Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1987 Feb 1;241(3):729–735. doi: 10.1042/bj2410729

The glucagon-induced activation of pyruvate dehydrogenase in hepatocytes is diminished by 4 beta-phorbol 12-myristate 13-acetate. A role for cytoplasmic Ca2+ in dehydrogenase regulation.

J M Staddon, R G Hansford
PMCID: PMC1147624  PMID: 3593219

Abstract

Phenylephrine, vasopressin and glucagon each increased the amount of active (dephospho) pyruvate dehydrogenase (PDHa) in isolated rat hepatocytes. Treatment with 4 beta-phorbol 12-myristate 13-acetate (PMA) opposed the increase in PDHa caused by both phenylephrine and glucagon, but had no effect on the response to vasopressin: PMA alone had no effect on PDHa. As PMA is known to prevent the phenylephrine-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and to diminish the increase [Ca2+]c caused by glucagon, while having no effect on the ability of vasopressin to increase [Ca2+]c, these data are consistent with the notion that in intact cells an increase in [Ca2+]c results in an increase in the mitochondrial free Ca2+ concentration, which in turn leads to the activation of PDH. In the presence of 2.5 mM-Ca2+, glucagon caused an increase in NAD(P)H fluorescence in hepatocytes. This increase is taken to reflect an enhanced activity of mitochondrial dehydrogenases. PMA alone had no effect on NAD(P)H fluorescence; it did, however, compromise the increase produced by glucagon. When the extracellular free [Ca2+] was decreased to 0.2 microM, glucagon could still increase NAD(P)H fluorescence. Vasopressin also increased fluorescence under these conditions; however, if vasopressin was added after glucagon, no further increase in fluorescence was observed. Treatment of the cells with PMA resulted in a smaller increase in NAD(P)H fluorescence on addition of glucagon: the subsequent addition of vasopressin now caused a further increase in fluorescence. Changes in [Ca2+]c corresponding to the changes in NAD(P)H fluorescence were observed, again supporting the idea that [Ca2+]c indirectly regulates intramitochondrial dehydrogenase activity in intact cells. PMA alone had no effect on pyruvate kinase activity, and the phorbol ester did not prevent the inactivation caused by glucagon. The latter emphasizes the different mechanisms by which the hormone influences mitochondrial and cytoplasmic metabolism.

Full text

PDF
729

Selected References

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

  1. Assimacopoulos-Jeannet F., McCormack J. G., Jeanrenaud B. Effect of phenylephrine on pyruvate dehydrogenase activity in rat hepatocytes and its interaction with insulin and glucagon. FEBS Lett. 1983 Aug 8;159(1-2):83–88. doi: 10.1016/0014-5793(83)80421-9. [DOI] [PubMed] [Google Scholar]
  2. Balaban R. S., Blum J. J. Hormone-induced changes in NADH fluorescence and O2 consumption of rat hepatocytes. Am J Physiol. 1982 Mar;242(3):C172–C177. doi: 10.1152/ajpcell.1982.242.3.C172. [DOI] [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. Berthon B., Binet A., Mauger J. P., Claret M. Cytosolic free Ca2+ in isolated rat hepatocytes as measured by quin2. Effects of noradrenaline and vasopressin. FEBS Lett. 1984 Feb 13;167(1):19–24. doi: 10.1016/0014-5793(84)80824-8. [DOI] [PubMed] [Google Scholar]
  5. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  6. Charest R., Blackmore P. F., Berthon B., Exton J. H. Changes in free cytosolic Ca2+ in hepatocytes following alpha 1-adrenergic stimulation. Studies on Quin-2-loaded hepatocytes. J Biol Chem. 1983 Jul 25;258(14):8769–8773. [PubMed] [Google Scholar]
  7. Cooper R. H., Coll K. E., Williamson J. R. Differential effects of phorbol ester on phenylephrine and vasopressin-induced Ca2+ mobilization in isolated hepatocytes. J Biol Chem. 1985 Mar 25;260(6):3281–3288. [PubMed] [Google Scholar]
  8. Cooper R. H., Randle P. J., Denton R. M. Stimulation of phosphorylation and inactivation of pyruvate dehydrogenase by physiological inhibitors of the pyruvate dehydrogenase reaction. Nature. 1975 Oct 30;257(5529):808–809. doi: 10.1038/257808a0. [DOI] [PubMed] [Google Scholar]
  9. Coore H. G., Denton R. M., Martin B. R., Randle P. J. Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones. Biochem J. 1971 Nov;125(1):115–127. doi: 10.1042/bj1250115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Denton R. M., McCormack J. G., Edgell N. J. Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria. Biochem J. 1980 Jul 15;190(1):107–117. doi: 10.1042/bj1900107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Denton R. M., McCormack J. G. On the role of the calcium transport cycle in heart and other mammalian mitochondria. FEBS Lett. 1980 Sep 22;119(1):1–8. doi: 10.1016/0014-5793(80)80986-0. [DOI] [PubMed] [Google Scholar]
  12. Denton R. M., Randle P. J., Martin B. R. Stimulation by calcium ions of pyruvate dehydrogenase phosphate phosphatase. Biochem J. 1972 Jun;128(1):161–163. doi: 10.1042/bj1280161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Denton R. M., Richards D. A., Chin J. G. Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues. Biochem J. 1978 Dec 15;176(3):899–906. doi: 10.1042/bj1760899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fisher R. A., Tanabe S., Buxton D. B., Olson M. S. The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver. J Biol Chem. 1985 Aug 5;260(16):9223–9229. [PubMed] [Google Scholar]
  15. García-Sáinz J. A., Mendlovic F., Martínez-Olmedo M. A. Effects of phorbol esters on alpha 1-adrenergic-mediated and glucagon-mediated actions in isolated rat hepatocytes. Biochem J. 1985 May 15;228(1):277–280. doi: 10.1042/bj2280277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gelfand E. W., Cheung R. K., Grinstein S. Mitogen-induced changes in Ca2+ permeability are not mediated by voltage-gated K+ channels. J Biol Chem. 1986 Sep 5;261(25):11520–11523. [PubMed] [Google Scholar]
  17. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  18. Hansford R. G., Cohen L. Relative importance of pyruvate dehydrogenase interconversion and feed-back inhibition in the effect of fatty acids on pyruvate oxidation by rat heart mitochondria. Arch Biochem Biophys. 1978 Nov;191(1):65–81. doi: 10.1016/0003-9861(78)90068-1. [DOI] [PubMed] [Google Scholar]
  19. Hansford R. G. Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart mitochondria. Biochem J. 1981 Mar 15;194(3):721–732. doi: 10.1042/bj1940721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hansford R. G. Relation between mitochondrial calcium transport and control of energy metabolism. Rev Physiol Biochem Pharmacol. 1985;102:1–72. doi: 10.1007/BFb0034084. [DOI] [PubMed] [Google Scholar]
  21. Hems D. A., McCormack J. G., Denton R. M. Activation of pyruvate dehydrogenase in the perfused rat liver by vasopressin. Biochem J. 1978 Nov 15;176(2):627–629. doi: 10.1042/bj1760627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heyworth C. M., Whetton A. D., Kinsella A. R., Houslay M. D. The phorbol ester, TPA inhibits glucagon-stimulated adenylate cyclase activity. FEBS Lett. 1984 May 7;170(1):38–42. doi: 10.1016/0014-5793(84)81364-2. [DOI] [PubMed] [Google Scholar]
  23. Holian A., Owen C. S., Wilson D. F. Control of respiration in isolated mitochondria: quantitative evaluation of the dependence of respiratory rates on [ATP], [ADP], and [Pi]. Arch Biochem Biophys. 1977 May;181(1):164–171. doi: 10.1016/0003-9861(77)90494-5. [DOI] [PubMed] [Google Scholar]
  24. Hucho F., Randall D. D., Roche T. E., Burgett M. W., Pelley J. W., Reed L. J. -Keto acid dehydrogenase complexes. XVII. Kinetic and regulatory properties of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase from bovine kidney and heart. Arch Biochem Biophys. 1972 Jul;151(1):328–340. doi: 10.1016/0003-9861(72)90504-8. [DOI] [PubMed] [Google Scholar]
  25. Lynch C. J., Charest R., Bocckino S. B., Exton J. H., Blackmore P. F. Inhibition of hepatic alpha 1-adrenergic effects and binding by phorbol myristate acetate. J Biol Chem. 1985 Mar 10;260(5):2844–2851. [PubMed] [Google Scholar]
  26. McCormack J. G. Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria. Biochem J. 1985 Nov 1;231(3):581–595. doi: 10.1042/bj2310581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McCormack J. G., Denton R. M. The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem J. 1979 Jun 15;180(3):533–544. doi: 10.1042/bj1800533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McCormack J. G. Studies on the activation of rat liver pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase by adrenaline and glucagon. Role of increases in intramitochondrial Ca2+ concentration. Biochem J. 1985 Nov 1;231(3):597–608. doi: 10.1042/bj2310597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oviasu O. A., Whitton P. D. Hormonal control of pyruvate dehydrogenase activity in rat liver. Biochem J. 1984 Nov 15;224(1):181–186. doi: 10.1042/bj2240181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pettit F. H., Pelley J. W., Reed L. J. Regulation of pyruvate dehydrogenase kinase and phosphatase by acetyl-CoA/CoA and NADH/NAD ratios. Biochem Biophys Res Commun. 1975 Jul 22;65(2):575–582. doi: 10.1016/s0006-291x(75)80185-9. [DOI] [PubMed] [Google Scholar]
  31. Reed L. J. Regulation of mammalian pyruvate dehydrogenase complex by a phosphorylation-dephosphorylation cycle. Curr Top Cell Regul. 1981;18:95–106. doi: 10.1016/b978-0-12-152818-8.50012-8. [DOI] [PubMed] [Google Scholar]
  32. Sies H., Graf P., Crane D. Decreased flux through pyruvate dehydrogenase during calcium ion movements induced by vasopressin, alpha-adrenergic agonists and the ionophore A23187 in perfused rat liver. Biochem J. 1983 May 15;212(2):271–278. doi: 10.1042/bj2120271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Siess E. A., Brocks D. G., Lattke H. K., Wieland O. H. Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate. Biochem J. 1977 Aug 15;166(2):225–235. doi: 10.1042/bj1660225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Siess E. A., Wieland O. H. Regulation of pyruvate dehydrogenase interconversion in isolated hepatocytes by the mitochondrial ATP/ADP ratio. FEBS Lett. 1975 Apr 1;52(2):226–230. doi: 10.1016/0014-5793(75)80811-8. [DOI] [PubMed] [Google Scholar]
  36. Sistare F. D., Picking R. A., Haynes R. C., Jr Sensitivity of the response of cytosolic calcium in Quin-2-loaded rat hepatocytes to glucagon, adenine nucleosides, and adenine nucleotides. J Biol Chem. 1985 Oct 15;260(23):12744–12747. [PubMed] [Google Scholar]
  37. Staddon J. M., Hansford R. G. 4 beta-Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes. Biochem J. 1986 Sep 15;238(3):737–743. doi: 10.1042/bj2380737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Staddon J. M., McGivan J. D. Distinct effects of glucagon and vasopressin on proline metabolism in isolated hepatocytes. The role of oxoglutarate dehydrogenase. Biochem J. 1984 Jan 15;217(2):477–483. doi: 10.1042/bj2170477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sugano T., Shiota M., Khono H., Shimada M., Oshino N. Effects of calcium ions on the activation of gluconeogenesis by norepinephrine in perfused rat liver. J Biochem. 1980 Feb;87(2):465–472. doi: 10.1093/oxfordjournals.jbchem.a132766. [DOI] [PubMed] [Google Scholar]
  40. Thomas A. P., Alexander J., Williamson J. R. Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2+ induced by vasopressin in isolated hepatocytes. J Biol Chem. 1984 May 10;259(9):5574–5584. [PubMed] [Google Scholar]
  41. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wieland O. H. The mammalian pyruvate dehydrogenase complex: structure and regulation. Rev Physiol Biochem Pharmacol. 1983;96:123–170. doi: 10.1007/BFb0031008. [DOI] [PubMed] [Google Scholar]

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

RESOURCES