Figure 5.

Reductive carboxylation of glutamine depends on medium depth. (A) Schematic detailing mitochondrial pathways for carbons from anapleurotic glutamine. These carbons enter the Krebs cycle as α-ketoglutarate and undergo either oxidative decarboxylation or reductive carboxylation. Both metabolic routes lead to the formation of citrate. If all carbons on glutamine are ¹³C, reductive carboxylation leads to an m+5 citrate isotopologue, whereas oxidative decarboxylation leads to an m+4 citrate isotopologue. (B) In hfRPE culture, ¹³C₅-glutamine is marginally consumed at lower medium depths. (C, D) Tracing glutamine fate demonstrates that more is converted to citrate through oxidative decarboxylation at lower medium column heights (C) while more is converted to citrate through reductive carboxylation at higher medium column heights (D). (E) Quantification of oxidative versus reductive pathways from glutamine to citrate as a function of medium volume (n = 3, donor 6). Media pooled from apical and basal chambers for analysis.