Neurochemical Research

, Volume 20, Issue 12, pp 1491–1501 | Cite as

Regulation of mitochondrial and cytosolic malic enzymes from cultured rat brain astrocytes

  • Mary C. McKenna
  • J. Tyson Tildon
  • J. H. Stevenson
  • Xueli Huang
  • Kenneth G. Kingwell
Original Articles


Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is converted to pyruvate and CO2 via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37°C in the presence of 0.5 mM malate was 4.15±0.47 and 11.61±0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean±SEM, n=18–19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. α-Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.

Key Words

Malate mitochondrial malic enzyme cytosolic malic enzyme astrocytes energy metabolism 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Lehninger, A. L. 1970. Biochemistry, Worth Publishers, Inc., New York.Google Scholar
  2. 2.
    Fitzpatrick, S. M., Cooper, A. J. L., and Duffy, T. E. 1983. Use of β-methylene-D, L-aspartate to assess the role of aspartate aminotransferase in cerebral oxidative metabolism. J. Neurochem. 41: 1370–1383.Google Scholar
  3. 3.
    Murthy, Ch. R. K., and Hertz, L. 1988. Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia. Neurochem. Res. 13:57–61.Google Scholar
  4. 4.
    Lai, J. C. K., Murthy, Ch. R. K., Cooper, A. J. L., Hertz, E., and Hertz, L. 1989. Differential effects of ammonia and β-methylene-D, L-aspartate on metabolism of glutamate and related amino acids by astrocytes and neurons in primary culture. Neurochem. Res. 14:377–389.Google Scholar
  5. 5.
    McKenna, M. C., Tildon, J. T., Couto, R., Stevenson, J. H., and Caprio, F. J. 1990. The metabolism of malate by cultured rat brain astrocytes. Neurochem. Res. 15:1211–1220.Google Scholar
  6. 6.
    Shank, R. P., and Campbell, G. Le. M. 1984. α-ketoglutarate and malate uptake and metabolism by synaptosomes: Further evidence for an astrocyte-to-neuron metabolic shuttle. J. Neurochem. 42: 1153–1161.Google Scholar
  7. 7.
    Shank, R. P., and Campbell, G. Le. M. 1984. Amino acid uptake, content and metabolism by neuronal and glial enriched cellular fractions from mouse cerebellum. J. Neurosci. 4:58–69.Google Scholar
  8. 8.
    Sonnewald, U., Westergaard, N., Hassel, B., Muller, T. B., Unsgard, G., Fonnum, F., Hertz, L., Schousboe, A., and Petersen, S. B. 1993. NMR spectroscopic studies of13C acetate and13C glucose metabolism in neocortical astrocytes: Evidence for mitochondrial heterogeneity. Develop. Neurosci. 15:351–358.Google Scholar
  9. 9.
    Salganicoff, L., and Koeppe, R. E. 1968. Subcellular distribution of pyruvate carboxylase, diphosphopyridine nucleotide and triphosphoryridine nucleotide isocitrate dehydrogenases and malate enzyme in rat brain. J. Biol. Chem. 243:3416–3420.Google Scholar
  10. 10.
    Frenkel, R. 1972. Isolation and some properties of a cytosol and a mitochondrial malic enzyme from bovine brain. Arch. Biochem. Biophys. 152:136–142.Google Scholar
  11. 11.
    McKenna, M. C., Tildon, J. T., Stevenson, J. H., and Kingwell, K. G. 1993. Regulation of astrocyte malic enzyme by metabolites. Transact. Am. Soc. Neurochem. 24, A54.Google Scholar
  12. 12.
    Zielke, H. R., Ozand, P. T., Tildon, J. T., Sevadalian, D. A., and Cornblath, M. 1978. Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts. J. Cell. Physiol. 95:41–48.Google Scholar
  13. 13.
    Tildon, J. T. 1983. Glutamine: A possible energy source for the brain. Pages 415–429, in Hertz, L., Kvamme, E., McGee, E. and Schousboe, A. (eds.), Metabolic Relationship Between Glutamine, Glutamate and GABA in the CNS, Alan R. Liss, New York.Google Scholar
  14. 14.
    Tildon, J. T., and Zielke, H. R. 1988. Glutamine: An energy source for mammalian tissues. Pages 167–182, in Kvamme, E. (ed.), Glutamine and Glutamate in Mammals. CRC Press, New York.Google Scholar
  15. 15.
    Sonnewald, U., Westergaard, N., Petersen, S. B., Unsgard, G., and Schousboe, A. 1993. Metabolism of [U-13C]glutamate in astrocytes studies by13C NMR spectroscopy: Incorporation of more label into lactate than into glutamine demonstrates the importance of the tricarboxylic acid cycle. J. Neurochem. 61:1179–1182.Google Scholar
  16. 16.
    Malik, P., McKenna, M. C., and Tildon, J. T. 1993. Regulation of malate dehydrogenases from neonatal, adolescent and mature rat brain. Neurochem. Res. 18:247–257.Google Scholar
  17. 17.
    McKenna, M. C., Tildon, J. T., Stevenson, J. H., Jr., Boatright, R., and Huang, S. 1993. Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: Differences revealed using aminooxyacetate. Develop. Neurosci. 15:320–329.Google Scholar
  18. 18.
    Booher, J., and Sensenbrenner, M. 1972. Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology 2:97–105.Google Scholar
  19. 19.
    Hertz, L., Juurlink, B. H. J., Fosmark, H., and Schousboe, A. 1982. Astrocytes in primary culture. Pages, 175–186, in Pfeiffer, S. E. (ed.), Neuroscience Approached Through Cell Culture, Vol. 1 CRC Press, Boca Raton.Google Scholar
  20. 20.
    Schousboe, A. 1988. Primary cultures of astrocytes from mammalian brain as a tool in neurochemical research. Cell Mol. Biol. 26:505–513.Google Scholar
  21. 21.
    Fedoroff, S., White, R., Subrahmanyan, L., and Kalnins, V. I. 1981. Properties of putative astrocytes in colony cultures of mouse neopallium. Pages 1–19, in Vidrio, E. A., and Fedoroff, S. (eds.), Eleventh International Congress of Anatomy. Glial and Neuronal Cell Biology. Alan R. Liss, New York.Google Scholar
  22. 22.
    Lai, J. C. K., and Clark, J. B. 1979. Preparation of synaptic and nonsynaptic mitochondria from the mammalian brain. Pages 51–59,in Fleischer, S., Packer, L. (eds.) Methods in Enzymology, Volume LV, Academy Press, New York.Google Scholar
  23. 23.
    Bergmeyer, H. U., and Bernt, E. 1974. Lactate dehydrogenase. Pages 574–579,in Bergmeyer, H. U. (ed.), Methods in Enzymatic Analysis, 2nd edition, Vol. 2, Academy Press, New York.Google Scholar
  24. 24.
    Pennington, R. J. 1961. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem. J. 80:649–654.Google Scholar
  25. 25.
    Clark, J. B., and Nicklas, W. J. 1970. The metabolism of rat brain mitochondria. J. Biol. Chem. 285:4724–4731.Google Scholar
  26. 26.
    Dennis, S. C., Lai, J. C. K., and Clark, J. B. 1977. Comparative studies on glutamate metabolism in synaptic and non synaptic rat brain mitochondria. Biochem. J. 164:727–736.Google Scholar
  27. 27.
    Hsu, R. Y., and Lardy, H. A. 1969. Malic enzyme. Pages 230–235, in Colwick, S. P., Kaplan, N. O. and Lowenstein, J. M., (eds.) Methods in Enzymology, Vol. 12, Academic Press, New York.Google Scholar
  28. 28.
    Smith, P. K., Khron, R. I. Hermanson, G. T., Mallia, A. K., Artner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., Klenk, D. C. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76–85.Google Scholar
  29. 29.
    Fitzpatrick, S. M., Cooper, A. J. L., and Hertz, L. 1988. Effects of ammonia and β-methylene-D, L-aspartate on the oxidation of glucose and pyruvate by neurons and astrocytes in primary culture. J. Neurochem. 51:1197–1203.Google Scholar
  30. 30.
    Snedecor, G. W., and Cochran W. G.: Statistical Methods. Iowa State University Press, Ames, Sixth Edition, 1967.Google Scholar
  31. 31.
    Mathews, C. K., and van Holde, K. E. 1990. Biochemistry. The Benjamin Cummings Publishing Company, Inc., Redwood City, pp. 381–401.Google Scholar
  32. 32.
    Zeffren, E., and Hall, P. L. 1973. Kinetics Ill-Reversible inhibition of enzyme action, in The Studh of Enzyme Mechanisms. John Wiley and Sons, New York, pp. 87–99.Google Scholar
  33. 33.
    Bukato, G., Kochan, Z., and Swierczynski, J. 1994. Subregional and intracellular distribution of NADP-linked malic enzyme in human brain. Biochem. Med. and Metabolic Biol. 51:43–50.Google Scholar
  34. 34.
    Yudkoff, M., Pleasure, D., Cregar, L., Lin, Z.-P., Nissim, I., Stern, J., and Nissim, I. 1990. Glutathione turnover in cultured astrocytes: Studies with [15N]glutamate. J. Neurochem. 55:137–145.Google Scholar
  35. 35.
    Makar, T. K., Nedergaard, M., Preuss, A., Gelbard, A. S., Perumal, A. S., and Cooper, A. J. L. 1994. Vitamin E, ascorbate glutathione, glutathione disulfide, and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons: Evidence that astrocytes play an important role in antioxidant processes in the brain. J. Neurochem. 62:45–53.Google Scholar
  36. 36.
    Jain, J., Martensson, J., Stole, E., Auld, P. A. M., and Meister, A. 1991. Glutathione deficiency leads to mitochondrial damage in brain. Proc. Nat. Acad. Sci. USA 88:1913–1917.Google Scholar
  37. 37.
    Fox, R. E., Kingwell, K. G., and Tildon, J. T. 1994. Malic enzyme activity in adult and newborn rat lung. Pediatr. Res. 35:589–593.Google Scholar
  38. 38.
    Cerdan, S., Kunnecke, B., and Seelig, J. 1990. Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro,13C NMR. J. Biol. Chem. 265:12916–12926.Google Scholar
  39. 39.
    McKenna, M. C., Sonnewald, U., Huang, X., Stevenson, J. H., and Zielke, H. R. 1996. Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J. Neurochem. (In Press).Google Scholar
  40. 40.
    Shank, R. P., and Aprison, M. H. 1988. Glutamate as a neurotransmitter. Pages 3–20,in Kvamme, E. (ed.), Glutamine and Glutamate in Mammals, Vol. II, CRC Press, Boca Raton.Google Scholar
  41. 41.
    Walz, W., and Mukerji, S. 1988. Lactate release from cultured astrocytes and neurons: A comparison. Glia 1:366–370.Google Scholar
  42. 42.
    Schurr, A., West, C. A., and Rigor, B. M. 1988. Lactate-supported synaptic function in the rat hippocampal slice preparation. Science 240:1326–1328.Google Scholar
  43. 43.
    Schurr, A., West, C. A., and Rigor, B. M. 1989. Electrophysiology of energy metabolism and neuronal function in the hippocampal slice preparation. J. Neurosci. Methods 28:7–13.Google Scholar
  44. 44.
    Bukato, G., Kochan, Z., and Swierczynski, J. 1992. Changes of malic enzyme activity in the developing rat brain are due to both the increase of mitochondrial protein content and the increase of specific activity. Int. J. Biochem. 24:267–273.Google Scholar
  45. 45.
    Martinez-Rodriguez, R., Arenas, G., Hidalgo, M. M., and Carnicero, M. B. 1989. Light and electron microscope immunolocalization of cytosolic malic enzyme-like activity in the rat's cerebellar cortex. J. Hirnforch. 30:291–296.Google Scholar
  46. 46.
    Kurz, G. M., Weisinger, H., and Hamprecht, B. 1993. Purification of cytosolic malic enzyme from bovine brain, generation of monoclonal antibodies, and immunohistochemical localization of the enzyme in glial cells of neural primary cultures. J. Neurochem. 60:1467–1474.Google Scholar
  47. 47.
    Saito, T., and Tomita, K. 1972. Malic enzyme activity in heart. J. Biochem. (Tokyo) 72:807–815.Google Scholar
  48. 48.
    Frenkel, R. 1975. Regulation and physiological functions of malic enzymes. Curr. Topics Cell Reg. 9:157–181.Google Scholar
  49. 49.
    Saito, T., and Tomita, K. 1973. Two types of soluble malic enzymes in rat tissues. J. Biochem. (Tokyo) 73:803–810.Google Scholar
  50. 50.
    Oh, Y. J., Markelonis, G. J., and Oh, T. H. 1991. Immunocytochemical localization of mitochondrial malate dehydrogenase in primary cultures of rat astrocytes and oligodendrocytes. J. Histochem. and Cytochem. 39:681–688.Google Scholar
  51. 51.
    Cesar, M., Schmoll, D., Hamprecht, B., Berg, P., Klein, R., and Bachmann, M. 1994. Co-localization of pyruvate carboxylase with pyruvate dehydrogenase in glial primary cultures and with fructose-1,6-bisphosphatase in brain slices. J. Neurochem. 63:S91B.Google Scholar
  52. 52.
    Sonnewald, U., Westergaard, N., Krane, J., Unsgard, G., Petersen, S. B., and Schousboe, A. 1991. First, direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes. Neurosci. Lett. 128: 235–239.Google Scholar
  53. 53.
    Westergaard, N., Sonnewald, U., Unsgard, G., Peng, L., Hertz, L., and Schousboe, A. 1994. Uptake, release and metabolism of citrate in neurons and astrocytes in primary cultures. J. Neurochem. 62:1727–1733.Google Scholar
  54. 54.
    Lai, J. C. K., Walsh, J. M., Dennis, S. C. and Clark, J. B. 1977. Synaptic and non-synaptic mitochondria from rat brain: Isolation and characterization. J. Neurochem. 28:625–631.Google Scholar
  55. 55.
    McKenna, M. C., Tildon, J. T., Stevenson, J. H., Hopkins, I. B., and Huang, X. 1994. Multiple metabolic compartments in rat brain astrocytes. Transact. Am. Soc. Neurochem. 25:A447.Google Scholar
  56. 56.
    Pryce, J. D., Gant, P. W., and Saul, K. J. 1970. Normal concentration of lactate, glucose and protein in the cerebral spinal fluid and the diagnostic implications of abnormal concentrations. Clin. Chem. 16:562–565.Google Scholar
  57. 57.
    Hawkins, R. A., and Mans, A. M. 1983. Intermediary metabolism of carbohydrates and other fuels. Pages 259–294, in Lajtha, A. (ed.) Handbook of Neurochemistry, Vol. 3, Second Edition, Plenum Press, New York.Google Scholar
  58. 58.
    Bachelard, H., Morris, P., and Taylor, A. 1994. Metabolism of U-[13C]glutamate to glutamine and lactate in cortical slices: an NMR study. J. Neurochem. 63:S48B.Google Scholar
  59. 59.
    Schousboe, A., Westergaard, N., Sonnewald, U., Petersen, S. B., Huang, R., Peng, L. and Hertz, L. 1993. Glutamate and glutamine metabolism and compartmentation in astrocytes. Develop. Neurosci. 15:359–366.Google Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Mary C. McKenna
    • 1
  • J. Tyson Tildon
    • 1
    • 2
  • J. H. Stevenson
    • 1
  • Xueli Huang
    • 1
  • Kenneth G. Kingwell
    • 1
  1. 1.Department of PediatricsUniversity of Maryland School of MedicineBaltimoreU.S.A.
  2. 2.Department of Biological ChemistryUniversity of Maryland School of MedicineBaltimoreU.S.A.

Personalised recommendations