Abstract
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 multiphoton 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 fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ2) 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 τ2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.
Highlights
Reduced nicotinamide adenine dinucleotide (NADH) is a fluorescent electron donor that binds to metabolic enzymes in the cytoplasm and mitochondria
We used NAD(P)H-enzyme solutions, metabolic inhibitors in cells, and alternate fuel sources for cells to determine if NAD(P)H Fluorescence lifetime imaging microscopy (FLIM) is sensitive to key enzymatic steps that control the path of carbon from glucose uptake to electron transport chain (ETC) activity
The measured lifetimes of NADH bound to lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in solution were distinct from each other (Fig. 1) and only slightly different than previously reported values[22,36]
Summary
Reduced nicotinamide adenine dinucleotide (NADH) is a fluorescent electron donor that binds to metabolic enzymes in the cytoplasm and mitochondria. Many studies have shown that NAD(P)H FLIM can detect changes in flux through glycolysis, the TCA cycle and ETC9,25,27,36–41 It remains unclear whether NAD(P)H FLIM can be used to detect metabolic alterations at other key enzymatic steps that control the path of carbon from glucose uptake to ETC activity. These carbon-diverting steps include the pentose phosphate pathway (PPP) via G6PDH, lactate production via LDH, and inhibited entry into the TCA cycle via pyruvate dehydrogenase kinase (PDK) (PDK inhibits PDH activity). Our results indicate that the fluorescence lifetime of protein-bound NAD(P)H is more sensitive to these key enzymatic steps than the NAD(P)H intensity, free NAD(P)H lifetime, or relative amounts of free and protein-bound NAD(P)H
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