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

Abstract

1H/ 15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2- 15N]glutamine, [5- 15N]glutamine, [2- 15N]glutamate, 15NH 4Cl, [2- 15N]alanine, and [1- 13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH 4 +, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/ 15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH 4 + but directly via an azaserine inhibitable amidotransfer reaction. In glutamine-free media 15NH 4 + was consumed and incorporated into alanine. 15NH 4 + was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1- 13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9–3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.