Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1983 Aug 15;214(2):395–404. doi: 10.1042/bj2140395

Measurement of the intramitochondrial volume in hepatocytes without cell disruption and its elevation by hormones and valinomycin.

P T Quinlan, A P Thomas, A E Armston, A P Halestrap
PMCID: PMC1152260  PMID: 6412700

Abstract

Methods have been developed to measure the lysophospholipid content and matrix volume of liver cell mitochondria in situ in order to test the hypothesis that these parameters may be important in the hormonal control of mitochondrial function [Armston, Halestrap & Scott (1982) Biochim. Biophys. Acta 681, 429-439]. No change in the labelling of mitochondrial lysophospholipids with [32P]Pi was detected after treatment of liver cells with glucagon, phenylephrine or vasopressin. Incorporation of [32P]Pi into mitochondrial phosphatidylinositol was enhanced by phenylephrine and vasopressin. Mitochondrial volumes were measured using rapid disruption of cells by sonication into 3H2O and [14C]sucrose or without cell disruption using 3H2O and [14C]mannitol. In control cells the two methods gave values of 1.09 and 0.40 microliters/mg of mitochondrial protein respectively, which represent 19 and 7% respectively of the total cell volume measured with 3H2O and inulin [14C]carboxylic acid. Both methods showed that glucagon, phenylephrine and 1 nm-valinomycin produced significant increases (13% and 26% using sucrose and mannitol respectively) in mitochondrial volume. The increase was coincident with the stimulation of gluconeogenesis from L-lactate and pyruvate and of mitochondrial respiratory chain activity. The effects of glucagon and phenylephrine were additive on both mitochondrial volume and respiratory chain activity, but not on gluconeogenesis. Liver cells exposed to gluconeogenic hormones or low concentrations of valinomycin showed a decrease in light scattering at 520 nM correlating with the change in mitochondrial volume but without a change in whole-cell volume. The time course and hormone sensitivity of this response were similar to those for the hormonal stimulation of gluconeogenesis. The light-scattering response to glucagon, phenylephrine and vasopressin, but not to valinomycin, were greatly reduced or abolished in Ca2+-free media.

Full text

PDF
397

Selected References

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

  1. Armston A. E., Halestrap A. P., Scott R. D. The nature of the changes in liver mitochondrial function induced by glucagon treatment of rats. The effects of intramitochondrial volume, aging and benzyl alcohol. Biochim Biophys Acta. 1982 Sep 15;681(3):429–439. doi: 10.1016/0005-2728(82)90185-2. [DOI] [PubMed] [Google Scholar]
  2. Barritt G. J., Thorne R. F., Hughes B. P. Effects of hormones and N6O2'-dibutyryl-adenosine 3' :5'-cyclic monophosphate, administered in vivo, on phosphate transport and metabolism in isolated rat liver mitochondria. Biochem J. 1978 Jun 15;172(3):577–585. doi: 10.1042/bj1720577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bryla J., Harris E. J., Plumb J. A. The stimulatory effect of glucagon and dibutyryl cyclic AMP on ureogenesis and gluconeogenesis in relation to the mitochondrial ATP content. FEBS Lett. 1977 Aug 15;80(2):443–448. doi: 10.1016/0014-5793(77)80494-8. [DOI] [PubMed] [Google Scholar]
  5. Exton J. H., Corbin J. G., Harper S. C. Control of gluconeogenesis in liver. V. Effects of fasting, diabetes, and glucagon on lactate and endogenous metabolism in the perfused rat liver. J Biol Chem. 1972 Aug 25;247(16):4996–5003. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Halestrap A. P., Quinlan P. T. The intramitochondrial volume measured using sucrose as an extramitochondrial marker overestimates the true matrix volume determined with mannitol. Biochem J. 1983 Aug 15;214(2):387–393. doi: 10.1042/bj2140387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Haynes R. C., Jr, Garrison J. C., Yamazaki R. K. Comparison of effects of glucagon and valinomycin on rat liver mitochondria and cells. Mol Pharmacol. 1974 May;10(3):381–388. [PubMed] [Google Scholar]
  11. Hensgens H. E., Verhoeven A. J., Meijer A. J. The relationship between intramitochondrial N-acetylglutamate and activity of carbamoyl-phosphate synthetase (ammonia). The effect of glucagon. Eur J Biochem. 1980;107(1):197–205. doi: 10.1111/j.1432-1033.1980.tb04640.x. [DOI] [PubMed] [Google Scholar]
  12. Joseph S. K., McGivan J. D., Meijer A. J. The stimulation of glutamine hydrolysis in isolated rat liver mitochondria by Mg2+ depletion and hypo-osmotic incubation conditions. Biochem J. 1981 Jan 15;194(1):35–41. doi: 10.1042/bj1940035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Joseph S. K., McGivan J. D. The effect of ammonium chloride and glucagon on the metabolism of glutamine in isolated liver cells from starved rats. Biochim Biophys Acta. 1978 Sep 21;543(1):16–28. doi: 10.1016/0304-4165(78)90450-6. [DOI] [PubMed] [Google Scholar]
  14. Kirk C. J., Michell R. H., Hems D. A. Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin. Biochem J. 1981 Jan 15;194(1):155–165. doi: 10.1042/bj1940155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kneer N. M., Wagner M. J., Lardy H. A. Regulation by calcium of hormonal effects on gluconeogenesis. J Biol Chem. 1979 Dec 10;254(23):12160–12168. [PubMed] [Google Scholar]
  16. Prpić V., Spencer T. L., Bygrave F. L. Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal. Biochem J. 1978 Dec 15;176(3):705–714. doi: 10.1042/bj1760705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Reinhart P. H., Taylor W. M., Bygrave F. L. Calcium ion fluxes induced by the action of alpha-adrenergic agonists in perfused rat liver. Biochem J. 1982 Dec 15;208(3):619–630. doi: 10.1042/bj2080619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Reinhart P. H., Taylor W. M., Bygrave F. L. Studies on alpha-adrenergic-induced respiration and glycogenolysis in perfused rat liver. J Biol Chem. 1982 Feb 25;257(4):1906–1912. [PubMed] [Google Scholar]
  19. Reinhart P. H., Taylor W. M., Bygrave F. L. Trifluoperazine, an inhibitor of calmodulin action, antagonises phenylephrine-induced metabolic responses and mitochondrial calcium fluxes in liver. FEBS Lett. 1980 Oct 20;120(1):71–74. doi: 10.1016/0014-5793(80)81049-0. [DOI] [PubMed] [Google Scholar]
  20. Siess E. A., Brocks D. G., Wieland O. H. Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats. Biochem J. 1978 Jun 15;172(3):517–521. doi: 10.1042/bj1720517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Siess E. A., Brocks D. G., Wieland O. H. Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats. Biochem J. 1978 Jun 15;172(3):517–521. doi: 10.1042/bj1720517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Siess E. A., Wieland O. H. Early kinetics of glucagon action in isolated hepatocytes at the mitochondrial level. Eur J Biochem. 1980 Sep;110(1):203–210. doi: 10.1111/j.1432-1033.1980.tb04856.x. [DOI] [PubMed] [Google Scholar]
  24. Srere P. A. The citrate enzymes: their structures, mechanisms, and biological functions. Curr Top Cell Regul. 1972;5:229–283. doi: 10.1016/b978-0-12-152805-8.50013-7. [DOI] [PubMed] [Google Scholar]
  25. Thomas A. P., Halestrap A. P. Computer stimulation of the effects of alpha-cyano-4-hydroxycinnamate on gluconeogenesis from L-lactate in rat liver cells. Biochem J. 1981 Sep 15;198(3):561–564. doi: 10.1042/bj1980561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Titheradge M. A., Haynes R. C., Jr The control of uncoupler-activated ATPase activity in rat liver mitochondria by adenine nucleotide transport. The effect of glucagon treatment. J Biol Chem. 1980 Feb 25;255(4):1471–1477. [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Tolbert M. E., Fain J. N. Studies on the regulation of gluconeogenesis in isolated rat liver cells by epinephrine and glucagon. J Biol Chem. 1974 Feb 25;249(4):1162–1166. [PubMed] [Google Scholar]
  30. Vargas A. M., Halestrap A. P., Denton R. M. The effects of glucagon, phenylephrine and insulin on the phosphorylation of cytoplasmic, mitochondrial and membrane-bound proteins of intact liver cells from starved rats. Biochem J. 1982 Oct 15;208(1):221–229. doi: 10.1042/bj2080221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Williamson J. R., Cooper R. H., Hoek J. B. Role of calcium in the hormonal regulation of liver metabolism. Biochim Biophys Acta. 1981 Dec 30;639(3-4):243–295. doi: 10.1016/0304-4173(81)90012-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES