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Cytotechnology

, Volume 17, Issue 1, pp 35–44 | Cite as

Changes in metabolism and hormone trafficking during exposure of endocrine cells to elevated ammonium

  • Jill J. Dyken-Young
  • Athanassios Sambanis
Article
  • 20 Downloads

Abstract

Endocrine cell cultures have potential in bioprocessing, for the production of biologically active hormones, and in tissue engineering, for the development of implantable artificial tissues for long-term restoration of endocrine function. To optimize such systems, it is necessary to develop a thorough understanding of how inherently present environmental stresses, such as nutrient depletion and metabolite accumulation, affect the cells. This work focuses on the effects of the metabolite ammonium on indicators of endocrine cell metabolism and on the processing, storage and secretion of regulated secretory proteins. Experiments were conducted on recombinant insulin-producing mouse pituitary AtT-20 cells and mouse insulinoma βTC3 cells. Exposure for 24–48 hours to 6 mM of exogenous ammonium resulted in higher rates of glucose consumption by both AtT-20 and βTC3 cells, while the formation of additional ammonium generally decreased relative to ammonium-free controls. When βTC3 cells were discharged of their intracellular insulin stores, the presence of ammonium during a subsequent recharge completely inhibited addition of new insulin-related peptides to the stores, as we had observed previously for both cell lines. There was a correlation between insulin-related peptides stored in βTC3 cells during recharging and the amount that could be released upon secretagogue stimulation. Using a combination of radioimmunoassay and high performance liquid chromatography, we found that intracellular insulin and insulin-related peptides changed in the same fashion. Intracellular mechanisms that may be producing the observed results are discussed.

Key words

AtT-20 βTC3 (pro)insulin processing insulin secretion ammonium metabolism 

Abbreviations

IRP

insulin-related peptides

HPLC

high performance liquid chromatography

DAMP

3-(2,4-dinitroanilino)-3′ amino-N-methyldipropylamine

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References

  1. Adema E (1989) Ammonium Toxicity in Mammalian Cell Culture. Ph.D. Thesis. Massachusetts Institute of Technology, Cambridge, Massachusetts.Google Scholar
  2. Chung K-N, Walter P, Aponte GW & Moore H-PH (1989) Molecular sorting in the secretory pathway. Science 243:192–197.Google Scholar
  3. Davidson HW, Rhodes CJ & Hutton JC (1988) Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic β cell via two distinct site-specific endopeptidases. Nature 333:93–96.Google Scholar
  4. de Lisle RC & Williams JA (1986) Regulation of membrane fusion in secretory exocytosis. Ann. Rev. Physiol. 48:225–238.Google Scholar
  5. Dyken JJ (1994) Effects of Ammonium Ion and Culturing Mode on Protein Production from Endocrine Cell Cultures. Ph.D. Thesis, Georgia Institute of Technology, Atlanta, Georgia.Google Scholar
  6. Dyken JJ & Sambanis A (1994) Ammonium selectively inhibits the regulated pathway of protein secretion in two endocrine cell lines. Enz. Microb. Technol. 16:90–98.Google Scholar
  7. Efrat S, Linde S, Kofod H, Spector D, Delannoy M, Grant S, Hanahan D & Baekkesskow S (1988) Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc. Natl. Acad. Sci. USA 85:9037–9041.Google Scholar
  8. Forgac M & Cantley L (1984) Characterization of the ATP-dependent proton pump of clathrin-coated vesicles. J. Biol. Chem. 259:8101–8105.Google Scholar
  9. Glacken MW, Adema E & Sinskey AJ (1988) Mathematical descriptions of hybridoma culture kinetics: I. Initial metabolic rates. Biotechnol. Bioeng. 32:491–506.Google Scholar
  10. Grampp GE (1992) Controlled Protein Secretion in Animal Cell Culture. Ph.D. thesis. Massachusetts Institute of Technology. Cambridge, Massachusetts.Google Scholar
  11. Halban PA, Rhodes CJ & Sheolson SE (1986) High-performance liquid chromatography (HPLC: a rapid, flexible, and sensitive method for separating islet proinsulin and insulin. Diabetologia 29:893–896.Google Scholar
  12. Halban PA Structural domains and molecular lifestyles of insulin and its precursors in the pancreatic beta cell. (1991) Diabetologia 34:767–778.Google Scholar
  13. Kurano N, Leist C, Messi F, Kurano S & Fiechter A (1990) Growth behavior of chinese hamster ovary cells in a compact loop bioreactor. 2 Effects of medium components and waste products. J. Biotechnol. 15:113–128.Google Scholar
  14. McQueen A & Bailey JE (1990) Effect of ammonium ion and extracellular pH on hybridoma cell metabolism and antibody production. Biotechnol. Bioeng. 35:1067–1077.Google Scholar
  15. Michael J, Carrol R, Swift HH & Steiner DF (1987) Studies on the molecular organization of rat insulin secretory granules. J. Biol. Chem. 262:16531–16535.Google Scholar
  16. Moore H-PH, Walker MD, Lee F & Kelly RB, Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation. (1983) Cell 35:531–538.Google Scholar
  17. Neerman-Arbez M & Halban PA (1993) Novel, non-crinophagic, degradation of connecting peptide in transformed pancreatic beta cells. J. Biol. Chem. 268:16248–16252.Google Scholar
  18. Orci L, Ravazzola M, Amherdt M, Madsen O, Perrelet A, Vassalli J-D & Anderson RGW (1986) Conversion of Proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. J. Cell Biol. 103:2273–2281Google Scholar
  19. Orci L, Ravazzola M, Amherdt M, Perrelet A, Powell SK, Quinn DL & Moore H-P (1987a) Thetrans-most cisternae of the Golgi complex: a compartment for sorting of secretory and plasma membrane proteins. Cell 51:1039–1051.Google Scholar
  20. Orci L, Ravazzola M, Storch M-J, Anderson RGW, Vassalli J-D, Perrelet A (1987b) Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell. 49:865–868.Google Scholar
  21. Orci L, Ravazzolla M, Amherdt M, Yanaihara C, Yanaihara N, Halban P, Renold AE & Perrelet A (1984) Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell. J. Cell Biol. 98:222–228.Google Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Jill J. Dyken-Young
    • 1
  • Athanassios Sambanis
    • 1
  1. 1.School of Chemical EngineeringGeorgia Institute of TechnologyAtlantaUSA

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