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

The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-cell line INS-1, major secretagogue-stimulated signaling pathways converge to activate 44-kDa mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin secretion was found to be associated with a small stimulatory effect on 44-kDa MAP kinase, which was synergistically enhanced by increased levels of intracellular cAMP and by the hormonal secretagogues glucagon-like peptide-1 and pituitary adenylate cyclase-activating polypeptide. Activation of 44-kDa MAP kinase by glucose was dependent on Ca2+ influx and may in part be mediated by MEK-1, a MAP kinase kinase. Stimulation of Ca2+ influx by KCl was in itself sufficient to activate 44-kDa MAP kinase and MEK-1. Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP kinase by both Ca(2+)-dependent and -independent pathways. Nerve growth factor, independently of changes in cytosolic Ca2+, efficiently stimulated 44-kDa MAP kinase without causing insulin release, indicating that activation of this kinase is not sufficient for secretion. In the presence of glucose, however, nerve growth factor potentiated insulin secretion. In INS-1 cells, activation of 44-kDa MAP kinase was partially correlated with the induction of early response genes junB, nur77, and zif268 but not with stimulation of DNA synthesis. Our findings suggest a role of 44-kDa MAP kinase in mediating some of the pleiotropic actions of secretagogues on the pancreatic beta-cell.
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PMID:Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1. 771 82

Hepatocyte-nuclear factor 3 beta (HNF-3 beta), a member of the HNF-3 gene family, is expressed in glucagon-producing islet cells and represses glucagon gene expression. We show here that at least three different HNF-3 beta transcripts that encode HNF-3 beta protein variants are present in glucagon-producing cells, HNF-3 beta 1, HNF-3 beta 2, and HNF-3 beta 3. Compared with the HNF-3 beta 1 cDNA, HNF-3 beta 2 cDNA lacks sequences of exon 1 while exons 1 and 4 are absent from the HNF-3 beta 3 cDNA. The deduced amino-acid (aa) sequence of HNF-3 beta 2 and HNF-3 beta 3 proteins differs from HNF-3 beta 1 by a 6-aa amino-terminal extension and by the absence of the first 30 aa, respectively. HNF-3 beta 1, HNF-3 beta 2, and HNF-3 beta 3 bind to the major enhancer of the rat glucagon gene G2 with similar affinity. By contrast to HNF-3 beta 1, which represses glucagon gene expression when overexpressed in the glucagon-producing cell line InR1G9, HNF-3 beta 2 and HNF-3 beta 3 do not affect transcriptional activity. Furthermore, cotransfection of HNF-3 beta 2 or HNF-3 beta 3 along with HNF-3 beta 1 decreases the negative effects of HNF-3 beta 1. We conclude that glucagon gene expression may be regulated by the relative abundance of the three different HNF-3 beta variants in alpha-cells.
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PMID:Hepatocyte-nuclear factor 3 beta gene transcripts generate protein isoforms with different transactivation properties on the glucagon gene. 777 82

Pancreatic expression of the glucagon gene depends on multiple transcription factors interacting with at least three DNA control elements: G1, the upstream promoter element, and G2 and G3, two enhancer-like sequences. We report here that the major enhancer of the rat glucagon gene, G2, interacts with three protein complexes, A1, A2, and A3. A2 is detected only in islet cells, and impairment of its binding to mutant G2 causes a marked decrease in transcriptional activity. We identify A1 as hepatocyte nuclear factor 3 beta (HNF-3 beta), a member of the HNF-3 DNA-binding protein family found in abundance in the liver which has been proposed to play a role in the formation of gut-related organs. HNF-3 beta binds G2 on a site which overlaps A2 and acts as a repressor of glucagon gene expression, as demonstrated by mutational analyses of G2 and by cotransfection of HNF-3 beta cDNA along with reporter genes containing G2 into glucagon-producing cells. Our data implicate HNF-3 beta in the control of glucagon gene expression and strengthen the idea of endodermal origin of the islet cells.
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PMID:Glucagon gene expression is negatively regulated by hepatocyte nuclear factor 3 beta. 816 96

Neuropeptide-Y (NPY) is a 36-amino acid peptide known to inhibit glucose-stimulated insulin secretion in various animal models in vitro and in vivo. NPY is thought to be one of the mediators of sympathetic action in the pancreas through nerve endings surrounding the islets, and it has recently been shown to be synthesized within the islets of Langerhans. To elucidate the potential role of NPY in the endocrine pancreas, we studied the expression and regulation of NPY secretion in a rat insulinoma cell line (INS-1). NPY mRNA and peptide are highly expressed and secreted by INS-1 cells. NPY levels were determined by a sensitive and specific two-site amplified enzyme-linked immunosorbent assay. Incubation of INS-1 cells with various glucose concentrations did not modify NPY secretion; however, stimulation of adenylate cyclase by forskolin induced a dose- and time-dependent increase in NPY release in the medium. The glucagon-like peptide-I-(7-36) amide (GLP-1), a known gluco-incretin in humans, induced at low concentration (10(-9) M) a similar expression of NPY mRNA and peptide secretion in INS-1 cells. On the other hand, the inhibition of cAMP accumulation by the alpha 2-adrenergic agonist clonidine decreased NPY secretion. In conclusion, 1) high levels of gene expression and secretion of NPY are found in a rat insulinoma cell line (INS-1). 2) Accumulation of cAMP induced by forskolin or a gluco-incretin (GLP-1) induces a further increase in NPY gene expression and release. 3) NPY secretion is not modulated by low or high glucose concentrations in the medium. 4) Induction of NPY, a known inhibitor of insulin secretion, may represent a novel counterregulatory mechanism of insulin secretion, limiting the stimulatory effect of GLP-1 on insulin secretion.
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PMID:Neuropeptide Y expression and regulation in a differentiated rat insulin-secreting cell line. 839 8

The use of primary beta-cells in biochemical and molecular research is limited by the availability of pancreatic endocrine tissue. Numerous investigators have attempted to establish an insulin-secreting cell line that retains normal regulation of insulin secretion. Different approaches have been used, including induction of pancreatic tumors by irradiation or viral infection, immortalization of beta-cells in vitro, and development of transgenic mice with targeted expression of a recombinant oncogene in the beta-cell. Few of these attempts have proven successful, because cell differentiation and proliferation capacities are mutually exclusive. The most widely used insulin-secreting cell lines are RIN, HIT, beta TC, MIN6 and INS-1 cells. These cells contain mainly insulin and small amounts of glucagon and somatostatin. RIN cells, except for the subclone RIN-38, are not glucose-responsive. HIT cells and beta TC cells secrete insulin in response to glucose, but their dose-response curve is markedly shifted to the left MIN6, INS-1 and a newly available subclone of beta TC cells (beta TC-6 F7) are reported to retain normal regulation of glucose-induced insulin secretion. Although the behaviour of none of these cell lines perfectly mimics primary beta-cell physiology, they are extremely valuable tools for the study of molecular events underlying beta-cell function and dysfunction. In addition, insulin-secreting cell lines represent a potential source of transplantable tissue to overcome the limited availability of primary islets for this procedure.
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PMID:Insulin-secreting cell lines: classification, characteristics and potential applications. 869 99

Glucagon is insulinotropic, but it remains uncertain whether the insulinotropic action is mediated directly by glucagon receptors expressed on beta-cells or by cross-binding to the insulinotropic glucagon-like peptide-1 (GLP-1) receptor known to be expressed on beta-cells. Binding of [125I]glucagon to GLP-1 receptors and not to glucagon receptors has been reported in tumor-derived beta-cells (15). The objectives of the current study were to use receptor-binding techniques and a glucagon receptor-specific antiserum to determine whether glucagon receptors are present on beta-cells. Specific binding (7.2 +/- 0.8%) of [125I]GLP-1 to beta TC-3 cells was displaced equivalently with GLP-1 and exendin-(9-39) )Kd = 0.9 and 0.4 nM. respectively), whereas approximately 700-fold higher concentrations of glucagon were required for equal displacement (Kd = 400 nM). Binding of [125I]glucagon to beta TC-3 cells (approximately 1%) was displaced equivalently with 1 microM glucagon, GLP-1, or exendin-(9-39). These observations support earlier findings that beta TC-3 cells do not express functional glucagon receptors. However, specific binding of [125I]glucagon was observed on INS-1 cells (2.3 +/- 0.2%); this was displaced with glucagon (Kd = 1 nM), but not 1 microM GLP-1 or exendin-(9-39). To examine the distribution of glucagon receptors on native beta-cells, dispersed cultured rat islets were immunostained for glucagon, somatostatin, or insulin in combination with a polyclonal rabbit antiserum raised to an extracellular portion of the glucagon receptor (KD-14). The glucagon receptor antiserum colocalized staining with approximately 97% of immunoreactive insulin cells, 9% of immunoreactive glucagon cells, and 11% of immunoreactive somatostatin cells. Perfusion of the rat pancreas with concentrations of glucagon as low as 10(-12) M resulted in significant insulin release. These results suggest that whereas the tumor-derived beta-cell line beta TC-3 does not express functional glucagon receptors, INS-1 cells and isolated rat pancreatic beta-cells have specific glucagon receptors, as do a subpopulation of alpha- and delta-cells. A model is proposed for the role of glucagon in islet hormone secretion during feeding and fasting.
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PMID:Distribution of glucagon receptors on hormone-specific endocrine cells of rat pancreatic islets. 889 86

Pancreatic beta cells (insulin-producing cells) and neuronal cells share a large number of similarities. Here, we investigate whether the same mechanisms could control the expression of neuronal genes in both neurons and insulin-producing cells. For that purpose, we tested the role of the transcriptional repressor neuron-restrictive silencing factor/repressor element silencing transciption factor (NRSF/REST) in the expression of a battery of neuronal genes in insulin-producing cells. NRSF/REST is a negative regulator of the neuronal fate. It is known to silence neuronal-specific genes in non-neuronal cells. We demonstrate that, as in the case of the neuronal pheochromocytoma cell line PC12, mRNA coding for NRSF/REST is absent from the insulinoma cell line INS-1 and from three other insulin- and glucagon-producing cell lines. NRSF/REST activity is also absent from insulin-producing cell lines. Transient expression of REST in insulin-producing cell lines is sufficient to silence a reporter gene containing a NRSF/REST binding site, demonstrating the role of NRSF/REST in the expression of neuronal markers in insulin-producing cells. Finally, by searching for the expression of NRSF/REST-regulated genes in insulin-producing cells, we increased the list of the genes expressed in both neurons and insulin-producing cells.
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PMID:Expression of neuronal traits in pancreatic beta cells. Implication of neuron-restrictive silencing factor/repressor element silencing transcription factor, a neuron-restrictive silencer. 899 82

The peptide hormone glucagon is expressed in A cells of the pancreatic islets due to an interaction between multiple regulatory elements within the 5'-flanking region of its gene directing glucagon gene transcription. An A-cell-specific enhancer-like element in the rat glucagon gene, G3, contains two domains, both of which are necessary for G3 activity. Domain A of the G3 element comprises a sequence motif, PISCES, that is also found in control elements of the rat insulin I and somatostatin genes exhibiting cell-specific transcriptional activities distinct from G3. In this study, the nuclear proteins binding to domain B of G3 were characterized. In electrophoretic mobility shift assays using nuclear extracts from a glucagon-producing islet cell line, it was observed that the binding specificity of G3-domain-B-binding proteins is related to that of winged helix proteins supporting the hypothesis that the proteins binding to domain B of G3 may belong to the winged helix protein family of transcription factors. The overexpression of a dominant-negative winged helix protein mutant (derived from HNF-3) virtually abolished the transcriptional activity of G3 in a glucagon-expressing islet cell line. These results suggest that the unique A-cell-specific basal transcriptional activity of the glucagon G3 element depends on a combination of at least two proteins, the islet specific PISCES-binding protein and a more widely expressed winged helix protein.
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PMID:Glucagon gene G3 enhancer: evidence that activity depends on combination of an islet-specific factor and a winged helix protein. 908 37

Previous studies in rat islets have suggested that anaplerosis plays an important role in the regulation of pancreatic beta cell function and growth. However, the relative contribution of islet beta cells versus non-beta cells to glucose-regulated anaplerosis is not known. Furthermore, the fate of glucose carbon entering the Krebs cycle of islet cells remains to be determined. The present study has examined the anaplerosis of glucose carbon in purified rat beta cells using specific 14C-labeled glucose tracers. Between 5 and 20 mM glucose, the oxidative production of CO2 from [3,4-14C]glucose represented close to 100% of the total glucose utilization by the cells. Anaplerosis, quantified as the difference between 14CO2 production from [3,4-14C]glucose and [6-14C]glucose, was strongly influenced by glucose, particularly between 5 and 10 mM. The dose dependence of glucose-induced insulin secretion correlated with the accumulation of citrate and malate in beta(INS-1) cells. All glucose carbon that was not oxidized to CO2 was recovered from the cells after extraction in trichloroacetic acid. This indirectly indicates that lactate output is minimal in beta cells. From the effect of cycloheximide upon the incorporation of 14C-glucose into the acid-precipitable fraction, it could be calculated that 25% of glucose carbon entering the Krebs cycle via anaplerosis is channeled into protein synthesis. In contrast, non-beta cells (approximately 80% glucagon-producing alpha cells) exhibited rates of glucose oxidation that were (1)/(3) to (1)/(6) those of the total glucose utilization and no detectable anaplerosis from glucose carbon. This difference between the two cell types was associated with a 7-fold higher expression of the anaplerotic enzyme pyruvate carboxylase in beta cells, as well as a 4-fold lower ratio of lactate dehydrogenase to FAD-linked glycerol phosphate dehydrogenase in beta cells versus alpha cells. Finally, glucose caused a dose-dependent suppression of the activity of the pentose phosphate pathway in beta cells. In conclusion, rat beta cells metabolize glucose essentially via aerobic glycolysis, whereas glycolysis in alpha cells is largely anaerobic. The results support the view that anaplerosis is an essential pathway implicated in beta cell activation by glucose.
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PMID:Metabolic fate of glucose in purified islet cells. Glucose-regulated anaplerosis in beta cells. 922 23

To study the regulation of growth and differentiated function of insulin-secreting cells, the rat insulinoma cell line INS-1 was cultured in a defined serum-free medium containing prolactin, IGF-I, and triiodothyronine, which was originally reported to maintain insulin secretion of islet cells. Growth and viability, as well as cellular insulin content of INS-1 cells in the defined medium, were comparable to the control cells cultured in the complete medium containing 10% fetal calf serum. However, after a 3-day culture in this medium, insulin secretion in response to glucose, pyruvate, and leucine was markedly blunted compared with the control cells (-78, -68, and -56%, respectively), whereas the response to 30 mmol/l K+ was only slightly decreased. In these cells: 1) nutrient metabolism assessed by tetrazolium salt reduction was reduced in response to pyruvate and leucine, which are mainly metabolized in the mitochondria; 2) oxidation of both [3,4-(14)C]glucose and [1-(14)C]pyruvate was decreased (-22 and -32%, respectively); 3) glucose failed to depolarize the membrane potential, whereas tolbutamide was fully active; 4) video imaging analysis of cytosolic Ca2+ showed a decrease in the population of glucose-responsive cells, while the response to 30 mmol/l K+ was preserved; 5) serum replenishment for 3 days restored glucose-induced insulin secretion. Interestingly, conditioned serum-free medium from rat islets maintained the insulin secretory function of INS-1 cells, although glucagon, somatostatin, and some other factors failed to restore the function. In contrast, conditioned media from HepG2, PC12, and human umbilical vein endothelial cells did not substitute for serum. Thus, the impaired insulin secretion of the cells cultured in the defined medium is best explained by defective mitochondrial metabolism. Islet cells, but not INS-1 cells, produce factors required for normal signal generation by nutrient secretagogues.
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PMID:Glucose-induced insulin secretion in INS-1 cells depends on factors present in fetal calf serum and rat islet-conditioned medium. 928 42


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