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. 1971 Sep 1;50(3):565–582. doi: 10.1083/jcb.50.3.565

MORPHOLOGICAL AND BIOCHEMICAL STUDIES OF B CELLS OF FETAL RAT ENDOCRINE PANCREAS IN ORGAN CULTURE

Evidence for (Pro)Insulin Biosynthesis

Lelio Orci 1, Andre E Lambert 1, Yasunori Kanazawa 1, Mylene Amherdt 1, Charles Rouiller 1, Albert E Renold 1
PMCID: PMC2108314  PMID: 5098861

Abstract

Fetal rat pancreases explanted on the 18th day of gestation and maintained in organ culture for 1–10 days were utilized for this series of studies. Ultrastructurally, at the time of explantation, the majority of fetal B cells was sparsely granulated and characterized by numerous free ribosomes and undeveloped rough endoplasmic reticulum (RER) and Golgi complexes. During the culture period, extensive development of the RER and Golgi complexes preceded an increasing accumulation of β-granules. This later increase in the number of β-granules and in the concentration of immunoreactive insulin was paralleled by a reduction of RER and Golgi complex activity. High resolution radioautographic studies of pulse-chase experiment over a 1 hr period demonstrated the shift of silver grains from the elements of the RER, through the Golgi region, and finally to the β-granules. Incubation with 14C-labeled leucine demonstrated the incorporation of radioactivity into molecules possessing the immunological and electrophoretic properties of insulin. These studies indicate that de novo synthesis of (pro)insulin occurs also during culture of fetal rat pancreas explanted relatively late in gestation.

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Selected References

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  1. BLACK L., COMOLLI R. Sur la différenciation in vitro de l'ébauche du pancréas de l'embryon de poulet; recherches préliminaires. Arch Anat Microsc Morphol Exp. 1954;43(3):276–281. [PubMed] [Google Scholar]
  2. Bauer G. E., Lindall A. W., Jr, Dixit P. K., Lester G., Lazarow A. Studies on insulin biosynthesis. Subcellular distribution of leucine-H3 radioactivity during incubation of goosefish islet tissue. J Cell Biol. 1966 Mar;28(3):413–421. doi: 10.1083/jcb.28.3.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bencosme S. A., Martinez-Palomo A. Formation of secretory granules in pancreatic islet B cells of cortisone-treated rabbits. Lab Invest. 1968 Jun;18(6):746–756. [PubMed] [Google Scholar]
  4. Burr I. M., Balant L., Stauffacher W., Renold A. E. Micro gel electrophoresis modification for dilute (protein) solutions. Anal Biochem. 1970 May;35(1):82–86. doi: 10.1016/0003-2697(70)90012-6. [DOI] [PubMed] [Google Scholar]
  5. CARO L. G., PALADE G. E. PROTEIN SYNTHESIS, STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL. AN AUTORADIOGRAPHIC STUDY. J Cell Biol. 1964 Mar;20:473–495. doi: 10.1083/jcb.20.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. CARO L. G., VAN TUBERGEN R. P., KOLB J. A. High-resolution autoradiography. I. Methods. J Cell Biol. 1962 Nov;15:173–188. doi: 10.1083/jcb.15.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. CHEN J. M. The cultivation in fluid medium of organised liver, pancreas and other tissues of foetal rats. Exp Cell Res. 1954 Nov;7(2):518–529. doi: 10.1016/s0014-4827(54)80096-6. [DOI] [PubMed] [Google Scholar]
  8. DIETERLEN-LIEVRE F. [Influence of embryonic pancreas on the formation of liver glycogen in chicks studied by the method of in vitro parabiosis]. C R Hebd Seances Acad Sci. 1960 Feb 15;250:1349–1351. [PubMed] [Google Scholar]
  9. GOLOSOW N., GROBSTEIN C. Epitheliomesenchymal interaction in pancreatic morphogenesis. Dev Biol. 1962 Apr;4:242–255. doi: 10.1016/0012-1606(62)90042-8. [DOI] [PubMed] [Google Scholar]
  10. HALES C. N., RANDLE P. J. Immunoassay of insulin with insulin-antibody precipitate. Biochem J. 1963 Jul;88:137–146. doi: 10.1042/bj0880137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Howell S. L., Kostianovsky M., Lacy P. E. Beta granule formation in isolated islets of langerhans: a study by electron microscopic radioautography. J Cell Biol. 1969 Sep;42(3):695–705. doi: 10.1083/jcb.42.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Howell S. L., Taylor K. W. Effects of glucose concentration on incorporation of [3H]leucine into insulin using isolated mammalian islets of Langerhans. Biochim Biophys Acta. 1966 Dec 28;130(2):519–521. doi: 10.1016/0304-4165(66)90250-9. [DOI] [PubMed] [Google Scholar]
  13. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J Cell Biol. 1967 Aug;34(2):577–596. doi: 10.1083/jcb.34.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J Cell Biol. 1967 Aug;34(2):597–615. doi: 10.1083/jcb.34.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. KALLMAN F., GROBSTEIN C. FINE STRUCTURE OF DIFFERENTIATING MOUSE PANCREATIC EXOCRINE CELLS IN TRANSFILTER CULTURE. J Cell Biol. 1964 Mar;20:399–413. doi: 10.1083/jcb.20.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. KARNOVSKY M. J. Simple methods for "staining with lead" at high pH in electron microscopy. J Biophys Biochem Cytol. 1961 Dec;11:729–732. doi: 10.1083/jcb.11.3.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kawanishi H. Electron microscopic studies on the secretory mechanism of pancreatic islet cells, with particular reference to beta cells. I. Normal secretion in mice, rats, rabbits and chickens, and secretion stimulated by a single intravenous injection of glucose or chlorpropamide in rats. Endocrinol Jpn. 1966 Dec;13(4):358–383. doi: 10.1507/endocrj1954.13.358. [DOI] [PubMed] [Google Scholar]
  18. LACY P. E. Electron microscopy of the beta cell of the pancreas. Am J Med. 1961 Dec;31:851–859. doi: 10.1016/0002-9343(61)90024-9. [DOI] [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lambert A. E., Jeanrenaud B., Junod A., Renold A. E. Organ culture of fetal rat pancreas. II. Insulin release induced by amino and organic acids, by hormonal peptides, by cationic alterations of the medium and by other agents. Biochim Biophys Acta. 1969 Sep 2;184(3):540–553. doi: 10.1016/0304-4165(69)90268-2. [DOI] [PubMed] [Google Scholar]
  22. Lambert A. E., Junod A., Stauffahcer W., Jeanrenaud B., Renold A. E. Organ culture of fetal rat pancreas. I. Insulin release induced by caffeine and by sugars and some derivatives. Biochim Biophys Acta. 1969 Sep 2;184(3):529–539. doi: 10.1016/0304-4165(69)90267-0. [DOI] [PubMed] [Google Scholar]
  23. Lazarus S. S., Volk B. W., Barden H. Localization of acid phosphatase activity and secretion mechanism in rabbit pancreatic B-cells. J Histochem Cytochem. 1966 Mar;14(3):233–246. doi: 10.1177/14.3.233. [DOI] [PubMed] [Google Scholar]
  24. Logothetopoulos J. Electron microscopy of the pancreatic islets of the rat. Effects of prolonged insulin injections. Diabetes. 1966 Nov;15(11):823–829. doi: 10.2337/diab.15.11.823. [DOI] [PubMed] [Google Scholar]
  25. Merlini D., Caramia F. Effect of dehydroascorbic acid on the islets of Langerhans of the rat pancreas. J Cell Biol. 1965 Jul;26(1):245–261. doi: 10.1083/jcb.26.1.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Meyer J., Bencosme S. A. The fine structure of normal rabbit pancreatic islet cells. Rev Can Biol. 1965 Sep;24(3):179–205. [PubMed] [Google Scholar]
  27. Munger B. L., Caramia F., Lacy P. E. The ultrastructural basis for the identification of cell types in the pancreatic islets. II. Rabbit, dog and opossum. Z Zellforsch Mikrosk Anat. 1965 Sep 17;67(6):776–798. doi: 10.1007/BF00339301. [DOI] [PubMed] [Google Scholar]
  28. Murrell L. R. Mammalian pancreatic islet tissue in organ culture. I. Methods of culture and in vitro histogenesis. Exp Cell Res. 1966 Feb;41(2):350–364. doi: 10.1016/s0014-4827(66)80143-x. [DOI] [PubMed] [Google Scholar]
  29. Orci L., Lambert A. E., Amherdt M., Cameron D., Kanazawa Y., Stauffacher W. [The autonomous nervous system and the B-cell: metabolic and morphological observations made in spiny mice (Acomys cahirinus) and in cultured fetal rat pancreas]. Acta Diabetol Lat. 1970 Sep;7 (Suppl 1):184–228. [PubMed] [Google Scholar]
  30. Orci L., Lambert A. E., Kanazawa Y., Renold A. E., Rouiller C. Organ culture of fetal-rat pancreas. 3. Ultrastructural changes occurring in B cells during stimulation of insulin release. Chem Biol Interact. 1970 Apr;1(4):341–359. doi: 10.1016/0009-2797(70)90003-7. [DOI] [PubMed] [Google Scholar]
  31. Orci L., Lambert A. E., Rouiller Ch, Renold A. E., Samols E. Evidence for the presence of A-cells in the endocrine fetal pancreas of the rat. Horm Metab Res. 1969 May;1(3):108–110. doi: 10.1055/s-0028-1095159. [DOI] [PubMed] [Google Scholar]
  32. Parsa I., Marsh W. H., Fitzgerald P. J. Chemically defined medium for organ culture differentiation of rat pancreas anlage. Exp Cell Res. 1970 Jan;59(1):171–175. doi: 10.1016/0014-4827(70)90640-3. [DOI] [PubMed] [Google Scholar]
  33. Renold A. E. Insulin biosynthesis and secretion--a still unsettled topic. N Engl J Med. 1970 Jan 22;282(4):173–182. doi: 10.1056/NEJM197001222820401. [DOI] [PubMed] [Google Scholar]
  34. SCHWEISTHAL M. R., CEAS M. P., WELLS L. J. DEVELOPMENT OF THE PANCREAS OF THE RAT EMBRYO IN VITRO: ISLETS AND ACINI. Anat Rec. 1963 Sep;147:149–161. doi: 10.1002/ar.1091470111. [DOI] [PubMed] [Google Scholar]
  35. SCHWEISTHAL M. R., WELLS L. J., CEAS M. P. DEVELOPMENT OF ISLETS AND ACINI FROM THE EXPLANTED PRIMORDIUM OF THE PANCREAS OF THE RAT EMBRYO. Anat Rec. 1965 Jan;151:93–105. doi: 10.1002/ar.1091510109. [DOI] [PubMed] [Google Scholar]
  36. Scott D. A., Fisher A. M. THE INSULIN AND THE ZINC CONTENT OF NORMAL AND DIABETIC PANCREAS. J Clin Invest. 1938 Nov;17(6):725–728. doi: 10.1172/JCI101000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Steiner D. F., Hallund O., Rubenstein A., Cho S., Bayliss C. Isolation and properties of proinsulin, intermediate forms, and other minor components from crystalline bovine insulin. Diabetes. 1968 Dec;17(12):725–736. doi: 10.2337/diab.17.12.725. [DOI] [PubMed] [Google Scholar]
  38. VANHEYNINGEN H. E. SECRETION OF PROTEIN BY THE ACINAR CELLS OF THE RAT PANCREAS AS STUDIED BY ELECTRON MICROSCOPIC RADIOAUTOGRAPHY. Anat Rec. 1964 Mar;148:485–497. doi: 10.1002/ar.1091480306. [DOI] [PubMed] [Google Scholar]
  39. Vecchio D., Gonet A. E. Culture d'organe de pancréas foetal de rat: I. Effets du glucose, d'autres composants du milieu de culture, et d'un sulfamidé hypoglycémiant. Helv Physiol Pharmacol Acta. 1967;25(1):103–122. [PubMed] [Google Scholar]
  40. WELLS L. J., BORGHESE E. Development of the pancreas of the mouse embryo in vitro: acini and islets. Gen Comp Endocrinol. 1963 Jun;3:265–273. doi: 10.1016/0016-6480(63)90021-2. [DOI] [PubMed] [Google Scholar]
  41. WESSELLS N. K. DNA SYNTHESIS, MITOSIS, AND DIFFERENTIATION IN PANCREATIC ACINAR CELLS IN VITRO. J Cell Biol. 1964 Mar;20:415–433. doi: 10.1083/jcb.20.3.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. WILLIAMSON J. R., LACY P. E., GRISHAM J. W. Ultrastructural changes in islets of the rat produced by tolbutamide. Diabetes. 1961 Nov-Dec;10:460–469. doi: 10.2337/diab.10.6.460. [DOI] [PubMed] [Google Scholar]
  43. Wells L. J., Schweisthal M. R., Marx R. N., McKay M., Saccoman F. M., Lazarow A. Effects of different levels of glucose upon the development of granulated beta cells in cultures on pancreatic primordia from normal rat embryos. Diabetes. 1967 Dec;16(12):839–845. doi: 10.2337/diab.16.12.839. [DOI] [PubMed] [Google Scholar]
  44. ZAGURY D. [The culture of the rat endocrine pancreas and insulogenesis]. C R Hebd Seances Acad Sci. 1959 Dec 21;249:2834–2835. [PubMed] [Google Scholar]

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