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. 1976 Feb 15;154(2):327–348. doi: 10.1042/bj1540327

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

A L Kerbey, P J Randle, R H Cooper, S Whitehouse, H T Pask, R M Denton
PMCID: PMC1172714  PMID: 180974

Abstract

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.

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Selected References

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  1. BREMER J. Carnitine in intermediary metabolism. The metabolism of fatty acid esters of carnitine by mitochondria. J Biol Chem. 1962 Dec;237:3628–3632. [PubMed] [Google Scholar]
  2. Bowman R. H. Effects of diabetes, fatty acids, and ketone bodies on tricarboxylic acid cycle metabolism in the perfused rat heart. J Biol Chem. 1966 Jul 10;241(13):3041–3048. [PubMed] [Google Scholar]
  3. Bremer J. Comparison of acylcarnitines and pyruvate as substrates for rat-liver mitochondria. Biochim Biophys Acta. 1966 Feb 1;116(1):1–11. doi: 10.1016/0005-2760(66)90087-7. [DOI] [PubMed] [Google Scholar]
  4. Bremer J. Pyruvate dehydrogenase, substrate specificity and product inhibition. Eur J Biochem. 1969 Apr;8(4):535–540. doi: 10.1111/j.1432-1033.1969.tb00559.x. [DOI] [PubMed] [Google Scholar]
  5. CHANCE B., HOLLUNGER G. The interaction of energy and electron transfer reactions in mitochondria. IV. The pathway of electron transfer. J Biol Chem. 1961 May;236:1562–1568. [PubMed] [Google Scholar]
  6. Carlson C. A., Kim K. H. Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation. Arch Biochem Biophys. 1974 Oct;164(2):478–489. doi: 10.1016/0003-9861(74)90058-7. [DOI] [PubMed] [Google Scholar]
  7. Chen R. F. Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem. 1967 Jan 25;242(2):173–181. [PubMed] [Google Scholar]
  8. Chiang P. K., Sacktor B. Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase. J Biol Chem. 1975 May 10;250(9):3399–3408. [PubMed] [Google Scholar]
  9. Cooper R. H., Randle P. J., Denton R. M. Regulation of heart muscle pyruvate dehydrogenase kinase. Biochem J. 1974 Dec;143(3):625–641. doi: 10.1042/bj1430625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Denton R. M., Randle P. J. Concentrations of glycerides and phospholipids in rat heart and gastrocnemius muscles. Effects of alloxan-diabetes and perfusion. Biochem J. 1967 Aug;104(2):416–422. doi: 10.1042/bj1040416. [DOI] [PMC free article] [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., Randle P. J. Measurement of flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol and glycerol in rat heart and epididymal adipose tissue. Effects of insulin, adrenaline and alloxan-diabetes. Biochem J. 1967 Aug;104(2):423–434. doi: 10.1042/bj1040423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. ERNSTER L., LEE C. P. BIOLOGICAL OXIDOREDUCTIONS. Annu Rev Biochem. 1964;33:729–790. doi: 10.1146/annurev.bi.33.070164.003501. [DOI] [PubMed] [Google Scholar]
  15. ESTABROOK R. W., MAITRA P. K. A fluorimetric method for the quantitative microanalysis of adenine and pyridine nucleotides. Anal Biochem. 1962 May;3:369–382. doi: 10.1016/0003-2697(62)90065-9. [DOI] [PubMed] [Google Scholar]
  16. Evans J. R., Opie L. H., Renold A. E. Pyruvate metabolism in the perfused rat heart. Am J Physiol. 1963 Nov;205(5):971–976. doi: 10.1152/ajplegacy.1963.205.5.971. [DOI] [PubMed] [Google Scholar]
  17. FRAENKEL G., FRIEDMAN S. Carnitine. Vitam Horm. 1957;15:73–118. doi: 10.1016/s0083-6729(08)60508-7. [DOI] [PubMed] [Google Scholar]
  18. Garland P. B., Newsholme E. A., Randle P. J. Regulation of glucose uptake by muscle. 9. Effects of fatty acids and ketone bodies, and of alloxan-diabetes and starvation, on pyruvate metabolism and on lactate-pyruvate and L-glycerol 3-phosphate-dihydroxyacetone phosphate concentration ratios in rat heart and rat diaphragm muscles. Biochem J. 1964 Dec;93(3):665–678. doi: 10.1042/bj0930665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Garland P. B., Randle P. J. Regulation of glucose uptake by muscles. 10. Effects of alloxan-diabetes, starvation, hypophysectomy and adrenalectomy, and of fatty acids, ketone bodies and pyruvate, on the glycerol output and concentrations of free fatty acids, long-chain fatty acyl-coenzyme A, glycerol phosphate and citrate-cycle intermediates in rat heart and diaphragm muscles. Biochem J. 1964 Dec;93(3):678–687. doi: 10.1042/bj0930678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Glynn I. M., Chappell J. B. A simple method for the preparation of 32-P-labelled adenosine triphosphate of high specific activity. Biochem J. 1964 Jan;90(1):147–149. doi: 10.1042/bj0900147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Halestrap A. P. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem J. 1975 Apr;148(1):85–96. doi: 10.1042/bj1480085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hjelmquist G., Andersson J., Edlund B., Engstroöm L. Amino acid sequence of a (32P) phosphopeptide from pig liver pyruvate kinase phosphorylated by cyclic 3',5'-AMP-stimulated protein kinase and gamma-(32P)ATP. Biochem Biophys Res Commun. 1974 Nov 27;61(2):559–563. doi: 10.1016/0006-291x(74)90993-0. [DOI] [PubMed] [Google Scholar]
  23. Häussinger D., Weiss L., Sies H. Activation of pyruvate dehydrogenase during metabolism of ammonium ions in hemoglobin-free perfused rat liver. Eur J Biochem. 1975 Apr 1;52(3):421–431. doi: 10.1111/j.1432-1033.1975.tb04010.x. [DOI] [PubMed] [Google Scholar]
  24. KLINGENBERG M., SCHOLLMEYER P. ATP controlled redox states of respiratory carriers under the influence of DPNH-hydrogen accepting substrates. Biochem Biophys Res Commun. 1961 Apr 7;4:323–327. doi: 10.1016/0006-291x(61)90211-x. [DOI] [PubMed] [Google Scholar]
  25. LaNoue K. F., Walajtys E. I., Williamson J. R. Regulation of glutamate metabolism and interactions with the citric acid cycle in rat heart mitochondria. J Biol Chem. 1973 Oct 25;248(20):7171–7183. [PubMed] [Google Scholar]
  26. Linn T. C., Pelley J. W., Pettit F. H., Hucho F., Randall D. D., Reed L. J. -Keto acid dehydrogenase complexes. XV. Purification and properties of the component enzymes of the pyruvate dehydrogenase complexes from bovine kidney and heart. Arch Biochem Biophys. 1972 Feb;148(2):327–342. doi: 10.1016/0003-9861(72)90151-8. [DOI] [PubMed] [Google Scholar]
  27. Linn T. C., Pettit F. H., Hucho F., Reed L. J. Alpha-keto acid dehydrogenase complexes. XI. Comparative studies of regulatory properties of the pyruvate dehydrogenase complexes from kidney, heart, and liver mitochondria. Proc Natl Acad Sci U S A. 1969 Sep;64(1):227–234. doi: 10.1073/pnas.64.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Linn T. C., Pettit F. H., Reed L. J. Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc Natl Acad Sci U S A. 1969 Jan;62(1):234–241. doi: 10.1073/pnas.62.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Martin B. R., Denton R. M., Pask H. T., Randle P. J. Mechanisms regulating adipose-tissue pyruvate dehydrogenase. Biochem J. 1972 Sep;129(3):763–773. doi: 10.1042/bj1290763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McAllister A., Allison S. P., Randle P. J. Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-hydroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo. Biochem J. 1973 Aug;134(4):1067–1081. doi: 10.1042/bj1341067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Randle P. J., Denton R. M., Pask H. T., Severson D. L. Calcium ions and the regulation of pyruvate dehydrogenase. Biochem Soc Symp. 1974;(39):75–88. [PubMed] [Google Scholar]
  33. Randle P. J., England P. J., Denton R. M. Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart. Biochem J. 1970 May;117(4):677–695. doi: 10.1042/bj1170677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Randle P. J., Garland P. B., Hales C. N., Newsholme E. A., Denton R. M., Pogson C. I. Interactions of metabolism and the physiological role of insulin. Recent Prog Horm Res. 1966;22:1–48. doi: 10.1016/b978-1-4831-9825-5.50004-x. [DOI] [PubMed] [Google Scholar]
  35. SCHREIBER G., KOHLHAW G., GOEDDE H. W., HOLZER H. DIE BIOSYNTHESE VON ACETOIN IN SCHWEINEHERZMUSKEL. Biochem Z. 1963 Oct 14;339:83–93. [PubMed] [Google Scholar]
  36. Severson D. L., Denton R. M., Bridges B. J., Randle P. J. Exchangeable and total calcium pools in mitochondria of rat epididymal fat-pads and isolated fat-cells. Role in the regulation of pyruvate dehydrogenase activity. Biochem J. 1976 Jan 15;154(1):209–223. doi: 10.1042/bj1540209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Severson D. L., Denton R. M., Pask H. T., Randle P. J. Calcium and magnesium ions as effectors of adipose-tissue pyruvate dehydrogenase phosphate phosphatase. Biochem J. 1974 May;140(2):225–237. doi: 10.1042/bj1400225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Slater E. C. The coupling between energy-yielding and energy-utilizing reactions in mitochondria. Q Rev Biophys. 1971 Feb;4(1):35–71. doi: 10.1017/s0033583500000391. [DOI] [PubMed] [Google Scholar]
  39. Soderling T. R., Park C. R. Recent advances in glycogen metabolism. Adv Cyclic Nucleotide Res. 1974;4(0):283–333. [PubMed] [Google Scholar]
  40. Stanley P. E. Determination of subpicomole levels of NADH and FMN using bacterial luciferase and the liquid scintillation spectrometer. Anal Biochem. 1971 Feb;39(2):441–453. doi: 10.1016/0003-2697(71)90434-9. [DOI] [PubMed] [Google Scholar]
  41. Stanley P. E., Williams S. G. Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem. 1969 Jun;29(3):381–392. doi: 10.1016/0003-2697(69)90323-6. [DOI] [PubMed] [Google Scholar]
  42. Stansbie D., Denton R. M., Bridges B. J., Pask H. T., Randle P. J. Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet. Biochem J. 1976 Jan 15;154(1):225–236. doi: 10.1042/bj1540225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. TABOR H., MEHLER A. H., STADTMAN E. R. The enzymatic acetylation of amines. J Biol Chem. 1953 Sep;204(1):127–138. [PubMed] [Google Scholar]
  44. Taylor S. I., Mukherjee C., Jungas R. L. Regulation of pyruvate dehydrogenase in isolated rat liver mitochondria. Effects of octanoate, oxidation-reduction state, and adenosine triphosphate to adenosine diphosphate ratio. J Biol Chem. 1975 Mar 25;250(6):2028–2035. [PubMed] [Google Scholar]
  45. Tsai C. S., Burgett M. W., Reed L. J. Alpha-keto acid dehydrogenase complexes. XX. A kinetic study of the pyruvate dehydrogenase complex from bovine kidney. J Biol Chem. 1973 Dec 25;248(24):8348–8352. [PubMed] [Google Scholar]
  46. Ullrich J., Mannschreck A. Studies on the properties of (--)-2-alpha-hydroxyethyl-thiamine pyrophosphate ("active acetaldehyde"). Eur J Biochem. 1967 Mar;1(1):110–116. doi: 10.1111/j.1432-1033.1967.tb00051.x. [DOI] [PubMed] [Google Scholar]
  47. WILLIAMSON J. R. GLYCOLYTIC CONTROL MECHANISMS. I. INHIBITION OF GLYCOLYSIS BY ACETATE AND PYRUVATE IN THE ISOLATED, PERFUSED RAT HEART. J Biol Chem. 1965 Jun;240:2308–2321. [PubMed] [Google Scholar]
  48. Walajtys E. I., Gottesman D. P., Williamson J. R. Regulation of pyruvate dehydrogenase in rat liver mitochondria by phosphorylation-dephosphorylation. J Biol Chem. 1974 Mar 25;249(6):1857–1865. [PubMed] [Google Scholar]
  49. Whitehouse S., Cooper R. H., Randle P. J. Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids. Biochem J. 1974 Sep;141(3):761–774. doi: 10.1042/bj1410761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Whitehouse S., Randle P. J. Activation of pyruvate dehydrogenase in perfused rat heart by dichloroacetate (Short Communication). Biochem J. 1973 Jun;134(2):651–653. doi: 10.1042/bj1340651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wieland O. H., Portenhauser R. Regulation of pyruvate-dehydrogenase interconversion in rat-liver mitochondria as related to the phosphorylation state of intramitochondrial adenine nucleotides. Eur J Biochem. 1974 Jun 15;45(2):577–588. doi: 10.1111/j.1432-1033.1974.tb03584.x. [DOI] [PubMed] [Google Scholar]
  52. Wieland O., Funcke H. v., Löffler G. Interconversion of pyruvate dehydrogenase in rat heart muscle upon perfusion with fatty acids or ketone bodies. FEBS Lett. 1971 Jul 1;15(4):295–298. doi: 10.1016/0014-5793(71)80641-5. [DOI] [PubMed] [Google Scholar]
  53. Wieland O., Siess E., Schulze-Wethmar F. H., von Funcke H. G., Winton B. Active and inactive forms of pyruvate dehydrogenase in rat heart and kidney: effect of diabetes, fasting, and refeeding on pyruvate dehydrogenase interconversion. Arch Biochem Biophys. 1971 Apr;143(2):593–601. doi: 10.1016/0003-9861(71)90244-x. [DOI] [PubMed] [Google Scholar]
  54. Wieland O., Von Jagow-Westermann B., Stukowski B. Kinetic and regulatory properties of heart muscle pyruvate dehydrogenase. Hoppe Seylers Z Physiol Chem. 1969 Mar;350(3):329–334. doi: 10.1515/bchm2.1969.350.1.329. [DOI] [PubMed] [Google Scholar]
  55. Wilson D. F., Erecinska M., Nicholls P. An energy-dependent transformation of a ferricytochrome of the mitochondrial respiratory chain. FEBS Lett. 1972 Jan 15;20(1):61–65. doi: 10.1016/0014-5793(72)80017-6. [DOI] [PubMed] [Google Scholar]

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