While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as a fuel source. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ_2) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ_2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.
It remains unclear whether NAD(P)H FLIM is sensitive to potential fates of glucose other than the TCA cycle and electron transport chain. We show that the lifetime of protein-bound NAD(P)H (τ_2) can distinguish the relative concentrations of malate dehydrogenase (TCA cycle enzyme) and lactate dehydrogenase (diverts carbon to/from lactate production) in solutions containing both enzymes. In cells, NAD(P)H FLIM was also sensitive to alterations in the path of carbon from glucose uptake to mitochondrial activity. Additionally, τ_2 was found to be the FLIM parameter best-suited for detecting these carbon-diverting shifts in cell metabolism, which may support mechanisms of drug resistance.