Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A highly differentiated rat glucose-responsive insulin producing cell line INS-1 expresses high levels of insulin-like growth factor-II (IGF-II). Basal levels of IGF-II gene mRNA were expressed in cells cultured at 1-6 mmol/l glucose. At glucose concentrations of 10-20 mmol/l, IGF-II mRNA was increased more than threefold after 44 h of incubation. Levels of IGF-II mRNA in INS-1 cells incubated at 5.6 and 20 mmol/l glucose in the presence of 4 micrograms/ml actinomycin D are comparable and are not reduced during 20 h of treatment, indicating the high stability of IGF-II mRNA in this cell line. From the three rat IGF-II promoters, promoter 3 is by far the most active in INS-1 cells. The IGF-II promoter 3 activity and IGF-II mRNA production at high glucose concentrations increased threefold over their respective levels at low glucose concentration, suggesting that the glucose-induced IGF-II gene expression in this beta-cell line might be transcriptionally controlled. The up-regulation of IGF-II mRNA by glucose was not due to the increased intracellular cyclic AMP levels or protein kinase C activation. A protein kinase C activator had no effect on IGF-II gene expression, and an adenylate cyclase activator (forskolin), suppressed the stimulatory effects of glucose on the IGF-II mRNA. Under all the experimental conditions examined, the IGF-II and insulin genes were differentially regulated in INS-1 cells. The IGF-II gene expression and DNA synthesis, however, were regulated in parallel, suggesting that these two cellular activities are closely associated.
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PMID:Insulin-like growth factor-II gene expression in a rat insulin-producing beta-cell line (INS-1) is regulated by glucose. 758 78

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

Despite many triumphs, a significant limitation of the usefulness of many of the available B-cell lines for the study of insulin secretion are either inappropriate or lack of responsiveness to glucose. Commonly employed cell lines generated prior to the 1990s following X-ray irradiation (RINm5F cells) or simian virus 40 B-cell transformation (HIT-T15 cells and BTC) fall into this category. More recent success has been achieved with the generation of INS-1 cells and MIN6 cells, but the production of these cell lines owes much to good fortune, dedication and hard work. In the present era, molecular biology techniques provide the opportunity to engineer novel pancreatic B-cell lines which possess many attributes of normal insulin-secreting cells. This review describes the electrofusion of normal NEDH rat pancreatic B-cells with immortal RINm5F cells to create three new glucose-responsive clonal insulin-secreting cells, designated BRIN-BG5, BRIN-BG7 and BRIN-BD11. These cell lines exhibit up to four-fold insulin-secretory responses to depolarization with 25 mmol/l K+, 7.68 mmol/l Ca2+, 10 mmol/l L-alanine, and activation of protein kinase C or adenylate cyclase with 10 nmol/l phorbol- 12-myristate-13-acetate or 25 micromol/l forskolin, respectively. The maximal insulin-secretory response of both BRIN-BG5 and BRIN-BG7 cells to glucose occurred at 8.4 mmol/l (1.9- and 1.8-fold increases, respectively). In contrast, 4.2-16.7 mmol/l glucose evoked a stepwise 2- to 3-fold of insulin release from BRIN-BD11 cells. The superior glucose responsiveness of BRIN-BD11 cells compared with BRIN-BG5 or BRIN-BG7 cells was associated with increased expression of GLUT-2 and a greater contribution of glucokinase to total glucose phosphorylating enzyme activity. Furthermore, BRIN-BD11 cells also showed appropriate responses to a diverse range of modulators of pancreatic B-cell function, including amino acids, neurotransmitters and sulphonylurea drugs. Collectively these observations indicate that genetic modification of insulin-secreting cells by electrofusion (or transfection with cDNA) offers a new avenue for generation of useful clonal glucose-responsive pancreatic B-cell lines for studies of insulin secretion and transplantation in insulin-dependent diabetes mellitus.
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PMID:Engineering cultured insulin-secreting pancreatic B-cell lines. 993 Sep 71

Glucagon-like peptide 1 (7-36)amide (GLP-1) is an insulinotropic intestinal peptide hormone with a potential role as antidiabetogenic therapeutic agent. It mediates a potentiation of glucose-induced insulin secretion, by activation of adenylate cyclase and subsequent elevation of cytosolic free calcium, [Ca2+]cyt. We investigated the role of protein kinase A (PKA) in GLP-1 signal transduction, using isolated mouse islets as well as the differentiated beta-cell line INS-1. Two specific inhibitors of PKA, (Rp)-adenosine cyclic 3',5'-phosporothioate (Rp-cAMPS, up to 3 mM) and KT5720 (up to 10 microM), did not inhibit the GLP-1-induced [Ca2+]cyt elevation. Another PKA inhibitor, H-89, reduced the [Ca2+]cyt elevation only when applied at high concentrations (10-40 microM), higher than sufficient for PKA inhibition in many cell types. Furthermore, at these concentrations, H-89 also inhibited presumably PKA-independent processes such as glucose-induced [Ca2+]cyt elevations and intracellular calcium storage. This suggests a PKA-independent action of H-89. Similarly to H-89, the potent but unselective protein kinase inhibitor staurosporine inhibited the GLP-1-induced [Ca2+]cyt elevation only at high concentrations, at which it also inhibited glucose-induced [Ca2+]cyt elevations. The same observations as with GLP-1 were made when adenylate cyclase was stimulated with forskolin, for selective examination of signal transduction downstream of receptor and G protein. Our results suggest that the GLP-1-induced [Ca2+]cyt elevation is mediated independently of PKA and thus belongs to the yet-little-characterized ensemble of effects that are mediated by binding of cAMP to other target proteins.
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PMID:Glucagon-like peptide 1 elevates cytosolic calcium in pancreatic beta-cells independently of protein kinase A. 1046 60

IA-2, a member of the protein tyrosine phosphatase family, represents a major target autoantigen in type 1 diabetes. To study the regulation of IA-2 gene expression, we used INS-1 insulinoma cells to analyze beta-cell signal transduction pathways as well as the effect of metabolic and hormonal factors involved in the regulation of the insulin secretory pathway. Quantitative competitive reverse transcriptase-polymerase chain reaction revealed that an increase of cellular cAMP mediated by forskolin (10 micromol/l, 24 h) or 3-isobutyl-1-methylxanthine (100 micromol/l, 24 h) induced maximal stimulation of IA-2 mRNA levels (451 +/- 85 and 338 +/- 86% compared with basal conditions; P < 0.001). In contrast, activation of protein kinase C (PKC) by short-term treatment with phorbol 12-myristate 13-acetate (PMA) (1 micromol/l, 6 h) did not alter IA-2 expression, whereas depletion of PKC by prolonged culturing (24 h) exerted a significant inhibition (57 +/- 24%; P < 0.05). cAMP-dependent upregulation was confirmed by the findings that glucagon (10 micromol/l, 24-48 h) increased levels of IA-2 mRNA (190 +/- 35%; P < 0.05), whereas short-term incubation with high glucose concentration showed no effect. However, prolonged incubation in high glucose (21 mmol/l) induced a time- and dose-dependent increase of IA-2 mRNA expression, reaching maximal values after 144 h (285 +/- 68%; P < 0.05). These studies demonstrate that stimuli of insulin secretion that operate by activation of adenylate cyclase generating cAMP significantly increase IA-2 gene expression. In contrast, activation of PKC by high glucose concentration or PMA exerted no effect, suggesting that IA-2 gene expression is not simply coupled to insulin secretion, but may be involved in the fine regulation of beta-cell function. These findings may be important to clarify the function of IA-2 in beta-cells and elucidate mechanisms involved in the induction of autoimmunity to IA-2.
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PMID:Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-cells. 1090 70

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

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), have been suggested to act as beta-cell growth factors and may therefore be of critical importance for the maintenance of a proper beta-cell mass. We have investigated the molecular mechanism of incretin-induced beta-cell replication in primary monolayer cultures of newborn rat islet cells. GLP-1, GIP and the long-acting GLP-1 derivative, liraglutide, increased beta-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 (approximately 90% increase) and was additive (approximately 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 beta-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 beta-cell replication in humans which would have significant impact on long-term diabetes treatment.
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PMID:Stimulation of pancreatic beta-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways. 1652 28

Although genistein, a soy isoflavone, has beneficial effects on various tissues, it is unclear whether it plays a role in physiological insulin secretion. Here, we present evidence that genistein increases rapid glucose-stimulated insulin secretion (GSIS) in both insulin-secreting cell lines (INS-1 and MIN6) and mouse pancreatic islets. Genistein elicited a significant effect at a concentration as low as 10 nmol/l with a maximal effect at 5 micromol/l. The effect of genistein on GSIS was not dependent on estrogen receptor and also not related to an inhibition of protein tyrosine kinase (PTK). Consistent with its effect on GSIS, genistein increases intracellular cAMP and activates protein kinase A (PKA) in both cell lines and the islets by a mechanism that does not involve estrogen receptor or PTK. The induced cAMP by genistein, at physiological concentrations, may result primarily from enhanced adenylate cyclase activity. Pharmacological or molecular intervention of PKA activation indicated that the insulinotropic effect of genistein is primarily mediated through PKA. These findings demonstrated that genistein directly acts on pancreatic beta-cells, leading to activation of the cAMP/PKA signaling cascade to exert an insulinotropic effect, thereby providing a novel role of soy isoflavones in the regulation of insulin secretion.
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PMID:Genistein acutely stimulates insulin secretion in pancreatic beta-cells through a cAMP-dependent protein kinase pathway. 1656 27

A novel neuroendocrine peptide, pituitary adenylate cyclase activating peptide (PACAP), was found to have an important role in carbohydrate or lipid metabolism and was susceptible to dipeptidyl peptidase IV degradation. It can not only mediate glucose-dependent insulin secretion and lower blood glucose by activating VPAC2 receptor, but also raise blood glucose by promoting glucagon production by VPAC1 receptor activation. Therefore, its therapeutic application is restricted by the exceedingly short-acting half-life and the stimulatory function for glycogenolysis. Herein, we generated novel peptide-conjugated selenium nanoparticles (SeNPs; named as SCD), comprising a 32-amino acid PACAP-derived peptide DBAYL that selectively binds to VPAC2, and chitosan-modified SeNPs (SeNPs-CTS, SC) as slow-release carrier. The circulating half-life of SCD is 14.12 h in mice, which is 168.4-and 7.1-fold longer than wild PACAP (~5 min) and DBAYL (~1.98 h), respectively. SCD (10 nmol/L) significantly promotes INS-1 cell proliferation, glucose uptake, insulin secretion, insulin receptor expression and also obviously reduces intracellular reactive oxygen species levels in H2O2-injured INS-1 cells. Furthermore, the biological effects of SCD are stronger than Exendin-4 (a clinically approved drug through its insulinotropic effect), DBAYL, SeNPs or SC. A single injection of SCD (20 nmol/kg) into db/db mice with type 2 diabetes leads to enhanced insulin secretion and sustained hypoglycemic effect, and the effectiveness and duration of SCD in enhancing insulin secretion and reducing blood glucose levels are much stronger than Exendin-4, SeNPs or SC. In db/db mice, chronic administration of SCD by daily injection for 12 weeks markedly improved glucose and lipid profiles, insulin sensitivity and the structures of pancreatic and adipose tissue. The results indicate that SC can play a role as a carrier for the slow release of bioactive peptides and SCD could be a hopeful therapeutic against type 2 diabetes through the synergy effects of DBAYL and SeNPs.
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PMID:A novel selective VPAC2 agonist peptide-conjugated chitosan modified selenium nanoparticles with enhanced anti-type 2 diabetes synergy effects. 2835 33