Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Feb 15;218(1):19–27. doi: 10.1042/bj2180019

Immunoreactive insulin from mouse brain cells in culture and whole rat brain.

N P Birch, D L Christie, A G Renwick
PMCID: PMC1153303  PMID: 6143553

Abstract

Foetal mouse brain cells were cultured as described previously [Sotelo, Gibbs, Gajdusek, Toh & Wurth (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 653-657] without added insulin and without foetal calf serum after 12 days in culture. Examination by phase-contrast microscopy showed that these modifications did not appear to affect growth and development of the cells adversely. Silver impregnation of the cultures and indirect immunofluorescence following reaction with tetanus toxin showed that a high proportion of the cells resembled neurones. Analysis of concentrated culture medium by radioimmunoassay and high-pressure liquid chromatography (h.p.l.c.) revealed that the cells produced two main forms of immunoreactive insulin which differed from authentic pancreatic insulin in retention time. Immunoreactive somatostatin was also produced in culture and this was resolved into at least three forms by h.p.l.c. Immunoreactive insulin was also extracted from whole rat brain by using two published procedures. The method of Havrankova, Schmechel, Roth & Brownstein [Proc. Natl. Acad. Sci. U.S.A. (1978) 75, 5737-5741] consistently gave greater yields of insulin than did that of Eng & Yalow [Diabetes (1980) 29, 105-109] and the concentration was about three times that of plasma. The extracted insulin was further characterized by h.p.l.c. in each case and was found to behave like authentic pancreatic insulin. The production of insulin and somatostatin by foetal mouse brain cells in culture suggests that they may be a useful model system for studies of neuropeptide biosynthesis.

Full text

PDF
19

Images in this article

23Fig. 1.
on p.23

Selected References

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

  1. BROWN H., SANGER F., KITAI R. The structure of pig and sheep insulins. Biochem J. 1955 Aug;60(4):556–565. doi: 10.1042/bj0600556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Binoux M., Hossenlopp P., Lassarre C., Hardouin N. Production of insulin-like growth factors and their carrier by rat pituitary gland and brain explants in culture. FEBS Lett. 1981 Feb 23;124(2):178–184. doi: 10.1016/0014-5793(81)80131-7. [DOI] [PubMed] [Google Scholar]
  3. Blundell T. L., Humbel R. E. Hormone families: pancreatic hormones and homologous growth factors. Nature. 1980 Oct 30;287(5785):781–787. doi: 10.1038/287781a0. [DOI] [PubMed] [Google Scholar]
  4. Bünzli H. F., Glatthaar B., Kunz P., Mülhaupt E., Humbel R. E. Amino acid sequence of the two insulins from mouse (Maus musculus). Hoppe Seylers Z Physiol Chem. 1972 Mar;353(3):451–458. doi: 10.1515/bchm2.1972.353.1.451. [DOI] [PubMed] [Google Scholar]
  5. Delfs J., Robbins R., Connolly J. L., Dichter M., Reichlin S. Somatostatin production by rat cerebral neurones in dissociated cell culture. Nature. 1980 Feb 14;283(5748):676–677. doi: 10.1038/283676a0. [DOI] [PubMed] [Google Scholar]
  6. Dray F., Louis J. C., Rougeot C., Mandel P., Bennett C. Presence of two molecular weight forms of somatostatin in neurons from chick embryo cerebral hemispheres in culture. FEBS Lett. 1982 Aug 16;145(1):123–127. doi: 10.1016/0014-5793(82)81219-2. [DOI] [PubMed] [Google Scholar]
  7. Eng J., Yalow R. S. Absence of pork-like insulin in guinea pig tissues. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2683–2685. doi: 10.1073/pnas.79.8.2683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eng J., Yalow R. S. Evidence against extrapancreatic insulin synthesis. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4576–4578. doi: 10.1073/pnas.78.7.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eng J., Yalow R. S. Insulin recoverable from tissues. Diabetes. 1980 Feb;29(2):105–109. doi: 10.2337/diab.29.2.105. [DOI] [PubMed] [Google Scholar]
  10. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gambetti P., Autilio Gambetti L., Papasozomenos S. C. Bodian's silver method stains neurofilament polypeptides. Science. 1981 Sep 25;213(4515):1521–1522. doi: 10.1126/science.6169146. [DOI] [PubMed] [Google Scholar]
  12. Havrankova J., Roth J., Brownstein M. J. Concentrations of insulin and insulin receptors in the brain are independent of peripheral insulin levels. Studies of obese and streptozotocin-treated rodents. J Clin Invest. 1979 Aug;64(2):636–642. doi: 10.1172/JCI109504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Havrankova J., Schmechel D., Roth J., Brownstein M. Identification of insulin in rat brain. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5737–5741. doi: 10.1073/pnas.75.11.5737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mirsky R., Wendon L. M., Black P., Stolkin C., Bray D. Tetanus toxin: a cell surface marker for neurones in culture. Brain Res. 1978 Jun 9;148(1):251–259. doi: 10.1016/0006-8993(78)90399-2. [DOI] [PubMed] [Google Scholar]
  15. Patel Y. C., Wheatley T., Ning C. Multiple forms of immunoreactive somatostatin: comparison of distribution in neural and nonneural tissues and portal plasma of the rat. Endocrinology. 1981 Dec;109(6):1943–1949. doi: 10.1210/endo-109-6-1943. [DOI] [PubMed] [Google Scholar]
  16. Peterfreund R. A., Vale W. High molecular weight somatostatin secretion by cultured rat brain cells. Brain Res. 1982 May 13;239(2):463–477. doi: 10.1016/0006-8993(82)90522-4. [DOI] [PubMed] [Google Scholar]
  17. Rosenzweig J. L., Havrankova J., Lesniak M. A., Brownstein M., Roth J. Insulin is ubiquitous in extrapancreatic tissues of rats and humans. Proc Natl Acad Sci U S A. 1980 Jan;77(1):572–576. doi: 10.1073/pnas.77.1.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sotelo J., Gibbs C. J., Jr, Gajdusek D. C., Toh B. H., Wurth M. Method for preparing cultures of central neurons: cytochemical and immunochemical studies. Proc Natl Acad Sci U S A. 1980 Jan;77(1):653–657. doi: 10.1073/pnas.77.1.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Uvnäs B., Uvnäs-Wallensten K. "Insulinergic" nerves to the skeletal muscles of the cat? Acta Physiol Scand. 1978 Jul;103(3):346–348. doi: 10.1111/j.1748-1716.1978.tb06225.x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES