Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1983 May 15;212(2):417–426. doi: 10.1042/bj2120417

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes.

E H Allan, A B Chisholm, M A Titheradge
PMCID: PMC1152062  PMID: 6411066

Abstract

A method is described for measuring rates of mitochondrial pyruvate carboxylation in hepatocytes treated with the polyene antibiotic, filipin, to render the plasma membrane permeable to substrates. With this approach it was possible to demonstrate that treatment of cells with glucagon or catecholamines results in a stimulation of mitochondrial CO2 fixation measured in situ comparable with that observed in the isolated mitochondria, in terms of time of onset of the response, hormone selectivity and sensitivity. In addition, angiotensin II and vasopressin were shown to enhance the activity of pyruvate carboxylase in both the intact mitochondria and filipin-treated cells, thus strengthening the postulate that this site is a major locus of hormone action in the control of gluconeogenesis. Addition of 3-mercaptopicolinic acid, to inhibit gluconeogenesis at the level of phosphoenolpyruvate carboxykinase, had no significant effect on the stimulation of pyruvate carboxylation by adrenaline, suggesting that the effect of the hormone at this site is independent of changes in activity of other enzymes further on in the pathway. The data presented preclude the possibility that acute effects of hormones on mitochondrial metabolism are solely artifacts of the preparation procedure.

Full text

PDF
420

Selected References

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

  1. Adam P. A., Haynes R. C., Jr Control of hepatic mitochondrial CO2 fixation by glucagon, epinephrine, and cortisol. J Biol Chem. 1969 Dec 10;244(23):6444–6450. [PubMed] [Google Scholar]
  2. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chan T. M., Bacon C. B., Hill S. A. Glucagon stimulation of liver mitochondrial CO2 fixation utilizing pyruvate generated inside the mitochondria. J Biol Chem. 1979 Sep 25;254(18):8730–8732. [PubMed] [Google Scholar]
  4. Colbeau A., Nachbaur J., Vignais P. M. Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim Biophys Acta. 1971 Dec 3;249(2):462–492. doi: 10.1016/0005-2736(71)90123-4. [DOI] [PubMed] [Google Scholar]
  5. Edmondson J. W., Lumeng L., Li T. K. Direct measurement of active transport systems for alanine in freshly isolated rat liver cells. Biochem Biophys Res Commun. 1977 Jun 6;76(3):751–757. doi: 10.1016/0006-291x(77)91564-9. [DOI] [PubMed] [Google Scholar]
  6. Gankema H. S., Laanen E., Groen A. K., Tager J. M. Characterization of isolated rat-liver cells made permeable with filipin. Eur J Biochem. 1981 Oct;119(2):409–414. doi: 10.1111/j.1432-1033.1981.tb05623.x. [DOI] [PubMed] [Google Scholar]
  7. Garrison J. C., Borland M. K. Regulation of mitochondrial pyruvate carboxylation and gluconeogenesis in rat hepatocytes via an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism. J Biol Chem. 1979 Feb 25;254(4):1129–1133. [PubMed] [Google Scholar]
  8. Garrison J. C., Haynes R. C., Jr The hormonal control of gluconeogenesis by regulation of mitochondrial pyruvate carboxylation in isolated rat liver cells. J Biol Chem. 1975 Apr 25;250(8):2769–2777. [PubMed] [Google Scholar]
  9. Halestrap A. P., Denton R. M. The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds. Biochem J. 1975 Apr;148(1):97–106. doi: 10.1042/bj1480097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Halestrap A. P. Pyruvate and ketone-body transport across the mitochondrial membrane. Exchange properties, pH-dependence and mechanism of the carrier. Biochem J. 1978 Jun 15;172(3):377–387. doi: 10.1042/bj1720377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halestrap A. P. Stimulation of pyruvate transport in metabolizing mitochondria through changes in the transmembrane pH gradient induced by glucagon treatment of rats. Biochem J. 1978 Jun 15;172(3):389–398. doi: 10.1042/bj1720389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Halestrap A. P. The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals. Biochem J. 1982 Apr 15;204(1):37–47. doi: 10.1042/bj2040037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haynes R. C., Jr The fixation of carbon dioxide by rat liver mitochondria and its relation to gluconeogenesis. J Biol Chem. 1965 Oct;240(10):4103–4106. [PubMed] [Google Scholar]
  14. Hems D. A., Whitton P. D. Stimulation by vasopressin of glycogen breakdown and gluconeogenesis in the perfused rat liver. Biochem J. 1973 Nov;136(3):705–709. doi: 10.1042/bj1360705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hutson N. J., Brumley F. T., Assimacopoulos F. D., Harper S. C., Exton J. H. Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells. J Biol Chem. 1976 Sep 10;251(17):5200–5208. [PubMed] [Google Scholar]
  16. Jorgenson R. A., Nordlie R. C. Multifunctional glucose-6-phosphatase studied in permeable isolated hepatocytes. J Biol Chem. 1980 Jun 25;255(12):5907–5915. [PubMed] [Google Scholar]
  17. Joseph S. K., Bradford N. M., McGivan J. D. Characteristics of the transport of alanine, serine and glutamine across the plasma membrane of isolated rat liver cells. Biochem J. 1978 Dec 15;176(3):827–836. doi: 10.1042/bj1760827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kinsky S. C., Luse S. A., Zopf D., van Deenen L. L., Haxby J. Interaction of filipin and derivatives with erythrocyte membranes and lipid dispersions: electron microscopic observations. Biochim Biophys Acta. 1967;135(5):844–861. doi: 10.1016/0005-2736(67)90055-7. [DOI] [PubMed] [Google Scholar]
  19. Martin A. D., Titheradge M. A. Hormonal stimulation of gluconeogenesis through increased mitochondrial metabolic flux. Biochem Soc Trans. 1983 Jan;11(1):78–81. doi: 10.1042/bst0110078. [DOI] [PubMed] [Google Scholar]
  20. Morton B. E., Lardy H. A. Cellular oxidative phosphorylation. 3. Measurement in chemically modified cells. Biochemistry. 1967 Jan;6(1):57–61. doi: 10.1021/bi00853a011. [DOI] [PubMed] [Google Scholar]
  21. Norman A. W., Spielvogel A. M., Wong R. G. Polyene antibiotic - sterol interaction. Adv Lipid Res. 1976;14:127–170. [PubMed] [Google Scholar]
  22. Scrutton M. C., White M. D. Pyruvate carboxylase. Inhibition of the mammalian and avian liver enzymes by alpha-ketoglutarate and L-glutamate. J Biol Chem. 1974 Sep 10;249(17):5405–5415. [PubMed] [Google Scholar]
  23. Siess E. A., Fahimi F. M., Wieland O. H. Evidence that glucagon stabilizes rather than activates mitochondrial functions in rat liver. Hoppe Seylers Z Physiol Chem. 1981 Dec;362(12):1643–1651. doi: 10.1515/bchm2.1981.362.2.1643. [DOI] [PubMed] [Google Scholar]
  24. Siess E. A., Wieland O. H. Isolated hepatocytes as a model for the study of stable glucagon effects on mitochondrial respiratory functions. FEBS Lett. 1979 May 15;101(2):277–281. doi: 10.1016/0014-5793(79)81025-x. [DOI] [PubMed] [Google Scholar]
  25. Titheradge M. A., Coore H. G. Hormonal regulation of liver mitochondrial pyruvate carrier in relation to gluconeogenesis and lipogenesis. FEBS Lett. 1976 Nov 15;72(1):73–78. doi: 10.1016/0014-5793(76)80901-5. [DOI] [PubMed] [Google Scholar]
  26. Titheradge M. A., Coore H. G. The mitochondrial pyruvate carrier, its exchange properties and its regulation by glucagon. FEBS Lett. 1976 Mar 15;63(1):45–50. doi: 10.1016/0014-5793(76)80191-3. [DOI] [PubMed] [Google Scholar]
  27. Titheradge M. A., Haynes R. C., Jr Glucagon treatment stimulates the oxidation of durohydroquinone by rat liver mitochondria. FEBS Lett. 1979 Oct 15;106(2):330–334. doi: 10.1016/0014-5793(79)80526-8. [DOI] [PubMed] [Google Scholar]
  28. Titheradge M. A., Haynes R. C., Jr The hormonal stimulation of ureogenesis in isolated hepatocytes through increases in mitochondrial ATP production. Arch Biochem Biophys. 1980 Apr 15;201(1):44–55. doi: 10.1016/0003-9861(80)90485-3. [DOI] [PubMed] [Google Scholar]
  29. Titheradge M. A., Stringer J. L., Haynes R. C., Jr The stimulation of the mitochondrial uncoupler-dependent ATPase in isolated hepatocytes by catecholamines and glucagon and its relationship to gluconeogenesis. Eur J Biochem. 1979 Dec;102(1):117–124. doi: 10.1111/j.1432-1033.1979.tb06271.x. [DOI] [PubMed] [Google Scholar]
  30. Wakat D. K., Haynes R. C., Jr Glucocorticoid-stimulated utilization of substrates in hepatic mitochondria. Arch Biochem Biophys. 1977 Dec;184(2):561–571. doi: 10.1016/0003-9861(77)90466-0. [DOI] [PubMed] [Google Scholar]
  31. Whitton P. D., Rodrigues L. M., Hems D. A. Stimulation by vasopressin, angiotensin and oxytocin of gluconeogenesis in hepatocyte suspensions. Biochem J. 1978 Dec 15;176(3):893–898. doi: 10.1042/bj1760893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yamazaki R. K. Glucagon stimulation of mitochondrial respiration. J Biol Chem. 1975 Oct 10;250(19):7924–7930. [PubMed] [Google Scholar]
  33. Yamazaki R. K., Haynes R. C., Jr Dissociation of pyruvate dehydrogenase from the glucagon stimulation of pyruvate carboxylation in rat liver mitochondria. Arch Biochem Biophys. 1975 Feb;166(2):575–583. doi: 10.1016/0003-9861(75)90422-1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES