title: Interrelationships of leucine and glutamate metabolism in cultured astrocytes.
abstract: The aim was to study the extent to which leucine furnishes alpha-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 alpha-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the "reverse" direction and intraastrocytic [glutamate] was reduced by approximately 50% in only 5 min. Extracellular concentrations of alpha-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 approximately 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 alpha-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary "buffering" of intracellular glutamate.
authors.affiliation: Division of Metabolism, Children's Hospital of Philadelphia, University of Pennsylvania, School of Medicine 19104.
authors.forename: M
authors.initials: M
authors.lastname: Yudkoff
authors.forename: Y
authors.initials: Y
authors.lastname: Daikhin
authors.forename: Z P
authors.initials: ZP
authors.lastname: Lin
authors.forename: I
authors.initials: I
authors.lastname: Nissim
authors.forename: J
authors.initials: J
authors.lastname: Stern
authors.forename: D
authors.initials: D
authors.lastname: Pleasure
authors.forename: I
authors.initials: I
authors.lastname: Nissim
mesh_terms: D000586:Amination; D000596:Amino Acids / Q000378:metabolism; D000597:Amino Acids, Branched-Chain / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002478:Cells, Cultured; D005110:Extracellular Space / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007651:Keto Acids / Q000378:metabolism; D007930:Leucine / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000596:Amino Acids; D000597:Amino Acids, Branched-Chain; D005971:Glutamates; D007651:Keto Acids; D018698:Glutamic Acid; D007930:Leucine