Reductively metabolized glutamine is a major carbon source for fatty acid synthesis during hypoxia or impaired mitochondrial respiration. Yet, a mechanistic understanding of the switch in flux direction and carbon source selection is missing. Here, we reveal the metabolic rearrangements that trigger reductive glutamine metabolism by measuring metabolite levels and metabolic flux contributions. We find that reductive glutamine metabolism is a consequence an increase in the a-ketoglutarate/citrate ratio. We identified various conditions where the a-ketoglutarate/citrate ratio was changed due to an altered acetyl-CoA to citrate conversion. Thereby, the alteration in acetyl-CoA to citrate conversion was regulated through at least two distinct mechanisms (pyruvate dehydrogenase kinase 1 activation (hypoxia), or decreased NAD+ to NADH ratio (complex I/III inhibition)). Thus, changes in the carbon source selection for fatty acid synthesis are a consequence of distinct alteration in the a-ketoglutarate/citrate ratio, caused by various perturbations.
