INHIBITION OF ASTROCYTE GLUTAMINE PRODUCTION BY ALPHA-KETOISOCAPROIC ACID

We have evaluated the effect of alpha-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used (NH4Cl)-N-15 as a metabolic tracer to measure the production of both [5-N-15]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-N-15]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [N-15]glutamate to [2-N-15]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-N-15]glutamine and stimulated the formation of [2-N-15]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-N-15]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-N-15]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through 15 glutaminase, which was measured using [2-N-15]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 (CO2)-C-14, from [U-C-14]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic alpha-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 similar to 7 mM.