Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR

H-1/N-15 and C-13 NMR were used to investigate metabolism in Spodoptera fru giperda (Sf9) cells. Labelled substrates ([2-N-15]glutamine, [5-N-15]glutam ine, [2-N-15]glutamate, (NH4Cl)-N-15, [2-N-15]alanine, and [1-C-13]glucose) were added to batch cultures and the concentration of labelled excreted me tabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified . Cultures with excess glucose and glutamine produce alanine as the main me tabolic by-product while no ammonium ions are released. H-1/N-15 NMR data s howed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not tra nsferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amidotrans fer reaction. In glutamine-free media (NH4+)-N-15 was consumed and incorpor ated into alanine. (NH4+)-N-15 was also incorporated into the amide-positio n of glutamine synthesised by the cells. These data suggest that the nitrog en assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT ), is active in glutamine-deprived cells. In cultures devoid of glucose, am monium is the main metabolic by-product while no alanine is formed. The amm onium ions stem both from the amide and amine-nitrogen of glutamine, most l ikely via glutaminase and glutamate dehydrogenase. C-13 NMR revealed that t he [1-C-13] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates o f the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermo re, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) an d ethanol (6 mM), thus highlighting the importance of sinks for reducing eq uivalents in maintaining the cytosolic redox balance. (C) 2000 Elsevier Sci ence B.V. All rights reserved.