Inhibition of astrocyte glutamine production by α-ketoisocaproic acid

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

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5- 15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-15N]glutamine, representing the reductive amination of 2- oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]glutamate to [2-15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-15N]glutamine and stimulated the formation of [2-15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U-14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K(m) of glutamine synthetase for glutamate, which we determined to be ~7 mM.

Original languageEnglish (US)
Pages (from-to)1508-1515
Number of pages8
JournalJournal of Neurochemistry
Volume63
Issue number4
StatePublished - Oct 1994
Externally publishedYes

Fingerprint

Glutamine
Astrocytes
Acids
Glutamic Acid
Glutamate-Ammonia Ligase
Fluxes
Glutaminase
Amination
Glutamate Dehydrogenase
Citric Acid Cycle
Poisons
L-Lactate Dehydrogenase
Leucine
Labeling
Brain
Sheep
Atoms

Keywords

  • Glutamate
  • Glutamine synthetase
  • Leucine
  • Mass spectrometry
  • Stable isotopes

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Yudkoff, M., Daikhin, Y., Nissim, I., Pleasure, D. E., Stern, J., & Nissim, I. (1994). Inhibition of astrocyte glutamine production by α-ketoisocaproic acid. Journal of Neurochemistry, 63(4), 1508-1515.

Inhibition of astrocyte glutamine production by α-ketoisocaproic acid. / Yudkoff, Marc; Daikhin, Yevgeny; Nissim, Ilana; Pleasure, David E; Stern, Janet; Nissim, Itzhak.

In: Journal of Neurochemistry, Vol. 63, No. 4, 10.1994, p. 1508-1515.

Research output: Contribution to journalArticle

Yudkoff, M, Daikhin, Y, Nissim, I, Pleasure, DE, Stern, J & Nissim, I 1994, 'Inhibition of astrocyte glutamine production by α-ketoisocaproic acid', Journal of Neurochemistry, vol. 63, no. 4, pp. 1508-1515.
Yudkoff M, Daikhin Y, Nissim I, Pleasure DE, Stern J, Nissim I. Inhibition of astrocyte glutamine production by α-ketoisocaproic acid. Journal of Neurochemistry. 1994 Oct;63(4):1508-1515.
Yudkoff, Marc ; Daikhin, Yevgeny ; Nissim, Ilana ; Pleasure, David E ; Stern, Janet ; Nissim, Itzhak. / Inhibition of astrocyte glutamine production by α-ketoisocaproic acid. In: Journal of Neurochemistry. 1994 ; Vol. 63, No. 4. pp. 1508-1515.
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N2 - We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5- 15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-15N]glutamine, representing the reductive amination of 2- oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]glutamate to [2-15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-15N]glutamine and stimulated the formation of [2-15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U-14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K(m) of glutamine synthetase for glutamate, which we determined to be ~7 mM.

AB - We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5- 15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-15N]glutamine, representing the reductive amination of 2- oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]glutamate to [2-15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-15N]glutamine and stimulated the formation of [2-15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U-14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K(m) of glutamine synthetase for glutamate, which we determined to be ~7 mM.

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