Fig. 6: KISS1R expression promotes glutaminolysis, increases glutamine flux to tricarboxylic acid (TCA) cycle metabolites, and stimulates glutamine-dependent nucleotide synthesis.

a Production rate of glutamate measured in the conditioned media after 4 or 16 h of culturing SKBR3pFLAG controls or SKBR3FLAG-KISS1R cells grown in serum-free media (unlabeled). b Schematic showing the transfer of carbon atoms of the ¹³C₅-labeled glutamine tracer used to detect glutamine flux into TCA cycle intermediates; labeled carbon atoms (red) and unlabeled carbon atoms (white). c Glutamate pool size from ¹³C₅ glutamine tracer in cells. d–h Relative fractions of ¹³C₅ glutamine tracer in TCA cycle metabolites d citrate, e aconitate, f fumarate, and g malate. Normalization of pool size was done to pack cell volume per dish. Mean ± SEM shown (n = 3). *p < 0.05, Student’s unpaired t test. h Schematic of the pyrimidine synthesis pathway showing the contribution of the amide nitrogen using a ¹⁵N-glutamine tracer. Diamonds indicate nitrogen, labeled for amide (red) and unlabeled (white). Diamond with subscript α denotes the alpha nitrogen of glutamine. i Representative western blot of the CAD enzyme complex in lysates from SKBR3FLAG-KISS1R and SKBR3pFLAG vector control cells. See Supplementary Fig. 2f for quantification of blots. Fraction of glutamine’s amide nitrogen contributing to pyrimidine synthesis metabolites: j UMP and k uracil in cultured SKBR3FLAG-KISS1R and SKBR3pFLAG control cells (n = 3). Levels of pyrimidine synthesis metabolites measured by LC–MS in orthotopic breast tumors and serum from xenografts: l serum uridine; primary tumor (m), UTP (n) ribose phosphate. Student’s unpaired t test, *p < 0.05. (n = 4 mice for SKBR3FLAG-KISS1R xenografts; n = 3 for SKBR3pFLAG control xenografts).