(584af) Cofactor Balance By Nicotinamide Nucleotide Transhydrogenase (NNT) Coordinates Reductive Carboxylation and Glucose Catabolism in the Tricarboxylic Acid (TCA) Cycle

Cancer and proliferating cells exhibit an increased demand for
glutamine-derived carbons to support anabolic processes. In addition,
reductive carboxylation of-ketoglutarate by isocitrate dehydrogenase
1 (IDH1) and 2 (IDH2) was recently shown to be a major
source of citrate synthesis from glutamine. The role of NAD(P)H/
NAD(P) cofactors in coordinating glucose and glutamine utilization
in the tricarboxylic acid (TCA) cycle is not well understood,
with the source(s) of NADPH for the reductive carboxylation reaction
remaining unexplored. Nicotinamide nucleotide transhydrogenase
(NNT) is a mitochondrial enzyme that transfers reducing
equivalents from NADH to NADPH. Using 13C isotopic tracing methods,
we show that knockdown of NNT inhibits the contribution of glutamine to the
TCA cycle and activates glucose catabolism in SkMel5 melanoma cells.
The increase in glucose oxidation partially occurred through pyruvate
carboxylase and rendered NNT knockdown cells more sensitive
to glucose deprivation. Importantly, knocking down NNT
inhibits reductive carboxylation in SkMel5 and 786-O renal carcinoma
cells. Overexpression of NNT is sufficient to stimulate glutamine
oxidation and reductive carboxylation, whereas it inhibits
glucose catabolism in the TCA cycle. These observations are supported
by an impairment of the NAD(P)H/NAD(P) ratios. Our
findings underscore the role of NNT in regulating central carbon
metabolism via redox balance, calling for other mechanisms that
coordinate substrate preference to maintain a functional TCA cycle.