Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1976 Apr;256(3):709–729. doi: 10.1113/jphysiol.1976.sp011347

An in Vitro system for studying insulin release caused by secretory granules-plasma membrane interaction: definition of the system.

B Davis, N R Lazarus
PMCID: PMC1309333  PMID: 178856

Abstract

1. An in vitro system is described in which insulin beta-granule-plasma membrane interaction can be studied. 2. The system shows an absolute requirement for physiological amounts of Ca2+ (2 muM) in order for insulin release to proceed. 3. ATP (5 muM) is abot to augment the Ca2+ effect. 4. Glucose (17 mM) alone does not cause insulin release but in the presence of Ca2+ is as effective as ATP. 5. When glucose, ATP and Ca2+ are added together a positive cooperative effect is produced with over 85% of the total insulin, added in the form of beta-granules, being released into the medium in 10 min. 6. The system responds to tolbutamide, in the presence of Ca2+ and ATP, by releasing insulin. 7. Diazoxide, a potent insulin inhibitor in vivo, demonstrates a similar activity in vitro. 8. Various control experiments utilizing alternative membranes, granules, nucleotides, sugars and phosphorylated intermediates of metabolism have all reinforced the specificity of the release mechanisms. 9. These results demonstrate that the in vitro system mimics responses found in the intact organism and can be utilized to dissect the mechanisms associated with exocytosis of insulin granules. 10. Preliminary experiments utilizing adrenaline granules-adrenal plasma membranes and pituitary granules-pituitary plasma membranes suggest that the in vitro system can be extended to all granule secreting processes.

Full text

PDF
709

Selected References

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

  1. An ATP pool with rapid turnover within the cell membrane. Biochem Biophys Res Commun. 1972 Aug 7;48(3):598–604. doi: 10.1016/0006-291x(72)90390-7. [DOI] [PubMed] [Google Scholar]
  2. Ashcroft S. J., Capito K., Hedeskov C. J. Time course studies of glucose-induced changes in glucose-6-phosphate and fructose-1,6-diphosphate content of mouse and rat pancreatic islets. Diabetologia. 1973 Aug;9(4):299–302. doi: 10.1007/BF01221858. [DOI] [PubMed] [Google Scholar]
  3. Atkins T., Matty A. J. [Six years' experience with a musculoplastic technic]. J Endocrinol. 1971 Sep;51(1):67–78. doi: 10.1677/joe.0.0510067. [DOI] [PubMed] [Google Scholar]
  4. Basabe J. C., Lopez N. L., Viktora J. K., Wolff F. W. Insulin secretion studied in the perfused rat pancreas. II. Effect of glucose, glucagon, 3'5' adenosine monophosphate, theophylline, imidazole and phenoxybenzamine; their interaction with diazoxide. Diabetes. 1971 Jul;20(7):457–466. doi: 10.2337/diab.20.7.457. [DOI] [PubMed] [Google Scholar]
  5. Bowen V., Lazaus N. R. Glucose-mediated insulin release: 3',5' cAMP phosphodiesterase. Diabetes. 1973 Oct;22(10):738–743. doi: 10.2337/diab.22.10.738. [DOI] [PubMed] [Google Scholar]
  6. Coore H. G., Hellman B., Pihl E., Täljedal I. B. Physicochemical characteristics of insulin secretion granules. Biochem J. 1969 Jan;111(1):107–113. doi: 10.1042/bj1110107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Curry D. L. Glucagon potentiation of insulin secretion by the perfused rat pancreas. Diabetes. 1970 Jun;19(6):420–428. doi: 10.2337/diab.19.6.420. [DOI] [PubMed] [Google Scholar]
  8. Curry D. L. Is there a common beta cell insulin compartment stimulated by glucose and tolbutamide? Am J Physiol. 1971 Feb;220(2):319–323. doi: 10.1152/ajplegacy.1971.220.2.319. [DOI] [PubMed] [Google Scholar]
  9. DPAVIS B., Lazarus N. R. Regulation of 3',5'-cyclic AMP-dependent protein kinase in the plasma membrane of cod (Gadus callarius) and mouse islets. J Membr Biol. 1975;20(3-4):301–318. doi: 10.1007/BF01870640. [DOI] [PubMed] [Google Scholar]
  10. Davis B., Lazarus N. R. Insulin release from mouse islets. Effect of glucose and hormones on adenylate cyclase. Biochem J. 1972 Sep;129(2):373–379. doi: 10.1042/bj1290373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Exton J. H., Park C. R. Control of gluconeogenesis in liver. II. Effects of glucagon, catecholamines, and adenosine 3',5'-monophosphate on gluconeogenesis in the perfused rat liver. J Biol Chem. 1968 Aug 25;243(16):4189–4196. [PubMed] [Google Scholar]
  12. GRODSKY G. M., BATTS A. A., BENNETT L. L., VCELLA C., MCWILLIAMS N. B., SMITH D. F. EFFECTS OF CARBOHYDRATES ON SECRETION OF INSULIN FROM ISOLATED RAT PANCREAS. Am J Physiol. 1963 Oct;205:638–644. doi: 10.1152/ajplegacy.1963.205.4.638. [DOI] [PubMed] [Google Scholar]
  13. Grant P. T., Reid K. B. Biosynthesis of an insulin precursor by islet tissue of cod (Gadus callarias). Biochem J. 1968 Nov;110(2):281–288. doi: 10.1042/bj1100281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grodsky G. M. A threshold distribution hypothesis for packet storage of insulin and its mathematical modeling. J Clin Invest. 1972 Aug;51(8):2047–2059. doi: 10.1172/JCI107011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grodsky G. M., Bennett L. L. Cation requirements for insulin secretion in the isolated perfused pancreas. Diabetes. 1966 Dec;15(12):910–913. doi: 10.2337/diab.15.12.910. [DOI] [PubMed] [Google Scholar]
  16. Hellman B., Idahl L. A., Lernmark A., Sehlin J., Täljedal I. B. The pancreatic beta-cell recognition of insulin secretagogues. Comparisons of glucose with glyceraldehyde isomers and dihydroxyacetone. Arch Biochem Biophys. 1974 Jun;162(2):448–457. doi: 10.1016/0003-9861(74)90204-5. [DOI] [PubMed] [Google Scholar]
  17. Hellman B., Idahl L. A., Lernmark A., Täljedal I. B. The pancreatic beta-cell recognition of insulin secretagogues: does cyclic AMP mediate the effect of glucose? Proc Natl Acad Sci U S A. 1974 Sep;71(9):3405–3409. doi: 10.1073/pnas.71.9.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kiehn E. D., Holland J. J. Membrane and nonmembrane proteins of mammalian cells. Synthesis, turnover, and size distribution. Biochemistry. 1970 Apr 14;9(8):1716–1728. doi: 10.1021/bi00810a010. [DOI] [PubMed] [Google Scholar]
  19. Landgraf R., Kotler-Brajtburg J., Matschinsky F. M. Kinetics of insulin release from the perfused rat pancreas caused by glucose, glucosamine, and galactose. Proc Natl Acad Sci U S A. 1971 Mar;68(3):536–540. doi: 10.1073/pnas.68.3.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lazarus N. R., Davis B. Model for extrusion of insulin beta granules. Lancet. 1975 Jan 18;1(7899):143–144. doi: 10.1016/s0140-6736(75)91434-8. [DOI] [PubMed] [Google Scholar]
  21. Lenzen S. The immediate insulin secretory response of the isolated perfused rat pancreas to tolbutamide and glucose. FEBS Lett. 1975 Jan 1;49(3):407–408. doi: 10.1016/0014-5793(75)80797-6. [DOI] [PubMed] [Google Scholar]
  22. Lindl T., Heinl-Sawaya M. C., Cramer H. Compartmentation of an ATP substrate pool for histamine and adrenaline sensitive adenylate cyclase in rat superior cervical ganglia. Biochem Pharmacol. 1975 May 1;24(9):947–950. doi: 10.1016/0006-2952(75)90424-4. [DOI] [PubMed] [Google Scholar]
  23. MONOD J., WYMAN J., CHANGEUX J. P. ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. J Mol Biol. 1965 May;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
  24. Malaisse W. J., Malaisse-Lagae F., Walker M. O., Lacy P. E. The stimulus-secretion coupling of glucose-induced insulin release. V. The participation of a microtubular-microfilamentous system. Diabetes. 1971 May;20(5):257–265. doi: 10.2337/diab.20.5.257. [DOI] [PubMed] [Google Scholar]
  25. Milner R. D., Hales C. N. The role of calcium and magnesium in insulin secretion from rabbit pancreas studied in vitro. Diabetologia. 1967 Mar;3(1):47–49. doi: 10.1007/BF01269910. [DOI] [PubMed] [Google Scholar]
  26. Ray T. K. A modified method for the isolation of the plasma membrane from rat liver. Biochim Biophys Acta. 1970 Jan 6;196(1):1–9. doi: 10.1016/0005-2736(70)90159-8. [DOI] [PubMed] [Google Scholar]
  27. Smith A. D., Winkler H. A simple method for the isolation of adrenal chromaffin granules on a large scale. Biochem J. 1967 May;103(2):480–482. doi: 10.1042/bj1030480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zawalich W. S., Karl R. C., Ferrendelli J. A., Matschinsky F. M. Factors governing glucose induced elevation of cyclic 3'5' AMP levels in pancreatic islets. Diabetologia. 1975 Jun;11(3):231–235. doi: 10.1007/BF00422327. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES