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Active glycolysis and glycogenolysis in early stages of primary cultured hepatocytes. Role of AMP and fructose 2.6-bisphosphate

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This study examines the factors involved in the rapid glycolysis and glycogenolysis that occur during the first stages of hepatocyte culture: a) Shortly after seeding glycolysis, estimated as lactate released to culture medium, increased 10 times in comparison to that reported in vivo. By 8 to 9 h of culture, hepatocytes were nearly glycogen-depleted even in the presence of insulin. b) 6-Phosphofructo-2-kinase remained 100% active during this period. The proportion of the initial active phosphorylase (87%) decreased to 57% by 7 h of culture. c) Fructose 2,6-bisphosphate content was initially similar to that found in liver of fed animals, decreased after seeding and increased thereafter up to four times the initial concentration. In spite of changes in the concentration of this activator, the glycolytic rate remained high and constant. d) ADP and AMP increased sharply after cell plating, reaching values 1.7 and 3.5 times higher. The rise in AMP levels may be involved in the activation of glycolysis and glycogenolysis, because this metabolite is known to act as an allosteric activator of phosphofrucktokinase and glycogen phosphorylase. This metabolic situation resembles that of cells under hypoxia.

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References

  1. Adam, H. Determination of adenosine-5′-diphosphate and adenosine-5′-monophosphate. In: Bergmeyer, H. U., ed. Methods in enzymatic analysis. New York: Academic Press; 1965:253–259.

    Google Scholar 

  2. Bartrons, R.; Hue, L.; Van Schaftingen, E., et al. Hormonal control of fructose 2,6 bisphosphate concentration in isolated rat hepatocytes. Biochem. J. 214:829–837; 1983.

    PubMed  CAS  Google Scholar 

  3. Bernaert, D.; Wanson, J. C.; Drochmens, P., et al. Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes. J. Cell. Biol. 74:878–900; 1977.

    Article  PubMed  CAS  Google Scholar 

  4. Berry, M. N.; Friend, D. S. High yield preparation of isolated rat liver parenchymal cell. J. Cell Biol. 43:506–520; 1969.

    Article  PubMed  CAS  Google Scholar 

  5. Bissell, M.; Hammaker, L. E.; Meyer, U. A. Parenchymal cells from adult rat liver in nonproliferating monolayer culture: I. Functional studies. J. Cell. Biol. 59:722–734; 1973.

    Article  PubMed  CAS  Google Scholar 

  6. Blaauboer, B. J.; Paine, A. J. Attachment of rat hepatocytes to plastic substrata in the absence of serum requires protein synthesis. Biochem. Biophys. Res. Com. 90:368–374; 1979.

    Article  PubMed  CAS  Google Scholar 

  7. Bollen, M.; De Ruysscher, D.; Stalmans, W. On the mechanism of hepatic glycogenolysis induced by anoxia or cyanide. Biochem. Biophys. Res. Com. 115:1033–1039; 1983.

    Article  PubMed  CAS  Google Scholar 

  8. Castell, J. V.; Gomez-Lechon, M. J.; Coloma, J., et al. Preservation of the adult functionality of hepatocytes in serumfree cultures. In: Fischer, G.; Wieser, R. J., eds. Hormonally defined media: a tool in cell biology. Berlin: Springer-Verlag; 1983:333–336.

    Google Scholar 

  9. Edwards, K.; Urban, J.; Schreiber, G. Relationships between protein synthesis and secretion in liver cells and the state of adenine nucleotide system. Aust J. Biol. Sci. 32:299–307; 1979.

    PubMed  CAS  Google Scholar 

  10. El-Maghrabi, M. N.; Pilkis, S. J. Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a review of relationships between the two activities of the enzyme. J. Cell. Biochem. 26:1–17; 1984.

    Article  PubMed  CAS  Google Scholar 

  11. Furuya, E.; Uyeda, K. An activation factor of liver phosphofructokinase. Proc. Natl. Acad. Sci. USA 77:5861–5864; 1980.

    Article  PubMed  CAS  Google Scholar 

  12. Gomez-Lechon, M. J.; Castell, J. V. The role of fetal calf serum during the first stages of hepatocyte culture. In: Fischer, G.; Wieser, R. J., eds. Homonally defined media: a tool in cell biology. Berlin: Springer-Verlag; 1983:340–343.

    Google Scholar 

  13. Gomez-Lechon, M. J.; Lopez, M. P.; Castell, J. V. Biochemical functionality and recovery of hepatocytes after deep-freezing storage. In Vitro 20:826–832; 1984.

    Article  PubMed  CAS  Google Scholar 

  14. Guguen-Guillouzo, C.; Guillouzo, A. Modulation of functional activities in cultured rat hepatocytes. Mol. Cell. Biochem. 53/54:35–56; 1983.

    Article  Google Scholar 

  15. Gutmann, I.; Wahlefeld, A. W. Determination ofl-(+)-lactate with lactate dehydrogenase and NAD. In: Bergmeyer, H. U., ed. Methods of enzymatic analysis, vol. 4. New York: Academic Press; 1974:1464–1468.

    Google Scholar 

  16. Hassid, W. Z.; Abraham, S. Chemical procedures for analysis of polysaccharides. In: Colowick, S. P.; Kaplan, N. O., eds. Methods in Enzymology, vol. 3. New York: Academic Press; 1957:37.

    Google Scholar 

  17. Hems, D. A.; Whitton, P. D. Control of hepatic glycogenolysis. Physiol. Rev. 60:1–50; 1980.

    PubMed  CAS  Google Scholar 

  18. Hers, H. G.; Hue, L. Gluconeogenesis and related aspects of glycolysis. Ann. Rev. Biochem. 52:617–653; 1983.

    Article  PubMed  CAS  Google Scholar 

  19. Hers, H. G.; Van Schaftingen, E. Fructose 2,6 bisphosphate 2 years after its discovery. Biochem. J. 206:1–12; 1982.

    PubMed  CAS  Google Scholar 

  20. Holzer, C.; Meier, P. Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: influence on cellular DNA and protein content monitored by flow cytometry. J. Cell. Physiol. 133:297–303; 1987.

    Article  PubMed  CAS  Google Scholar 

  21. Horiuti, Y.; Nakamura, T.; Ichihara, A. Role of serum in the maintenance of functional hepatocytes in primary culture. J. Biochem. 92:1985–1994; 1982.

    PubMed  CAS  Google Scholar 

  22. Hue, L. Role of fructose 2,6 bisphosphate in the stimulation of glycolysis by anoxia in isolated hepatocytes. Biochem. J. 206:359–365; 1982.

    PubMed  CAS  Google Scholar 

  23. Hue, E.; Blackmore, P. F.; Shikana, H., et al. Regulation of fructose 2,6 bisphosphate content in rat hepatocytes, perfused hearts and perfused hindlimbs. J. Biol. Chem. 257:4308–4313; 1982.

    PubMed  CAS  Google Scholar 

  24. Ichihara, A.; Nakamura, T.; Tanaka, K., et al. Biochemical functions of adult rat hepatocytes in primary culture. Ann. N.Y. Acad. Sci. 349:77–86; 1980.

    Article  PubMed  CAS  Google Scholar 

  25. Jauregui, H. O.; McMillan, P. N.; Driscoll, J., et al. Attachment and long-term survival of adult rat hepatocytes in primary monolayer cultures: comparison of different substrata and tissue culture media formations. In Vitro 22:13–22; 1986.

    CAS  Google Scholar 

  26. Jeejeebhoy, K. N.; Phillips, M. J.; Ho, J., et al. Ultrastructural and functional studies of cultured adult rat hepatocytes. A. Ann. N.Y. Acad. Sci. 349:18–27; 1980.

    Article  CAS  Google Scholar 

  27. Katz, J.; McGarry, J. D. The glucose paradox. Is glucose a substrate for liver metabolism?. J. Clin. Invest. 74:1901–1909; 1984.

    Article  PubMed  CAS  Google Scholar 

  28. Katz, J.; Wals, P. A.; Golden, S., et al. Recycling of glucose by rat hepatocytes. Eur. J. Biochem. 60:91–101; 1975.

    Article  PubMed  CAS  Google Scholar 

  29. Laishes, B. A.; Williams, G. M. Conditions affecting primary cells cultures of functional adult rat hepatocytes: 1. Effect of insulin. In Vitro 12:521–532; 1976.

    PubMed  CAS  Google Scholar 

  30. Lampretch, N.; Trautschold I. ATP determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer, H. U., ed. Methods of enzymatic analysis. New York: Academic Press; 1965:543–551.

    Google Scholar 

  31. López, M. P.; Gómez-Lechón, M. J.; Castell, J. V. Glycogen synthesis in serum free cultured hepatocytes in response to insulin and dexamethasone. In Vitro 20:923–931; 1984.

    Article  PubMed  Google Scholar 

  32. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L., et al. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.

    PubMed  CAS  Google Scholar 

  33. Lutaya, G.; Sharma, R. J.; Griffiths, J. R. Glycogenolysis in liver of phosphorylase kinase-deficient rats during liver perfusion and ischaemia. Biochem. J. 214:645–648; 1983.

    PubMed  CAS  Google Scholar 

  34. Neely, P.; El-Maghrabi, M. R.; Pilkis, S. J. et al. Effect of diabetes, insulin, starvation and refeeding on the level of rat hepatic fructose 2,6 bisphosphate. Diabetes 30:1062–1064; 1981.

    Article  PubMed  CAS  Google Scholar 

  35. Pilkis, S. J.; Chrisam, J. D.; El-Maghrabi, M. R., et al. The action of insulin on hepatic fructose 2,6 bisphosphate metabolism. J. Biol. Chem. 258:1495–1503; 1983.

    PubMed  CAS  Google Scholar 

  36. Probst, I.; Jungermann, K. Short-term regulation of glycolysis by insulin and dexamethasone in cultured rat hepatocytes. Eur. J. Biochem. 135:151–155; 1983.

    Article  PubMed  CAS  Google Scholar 

  37. Probst, I.; Schwartz, P.; Jungermann, K. Induction in primary culture of gluconeogenic and glycolytic hepatocytes resembling periportal and perivenous cells. Eur. J. Biochem. 126:271–278; 1982.

    Article  PubMed  CAS  Google Scholar 

  38. Probst, I.; Unthan-Fechner, k. Activation of glycolysis by insulin with a sequential increase of the 6-phosphofructo-2-kinase activity, fructose 2,6 bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes. Eur. J. Biochem. 153:347–353; 1985.

    Article  PubMed  CAS  Google Scholar 

  39. Richards, C. S.; Furuya, E.; Uyeda, K. Regulation of fructose 2,6 bisphosphate concentration in isolated hepatocytes. Biochem. Biophys. Res. Com. 100:1673–1679; 1981.

    Article  PubMed  CAS  Google Scholar 

  40. Schudt, C. Regulation of glycogen synthesis in rat hepatocyte cultures by glucose, insulin and glucocorticoids. Eur. J. Biochem. 97:155–160; 1979.

    Article  PubMed  CAS  Google Scholar 

  41. Schudt, C. Influence of insulin, glucocorticoids and glucose on glycogen synthase activity in hepatocyte cultures. Biochim. Biophys. Acta 629: 499–509; 1980.

    PubMed  CAS  Google Scholar 

  42. Schwarze, P. E.; Solheim, A. E.; Seglen, P. O. Aminoacid and energy requirements for rat hepatocyte in primary culture. In Vitro 18:43–54; 1982.

    PubMed  CAS  Google Scholar 

  43. Sharma, R. J.; Rodrigues, L. M.; Whitton, P. D., et al. Control mechanism in the acceleration of hepatic glycogen degradation during hypoxia. Biochim. Biophys. Acta 630:414–424; 1980.

    PubMed  CAS  Google Scholar 

  44. Stalmans, W.; Hers, H. G. The stimulation of liver phosphorylase b by AMP, fluoride and sulfate; a technical note on the specific determination of the a and b forms of liver glycogen phosphorylase. Eur. J. Biochem. 54:341–350; 1975.

    Article  PubMed  CAS  Google Scholar 

  45. Trowell, D. A. Isolated liver perfusion in the study of hepatic function. J. Physiol. 100:432–435; 1942.

    PubMed  CAS  Google Scholar 

  46. Van Sohaftingen, E.; Hers, H. G. Phosphofructokinase 2, the enzyme that forms fructose 2,6 bisphosphate from fructose-6-phosphate and ATP. Biochem. Biophys. Res. Com. 101:1078–1084; 1981.

    Article  Google Scholar 

  47. Van Schaftingen, E.; Jett, M. F.; Hue, L., et al. Control of liver 6-phosphofructokinase by fructose 2,6 bisphosphate and other effectors. Proc. Natl. Acad. Sci. USA 78:3483–3486; 1981.

    Article  PubMed  Google Scholar 

  48. Van Schaftingen, E.; Lederer, B.; Bartrons, R., et al. A kinetic study of pyrophosphate:fructose-6-phosphate phosphotransferase from potato tubers. Eur. J. Biochem. 129:191–195; 1982.

    Article  PubMed  Google Scholar 

  49. Williams, G. M.; Bermudez, E.; Scaramuzziono, D. Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13:809–816; 1977.

    Article  PubMed  CAS  Google Scholar 

  50. Williamson, D. H.; Brosnan, J. T. Concentrations of metabolites in animal tissues. In: Bergmeyer, H. U., ed. Methods of enzymatic analysis, vol. 4. New York: Academic Press; 1974:2266–2302.

    Google Scholar 

  51. Wirthensohn, K.; Barth, C. A. Influence of hormones and growth factors on viability, DNA and protein content of adult hepatocytes in primary culture. In Vitro 21:346–351; 1985.

    Google Scholar 

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Part of this work was presented at the 38th Annual Meeting of the Tissue Culture Association, Washington, DC, May 1987.

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López, M.P., Gómez-Lechón, M.J. & Castell, J.V. Active glycolysis and glycogenolysis in early stages of primary cultured hepatocytes. Role of AMP and fructose 2.6-bisphosphate. In Vitro Cell Dev Biol 24, 511–517 (1988). https://doi.org/10.1007/BF02629084

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