The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Maja L. Johansen, Lasse K. Bak, Arne Schousboe, Peter Iversen, Michael Sørensen, Susanne Keiding, Hendrik Vilstrup, Albert Gjedde, Peter Ott, Helle S. Waagepetersen

Research output: Contribution to journalArticle

Abstract

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and α-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the α-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the α-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-13C]glucose (2.5 mM) and isoleucine (1 mM) or [U-13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-13C]glucose. Labeling in glutamate and aspartate from [U-13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-13C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.

Original languageEnglish (US)
Pages (from-to)1042-1051
Number of pages10
JournalNeurochemistry International
Volume50
Issue number7-8
DOIs
StatePublished - Jun 2007
Externally publishedYes

Fingerprint

Isoleucine
Ammonia
Astrocytes
Ammonium Compounds
Neurons
Glutamic Acid
Citric Acid Cycle
Glutamine
Aspartic Acid
Glucose
Pyruvic Acid
Hyperammonemia
Oxidoreductases
Skeleton
Alanine
Carbon
Ammonium Chloride
Branched Chain Amino Acids
Glutamate Dehydrogenase
Glutamate-Ammonia Ligase

Keywords

  • Aspartate
  • Energy
  • Glucose
  • Glutamate
  • Glutamine
  • Hepatic encephalopathy
  • Metabolism
  • TCA-cycle

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Johansen, M. L., Bak, L. K., Schousboe, A., Iversen, P., Sørensen, M., Keiding, S., ... Waagepetersen, H. S. (2007). The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes. Neurochemistry International, 50(7-8), 1042-1051. https://doi.org/10.1016/j.neuint.2007.01.009

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes. / Johansen, Maja L.; Bak, Lasse K.; Schousboe, Arne; Iversen, Peter; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Gjedde, Albert; Ott, Peter; Waagepetersen, Helle S.

In: Neurochemistry International, Vol. 50, No. 7-8, 06.2007, p. 1042-1051.

Research output: Contribution to journalArticle

Johansen, ML, Bak, LK, Schousboe, A, Iversen, P, Sørensen, M, Keiding, S, Vilstrup, H, Gjedde, A, Ott, P & Waagepetersen, HS 2007, 'The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes', Neurochemistry International, vol. 50, no. 7-8, pp. 1042-1051. https://doi.org/10.1016/j.neuint.2007.01.009
Johansen, Maja L. ; Bak, Lasse K. ; Schousboe, Arne ; Iversen, Peter ; Sørensen, Michael ; Keiding, Susanne ; Vilstrup, Hendrik ; Gjedde, Albert ; Ott, Peter ; Waagepetersen, Helle S. / The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes. In: Neurochemistry International. 2007 ; Vol. 50, No. 7-8. pp. 1042-1051.
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AU - Bak, Lasse K.

AU - Schousboe, Arne

AU - Iversen, Peter

AU - Sørensen, Michael

AU - Keiding, Susanne

AU - Vilstrup, Hendrik

AU - Gjedde, Albert

AU - Ott, Peter

AU - Waagepetersen, Helle S.

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N2 - Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and α-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the α-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the α-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-13C]glucose (2.5 mM) and isoleucine (1 mM) or [U-13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-13C]glucose. Labeling in glutamate and aspartate from [U-13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-13C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.

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KW - Glutamine

KW - Hepatic encephalopathy

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