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. 2002 Dec 1;368(Pt 2):397–404. doi: 10.1042/BJ20020999

Protein kinase A translocation and insulin secretion in pancreatic beta-cells: studies with adenylate cyclase toxin from Bordetella pertussis.

Zhiyong Gao 1, Robert A Young 1, Matteo M Trucco 1, Scott R Greene 1, Erik L Hewlett 1, Franz M Matschinsky 1, Bryan A Wolf 1
PMCID: PMC1223000  PMID: 12180908

Abstract

Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in the beta-cell. Adenylate cyclase toxin (ACT), a bacterial exotoxin with adenylate cyclase activity, and forskolin, an activator of adenylate cyclase, both dose-dependently increased insulin secretion in the presence, but not the absence, of glucose in insulin-secreting betaTC3 cells. The stimulation of cAMP release by either agent was dose-dependent but glucose-independent. Omission of extracellular Ca(2+) totally abolished the effects of ACT on insulin secretion and cytosolic cAMP accumulation. ACT and forskolin caused rapid and dramatic increases in cytosolic Ca(2+), which were blocked by nifedipine and the omission of extracellular Ca(2+). Omission of glucose completely blocked the effects of forskolin and partially blocked the effects of ACT on cytosolic Ca(2+). PKA alpha, beta and gamma catalytic subunits (Calpha, Cbeta and Cgamma respectively) were identified in betaTC6 cells by confocal microscopy. Glucose and glucagon-like polypeptide-1 (GLP-1) caused translocation of Calpha to the nucleus and of Cbeta to the plasma membrane and the nucleus, but did not affect the distribution of Cgamma. In conclusion, glucose and GLP-1 amplify insulin secretion via cAMP production and PKAbeta activation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Comb M., Birnberg N. C., Seasholtz A., Herbert E., Goodman H. M. A cyclic AMP- and phorbol ester-inducible DNA element. 1986 Sep 25-Oct 1Nature. 323(6086):353–356. doi: 10.1038/323353a0. [DOI] [PubMed] [Google Scholar]
  2. Daniel P. B., Walker W. H., Habener J. F. Cyclic AMP signaling and gene regulation. Annu Rev Nutr. 1998;18:353–383. doi: 10.1146/annurev.nutr.18.1.353. [DOI] [PubMed] [Google Scholar]
  3. Diedrich T., Knepel W. Interaction of the transcription factor CREB with pancreatic islet cell-specific enhancer elements. Biol Chem Hoppe Seyler. 1995 Jan;376(1):39–44. doi: 10.1515/bchm3.1995.376.1.39. [DOI] [PubMed] [Google Scholar]
  4. Eckert B., Schwaninger M., Knepel W. Calcium-mobilizing insulin secretagogues stimulate transcription that is directed by the cyclic adenosine 3',5'-monophosphate/calcium response element in a pancreatic islet beta-cell line. Endocrinology. 1996 Jan;137(1):225–233. doi: 10.1210/endo.137.1.8536617. [DOI] [PubMed] [Google Scholar]
  5. Edwards A. S., Scott J. D. A-kinase anchoring proteins: protein kinase A and beyond. Curr Opin Cell Biol. 2000 Apr;12(2):217–221. doi: 10.1016/s0955-0674(99)00085-x. [DOI] [PubMed] [Google Scholar]
  6. Efrat S., Surana M., Fleischer N. Glucose induces insulin gene transcription in a murine pancreatic beta-cell line. J Biol Chem. 1991 Jun 15;266(17):11141–11143. [PubMed] [Google Scholar]
  7. Furukawa N., Shirotani T., Araki E., Kaneko K., Todaka M., Matsumoto K., Tsuruzoe K., Motoshima H., Yoshizato K., Kishikawa H. Possible involvement of atypical protein kinase C (PKC) in glucose-sensitive expression of the human insulin gene: DNA-binding activity and transcriptional activity of pancreatic and duodenal homeobox gene-1 (PDX-1) are enhanced via calphostin C-sensitive but phorbol 12-myristate 13-acetate (PMA) and Gö 6976-insensitive pathway. Endocr J. 1999 Feb;46(1):43–58. doi: 10.1507/endocrj.46.43. [DOI] [PubMed] [Google Scholar]
  8. Gamm D. M., Baude E. J., Uhler M. D. The major catalytic subunit isoforms of cAMP-dependent protein kinase have distinct biochemical properties in vitro and in vivo. J Biol Chem. 1996 Jun 28;271(26):15736–15742. doi: 10.1074/jbc.271.26.15736. [DOI] [PubMed] [Google Scholar]
  9. Gao Z. Y., Li G., Najafi H., Wolf B. A., Matschinsky F. M. Glucose regulation of glutaminolysis and its role in insulin secretion. Diabetes. 1999 Aug;48(8):1535–1542. doi: 10.2337/diabetes.48.8.1535. [DOI] [PubMed] [Google Scholar]
  10. Gao Z., Konrad R. J., Collins H., Matschinsky F. M., Rothenberg P. L., Wolf B. A. Wortmannin inhibits insulin secretion in pancreatic islets and beta-TC3 cells independent of its inhibition of phosphatidylinositol 3-kinase. Diabetes. 1996 Jul;45(7):854–862. doi: 10.2337/diab.45.7.854. [DOI] [PubMed] [Google Scholar]
  11. Gao Z., Reavey-Cantwell J., Young R. A., Jegier P., Wolf B. A. Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet beta -cells. J Biol Chem. 2000 Nov 17;275(46):36079–36085. doi: 10.1074/jbc.M004284200. [DOI] [PubMed] [Google Scholar]
  12. German M. S., Wang J. The insulin gene contains multiple transcriptional elements that respond to glucose. Mol Cell Biol. 1994 Jun;14(6):4067–4075. doi: 10.1128/mcb.14.6.4067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goodison S., Kenna S., Ashcroft S. J. Control of insulin gene expression by glucose. Biochem J. 1992 Jul 15;285(Pt 2):563–568. doi: 10.1042/bj2850563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gray M., Szabo G., Otero A. S., Gray L., Hewlett E. Distinct mechanisms for K+ efflux, intoxication, and hemolysis by Bordetella pertussis AC toxin. J Biol Chem. 1998 Jul 17;273(29):18260–18267. doi: 10.1074/jbc.273.29.18260. [DOI] [PubMed] [Google Scholar]
  15. Gromada J., Holst J. J., Rorsman P. Cellular regulation of islet hormone secretion by the incretin hormone glucagon-like peptide 1. Pflugers Arch. 1998 Apr;435(5):583–594. doi: 10.1007/s004240050558. [DOI] [PubMed] [Google Scholar]
  16. Hackett M., Guo L., Shabanowitz J., Hunt D. F., Hewlett E. L. Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science. 1994 Oct 21;266(5184):433–435. doi: 10.1126/science.7939682. [DOI] [PubMed] [Google Scholar]
  17. Hansson V., Skålhegg B. S., Taskén K. Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA. J Steroid Biochem Mol Biol. 1999 Apr-Jun;69(1-6):367–378. doi: 10.1016/s0960-0760(99)00077-1. [DOI] [PubMed] [Google Scholar]
  18. Hansson V., Skålhegg B. S., Taskén K. Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA. J Steroid Biochem Mol Biol. 2000 May;73(1-2):81–92. [PubMed] [Google Scholar]
  19. Henquin J. C., Jonas J. C., Gilon P. Functional significance of Ca2+ oscillations in pancreatic beta cells. Diabetes Metab. 1998 Feb;24(1):30–36. [PubMed] [Google Scholar]
  20. Hewlett E. L., Urban M. A., Manclark C. R., Wolff J. Extracytoplasmic adenylate cyclase of Bordetella pertussis. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1926–1930. doi: 10.1073/pnas.73.6.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Holz G. G., 4th, Kühtreiber W. M., Habener J. F. Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7-37). Nature. 1993 Jan 28;361(6410):362–365. doi: 10.1038/361362a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Holz G. G., 4th, Leech C. A., Habener J. F. Activation of a cAMP-regulated Ca(2+)-signaling pathway in pancreatic beta-cells by the insulinotropic hormone glucagon-like peptide-1. J Biol Chem. 1995 Jul 28;270(30):17749–17757. [PMC free article] [PubMed] [Google Scholar]
  23. Holz G. G., Habener J. F. Signal transduction crosstalk in the endocrine system: pancreatic beta-cells and the glucose competence concept. Trends Biochem Sci. 1992 Oct;17(10):388–393. doi: 10.1016/0968-0004(92)90006-u. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Inagaki N., Gonoi T., Clement J. P., 4th, Namba N., Inazawa J., Gonzalez G., Aguilar-Bryan L., Seino S., Bryan J. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science. 1995 Nov 17;270(5239):1166–1170. doi: 10.1126/science.270.5239.1166. [DOI] [PubMed] [Google Scholar]
  25. Inagaki N., Gonoi T., Clement J. P., Wang C. Z., Aguilar-Bryan L., Bryan J., Seino S. A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron. 1996 May;16(5):1011–1017. doi: 10.1016/s0896-6273(00)80124-5. [DOI] [PubMed] [Google Scholar]
  26. Jones P. M., Persaud S. J. Protein kinases, protein phosphorylation, and the regulation of insulin secretion from pancreatic beta-cells. Endocr Rev. 1998 Aug;19(4):429–461. doi: 10.1210/edrv.19.4.0339. [DOI] [PubMed] [Google Scholar]
  27. Kennedy H. J., Rafiq I., Pouli A. E., Rutter G. A. Glucose enhances insulin promoter activity in MIN6 beta-cells independently of changes in intracellular Ca2+ concentration and insulin secretion. Biochem J. 1999 Sep 1;342(Pt 2):275–280. [PMC free article] [PubMed] [Google Scholar]
  28. Konrad R. J., Young R. A., Record R. D., Smith R. M., Butkerait P., Manning D., Jarett L., Wolf B. A. The heterotrimeric G-protein Gi is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis. J Biol Chem. 1995 May 26;270(21):12869–12876. doi: 10.1074/jbc.270.21.12869. [DOI] [PubMed] [Google Scholar]
  29. Leibiger I. B., Leibiger B., Moede T., Berggren P. O. Exocytosis of insulin promotes insulin gene transcription via the insulin receptor/PI-3 kinase/p70 s6 kinase and CaM kinase pathways. Mol Cell. 1998 May;1(6):933–938. doi: 10.1016/s1097-2765(00)80093-3. [DOI] [PubMed] [Google Scholar]
  30. Leiser M., Fleischer N. cAMP-dependent phosphorylation of the cardiac-type alpha 1 subunit of the voltage-dependent Ca2+ channel in a murine pancreatic beta-cell line. Diabetes. 1996 Oct;45(10):1412–1418. doi: 10.2337/diab.45.10.1412. [DOI] [PubMed] [Google Scholar]
  31. Lin Y. F., Jan Y. N., Jan L. Y. Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells. EMBO J. 2000 Mar 1;19(5):942–955. doi: 10.1093/emboj/19.5.942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MacDougald O. A., Lane M. D. Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem. 1995;64:345–373. doi: 10.1146/annurev.bi.64.070195.002021. [DOI] [PubMed] [Google Scholar]
  33. Malaisse W. J., Garcia-Morales P., Dufrane S. P., Sener A., Valverde I. Forskolin-induced activation of adenylate cyclase, cyclic adenosine monophosphate production and insulin release in rat pancreatic islets. Endocrinology. 1984 Nov;115(5):2015–2020. doi: 10.1210/endo-115-5-2015. [DOI] [PubMed] [Google Scholar]
  34. Mojsov S., Weir G. C., Habener J. F. Insulinotropin: glucagon-like peptide I (7-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J Clin Invest. 1987 Feb;79(2):616–619. doi: 10.1172/JCI112855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ohneda K., Ee H., German M. Regulation of insulin gene transcription. Semin Cell Dev Biol. 2000 Aug;11(4):227–233. doi: 10.1006/scdb.2000.0171. [DOI] [PubMed] [Google Scholar]
  36. Philippe J., Missotten M. Functional characterization of a cAMP-responsive element of the rat insulin I gene. J Biol Chem. 1990 Jan 25;265(3):1465–1469. [PubMed] [Google Scholar]
  37. Rothenberg P. L., Willison L. D., Simon J., Wolf B. A. Glucose-induced insulin receptor tyrosine phosphorylation in insulin-secreting beta-cells. Diabetes. 1995 Jul;44(7):802–809. doi: 10.2337/diab.44.7.802. [DOI] [PubMed] [Google Scholar]
  38. Ruppert S., Cole T. J., Boshart M., Schmid E., Schütz G. Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes. EMBO J. 1992 Apr;11(4):1503–1512. doi: 10.1002/j.1460-2075.1992.tb05195.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schuit F. C., Pipeleers D. G. Regulation of adenosine 3',5'-monophosphate levels in the pancreatic B cell. Endocrinology. 1985 Sep;117(3):834–840. doi: 10.1210/endo-117-3-834. [DOI] [PubMed] [Google Scholar]
  40. Sebo P., Glaser P., Sakamoto H., Ullmann A. High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system. Gene. 1991 Jul 31;104(1):19–24. doi: 10.1016/0378-1119(91)90459-o. [DOI] [PubMed] [Google Scholar]
  41. Skalhegg B. S., Tasken K. Specificity in the cAMP/PKA signaling pathway. Differential expression,regulation, and subcellular localization of subunits of PKA. Front Biosci. 2000 Aug 1;5:D678–D693. doi: 10.2741/skalhegg. [DOI] [PubMed] [Google Scholar]
  42. Skoglund G., Hussain M. A., Holz G. G. Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element. Diabetes. 2000 Jul;49(7):1156–1164. doi: 10.2337/diabetes.49.7.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sugden M. C., Ashcroft S. J., Sugden P. H. Protein kinase activities in rat pancreatic islets of Langerhans. Biochem J. 1979 Apr 15;180(1):219–229. doi: 10.1042/bj1800219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Szabo G., Gray M. C., Hewlett E. L. Adenylate cyclase toxin from Bordetella pertussis produces ion conductance across artificial lipid bilayers in a calcium- and polarity-dependent manner. J Biol Chem. 1994 Sep 9;269(36):22496–22499. [PubMed] [Google Scholar]
  45. Taylor S. S., Buechler J. A., Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem. 1990;59:971–1005. doi: 10.1146/annurev.bi.59.070190.004543. [DOI] [PubMed] [Google Scholar]
  46. Xu G. G., Gao Z. Y., Borge P. D., Jr, Wolf B. A. Insulin receptor substrate 1-induced inhibition of endoplasmic reticulum Ca2+ uptake in beta-cells. Autocrine regulation of intracellular ca2+ homeostasis and insulin secretion. J Biol Chem. 1999 Jun 18;274(25):18067–18074. doi: 10.1074/jbc.274.25.18067. [DOI] [PubMed] [Google Scholar]
  47. Yoshida A., Takahashi M., Nishimura S., Takeshima H., Kokubun S. Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca channel. FEBS Lett. 1992 Sep 14;309(3):343–349. doi: 10.1016/0014-5793(92)80804-p. [DOI] [PubMed] [Google Scholar]
  48. de Vargas L. M., Sobolewski J., Siegel R., Moss L. G. Individual beta cells within the intact islet differentially respond to glucose. J Biol Chem. 1997 Oct 17;272(42):26573–26577. doi: 10.1074/jbc.272.42.26573. [DOI] [PubMed] [Google Scholar]

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