccnd1 (cyclin D1) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in the pancreatic beta-cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between -73 and -46 for the activation of EGR-1 in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5'-GTACGTCA-3') located at -69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at -139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP response element-binding protein (CREB) phosphorylated on Ser(133) to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser(133) or a dominant negative CREB mutant that inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD-98059, indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1. Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser(133) is necessary for induction of the EGR-1 transcription by exendin-4.
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PMID:Proximal cyclic AMP response element is essential for exendin-4 induction of rat EGR-1 gene. 1692 76

Pancreatic beta-cells are the major extraneural site of glutamate decarboxylase expression (GAD). During culture of isolated beta-cells, the GAD product gamma-aminobutyrate (GABA) is rapidly released in the medium, independently of insulin. It is considered as a possible mediator of beta-cell influences on alpha-cells, acinar cells, and/or infiltrating lymphocytes. In this perspective, we investigated the regulation of GABA release by rat beta-cells during a 24-h culture period. Glucose was previously reported to inhibit GABA release by diverting cellular GABA to mitochondrial breakdown through activation of GABA transferase (GABA-T). In the present study, glucagon-like peptide-1 (GLP-1) was shown to stimulate GABA formation at the level of GAD, its effect being suppressed by the GAD inhibitor allylglycine and remaining unaltered by the GABA-T inhibitor gamma-vinyl-GABA. The stimulatory action of GLP-1 is cAMP dependent, being reproduced by the adenylate cyclase activator forskolin and the cAMP analog N(6)-benzoyladenosine-3',5'-cAMP and inhibited by a PKA inhibitor. It is dependent on protein synthesis and associated with an increased expression of GAD67 but not GAD65. The GLP-1-induced stimulation of GAD activity in beta-cells can elevate medium GABA levels in conditions of glucose-driven intracellular GABA breakdown and thus maintain GABA-mediated beta-cell influences on neighboring cells.
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PMID:Glucagon-like peptide-1 stimulates GABA formation by pancreatic beta-cells at the level of glutamate decarboxylase. 1719 Sep 4

Glucagon-like peptide 1 (GLP-1) is a potent inhibitor of food intake. GLP-1 receptor mRNA is densely expressed in hypothalamic arcuate nucleus (ARC) and precisely overlaps the area occupied by proopiomelanocortin (POMC) neurons. Activation of POMC neurons suppresses appetite, and lack of POMC-derived peptides or inhibition of POMC neuronal firing causes obesity. Here, we identify living POMC cells in mouse ARC brain slices by targeted expression of green fluorescent protein. Using whole-cell patch-clamp recordings, we show that GLP-1 increases the spontaneous action-potential firing of POMC neurons. The stimulatory effect of GLP-1 was mimicked by GLP-1 receptor agonist exendin-4 and abolished by the receptor antagonist exendin 9-39. The effect of GLP-1 was unchanged in the presence of the synaptic blockers DAP5 (D(-)-2-amino-5-phosphonopentanoic acid)/CNQX (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt) and picrotoxin. These results suggest that GLP-1 excites POMC neurons postsynaptically, via interaction with GLP-1 receptors on POMC cells. Whole-cell Ca2+ currents increased approximately 70% in the presence of GLP-1, and this effect was abolished by L-type Ca2+ channel antagonist nifedipine. Forskolin (which activates cAMP) mimicked the effects of GLP-1 and the PKA inhibitor Rp-8-Bromo-cAMPS (8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer) blocked GLP-1 action. These data indicate that GLP-1 stimulates the electrical activity of hypothalamic POMC neurons by activation of PKA and a subsequent increase in L-type Ca2+ current. This effect may contribute to the anorectic action of GLP-1, because excitation of POMC cells is well established to reduce food intake.
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PMID:Glucagon-like peptide 1 stimulates hypothalamic proopiomelanocortin neurons. 2165 75

The nuclear PXR (pregnane X receptor) was originally characterized as a key transcription factor that activated hepatic genes encoding drug-metabolizing enzymes. We have now demonstrated that PXR also represses glucagon-activated transcription of the G6Pase (glucose-6-phosphatase) gene by directly binding to CREB [CRE (cAMP-response element)-binding protein]. Adenoviral-mediated expression of human PXR (hPXR) and its activation by rifampicin strongly repressed cAMP-dependent induction of the endogenous G6Pase gene in Huh7 cells. Using the -259 bp G6Pase promoter construct in cell-based transcription assays, repression by hPXR of PKA (cAMP-dependent protein kinase)-mediated promoter activation was delineated to CRE sites. GST (glutathione transferase) pull-down and immunoprecipitation assays were employed to show that PXR binds directly to CREB, while gel-shift assays were used to demonstrate that this binding prevents CREB interaction with the CRE. These results are consistent with the hypothesis that PXR represses the transcription of the G6Pase gene by inhibiting the DNA-binding ability of CREB. In support of this hypothesis, treatment with the mouse PXR activator PCN (pregnenolone 16alpha-carbonitrile) repressed cAMP-dependent induction of the G6Pase gene in primary hepatocytes prepared from wild-type, but not from PXR-knockout, mice, and also in the liver of fasting wild-type, but not PXR-knockout, mice. Moreover, ChIP (chromatin immunoprecipitation) assays were performed to show a decreased CREB binding to the G6Pase promoter in fasting wild-type mice after PCN treatment. Thus drug activation of PXR can repress the transcriptional activity of CREB, down-regulating gluconeogenesis.
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PMID:Human nuclear pregnane X receptor cross-talk with CREB to repress cAMP activation of the glucose-6-phosphatase gene. 1763 6

Plasminogen activator inhibitor-1 (PAI-1) controls the regulation of the fibrinolytic system in blood by inhibiting both urokinase-type and tissue-type plasminogen activators. Enhanced levels of PAI-1 are related to pathological conditions associated with hypoxia or hyperinsulinemia. In this study, we investigated the regulation of PAI-1 expression by glucagon and the cAMP/PKA/CREB signalling pathway in the liver. Stimulation of the cAMP/PKA/CREB signalling cascade by starvation in vivo or glucagon in vitro induced PAI-1 gene expression in liver. Furthermore, this response was associated with enhanced phosphorylation of CREB. By using EMSAs we found that three promoter elements, the HRE2, E-box 4 and E-box 5, were able to bind CREB but only the HRE2 and E5 appeared to be functionally active. Reporter gene assays confirmed that cAMP induced PAI-1 gene transcription via the same element in both human and rat promoters. Interestingly, although the HRE2 was involved, the glucagon/cAMP pathway had no influence on hypoxia-inducible factor-1 (HIF-1) mRNA and protein levels. Thus, CREB binding to the HIF-1 responsive elements in PAI-1 promoter mediates the glucagon effect in the liver.
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PMID:CREB binding to the hypoxia-inducible factor-1 responsive elements in the plasminogen activator inhibitor-1 promoter mediates the glucagon effect. 1772 10

Glucagon-like peptide-1 (GLP-1) induces several immediate early response genes such as c-fos, c-jun, and early growth response-1 (Egr-1), which are involved in cell proliferation and differentiation. We recently reported that exendin-4 (EX-4), a potent GLP-1 agonist, upregulated Egr-1 expression via phosphorylation of CREB, a transcription factor in INS-1 beta-cells. This study was designed to investigate the role of another transcription factors, serum response factor (SRF) and Yin Yang-1 (YY1), in EX-4-induced Egr-1 expression. EX-4 significantly increased Egr-1 mRNA and subsequently its protein level. EX-4-induced Egr-1 expression was inhibited by pretreatment with a PKA inhibitor, H-89, and an MEK inhibitor, PD 98059. The siRNA-mediated inhibition of PKA and ERK1 resulted in significant reduction of EX-4-induced Egr-1 expression. Promoter analyses showed that SRE clusters were essential for Egr-1 transcription, and YY1 overexpression did not affect Egr-1 promoter activity. EMSA results demonstrated that EX-4-induced transient increase in DNA-protein complex on SRE site, and that both SRF and phospho-SRF were bound to this site. Treatment of either YY1 consensus oligonucleotide or YY1 antibody did not effect the change of density or migration of the DNA-protein complex. Collectively, EX-4-induced Egr-1 expression is largely dependent on cAMP-mediated extracellular signal-regulated kinase activation, and EX-4 induces Egr-1 transcription via the interaction of SRF and phospho-SRF to SRE sites.
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PMID:Exendin-4 induction of Egr-1 expression in INS-1 beta-cells: interaction of SRF, not YY1, with SRE site of rat Egr-1 promoter. 1844 85

Both Epac and PKA are effectors of the second messenger cAMP. Utilizing an exchange protein directly activated by cAMP (Epac) pathway-specific cAMP analog (ESCA), we previously reported that Epac signaling regulates proglucagon gene (gcg) expression in the glucagon-like peptide-1 (GLP-1)-producing intestinal endocrine L-cell lines GLUTag and STC-1. We now show that Epac-2 is also expressed in glucagon-producing pancreatic alpha-cell lines, including PKA-deficient InR1-G9 cells, and that ESCA stimulates gcg promoter and mRNA expression in the InR1-G9 cells. Using a dominant-negative Epac-2 expression plasmid (Epac-2DN), we found that Epac inhibition attenuated forskolin-stimulated gcg promoter expression in the PKA-active STC-1 cell line and blocked forskolin-stimulated gcg promoter expression in the InR1-G9 cells. Consistently, ESCA was shown to stimulate glucagon and GLP-1 production in the InR1-G9 and GLUTag cell lines, respectively. Surprisingly, ESCA treatment did not show a notable stimulation of glucagon or GLP-1 secretion from these two cell lines. This is in contrast to its ability to stimulate insulin secretion from the pancreatic INS-1 beta-cell line. Our findings suggest that Epac is selectively involved in peptide hormone secretion in pancreatic and intestinal endocrine cells and that distinct signaling cascades are involved in stimulating production vs. secretion of glucagon and GLP-1 in response to cAMP elevation.
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PMID:Epac is involved in cAMP-stimulated proglucagon expression and hormone production but not hormone secretion in pancreatic alpha- and intestinal L-cell lines. 1885 29

Stimulation of numerous G protein-coupled receptors leads to the elevation of intracellular concentrations of cAMP, which subsequently activates the PKA pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network that directs the spatiotemporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interactions of PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide designated "AKAPis" has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of the AKAPis inhibitor and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting pancreatic beta-cells (INS-1 cells), we showed that TAT-AKAPis (at a micromolar range) dose dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescent analysis also indicated that TAT-AKAPis significantly affected PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated glucagon-induced phosphorylations of p44/p42 MAPKs and cAMP response element binding protein, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic beta-cells. More interestingly, our data highlight the effectiveness of the cell-permeable peptide-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.
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PMID:Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring. 1907 98

Glucagon stimulates the vesicle trafficking of aquaporin-8 (AQP8) water channels to the rat hepatocyte canalicular membranes, a process thought to be relevant to glucagon-induced bile secretion. In this study, we investigated whether glucagon is able to modulate the gene expression of hepatocyte AQP8. Glucagon was administered to rats at 0.2 mg/100 g body wt ip in 2, 3, or 6 equally spaced doses for 8, 16, and 36 h, respectively. Immunoblotting analysis showed that hepatic 34-kDa AQP8 was significantly increased by 79 and 107% at 16 and 36 h, respectively. Hepatic AQP9 protein expression remained unaltered. AQP8 mRNA expression, assessed by real-time PCR, was not modified over time, suggesting a posttranscriptional mechanism of AQP8 protein increase. Glucagon effects on AQP8 were directly studied in primary cultured rat hepatocytes. Immunoblotting and confocal immunofluorescence microscopy confirmed the specific glucagon-induced AQP8 upregulation. The RNA polymerase II inhibitor actinomycin D was unable to prevent glucagon effect, providing additional support to the nontranscriptional upregulation of AQP8. Cycloheximide also showed no effect, suggesting that glucagon-induced AQP8 expression does not depend on protein synthesis but rather on protein degradation. Inhibitory experiments suggest that a reduced calpain-mediated AQP8 proteolysis could be involved. The action of glucagon on hepatocyte AQP8 was mimicked by dibutyryl cAMP and suppressed by PKA or phosphatidylinositol-3-kinase (PI3K) inhibitors. In conclusion, our data suggest that glucagon induces the gene expression of rat hepatocyte AQP8 by reducing its degradation, a process that involves cAMP-PKA and PI3K signal pathways.
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PMID:Glucagon induces the gene expression of aquaporin-8 but not that of aquaporin-9 water channels in the rat hepatocyte. 1919 45


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