INHIBITION OF ASTROCYTE GLUTAMINE PRODUCTION BY ALPHA-KETOISOCAPROIC ACID

We have evaluated the effect of alpha-ketoisocaproic acid (KIC), the k etoacid of leucine, on the production of glutamine by cultured astrocy tes. We used (NH4Cl)-N-15 as a metabolic tracer to measure the product ion of both [5-N-15]glutamine, reflecting amidation of glutamate via g lutamine synthetase, and [2-N-15]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequen t 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 an d stimulated the formation of [2-N-15]glutamine with the overall resul t being a significant inhibition of net glutamine synthesis. An extern al 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% exces s) of [2-N-15]glutamine. Higher concentrations of KIC in the medium ha d correspondingly larger effects. The presence of KIC in the medium di d not affect flux through 15 glutaminase, which was measured using [2- N-15]glutamine as a tracer. Nor did KIC inhibit the activity of glutam ine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from t he 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 tha t 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, wi th the exception of hydroxypyruvate. The data indicate that KIC dimini shes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K-m of glutamine synthetase for glu tamate, which we determined to be similar to 7 mM.