Interrelationships of leucine and glutamate metabolism in cultured astrocytes

Marc Yudkoff, Yevgeny Daikhin, Zhi Ping Lin, Ilana Nissim, Janet Stern, David E Pleasure, Itzhak Nissim

Research output: Contribution to journalArticle

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Abstract

The aim was to study the extent to which leucine furnishes α-NH2 groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2-15N]glutamine/[15N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 15N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [15N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α- ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the 'reverse' direction and intraastrocytic [glutamate] was reduced by ~50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was ~17 times greater than the rate of [1-14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of α-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary 'buffering' of intracellular glutamate.

Original languageEnglish (US)
Pages (from-to)1192-1202
Number of pages11
JournalJournal of Neurochemistry
Volume62
Issue number3
StatePublished - Mar 1994
Externally publishedYes

Fingerprint

Metabolism
Leucine
Astrocytes
Glutamic Acid
Amino Acids
Isoleucine
Nitrogen
Branched Chain Amino Acids
Valine
Glutamine
Alanine
Gas chromatography
Gas Chromatography-Mass Spectrometry
Labeling
Serine
Dilution
Mass spectrometry
Labels

Keywords

  • Astrocytes
  • Glutamate
  • Leucine
  • Mass spectrometry
  • Stable isotopes

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Yudkoff, M., Daikhin, Y., Lin, Z. P., Nissim, I., Stern, J., Pleasure, D. E., & Nissim, I. (1994). Interrelationships of leucine and glutamate metabolism in cultured astrocytes. Journal of Neurochemistry, 62(3), 1192-1202.

Interrelationships of leucine and glutamate metabolism in cultured astrocytes. / Yudkoff, Marc; Daikhin, Yevgeny; Lin, Zhi Ping; Nissim, Ilana; Stern, Janet; Pleasure, David E; Nissim, Itzhak.

In: Journal of Neurochemistry, Vol. 62, No. 3, 03.1994, p. 1192-1202.

Research output: Contribution to journalArticle

Yudkoff, M, Daikhin, Y, Lin, ZP, Nissim, I, Stern, J, Pleasure, DE & Nissim, I 1994, 'Interrelationships of leucine and glutamate metabolism in cultured astrocytes', Journal of Neurochemistry, vol. 62, no. 3, pp. 1192-1202.
Yudkoff M, Daikhin Y, Lin ZP, Nissim I, Stern J, Pleasure DE et al. Interrelationships of leucine and glutamate metabolism in cultured astrocytes. Journal of Neurochemistry. 1994 Mar;62(3):1192-1202.
Yudkoff, Marc ; Daikhin, Yevgeny ; Lin, Zhi Ping ; Nissim, Ilana ; Stern, Janet ; Pleasure, David E ; Nissim, Itzhak. / Interrelationships of leucine and glutamate metabolism in cultured astrocytes. In: Journal of Neurochemistry. 1994 ; Vol. 62, No. 3. pp. 1192-1202.
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N2 - The aim was to study the extent to which leucine furnishes α-NH2 groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2-15N]glutamine/[15N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 15N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [15N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α- ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the 'reverse' direction and intraastrocytic [glutamate] was reduced by ~50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was ~17 times greater than the rate of [1-14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of α-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary 'buffering' of intracellular glutamate.

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