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Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

Scientific reports (2018-04-05)
Joe T Sharick, Peter F Favreau, Amani A Gillette, Sophia M Sdao, Matthew J Merrins, Melissa C Skala
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cholera Toxin from Vibrio cholerae, ≥90% (SDS-PAGE), lyophilized powder
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Lactate Dehydrogenase Activity Assay Kit, sufficient for 500 colorimetric tests
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Insulin from bovine pancreas, γ-irradiated, BioXtra, suitable for cell culture, potency: ≥20 units/mg (USP units), lyophilized powder
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Glucose-6-phosphate Dehydrogenase from baker's yeast (S. cerevisiae), Type XV, lyophilized powder, 200-400 units/mg protein (modified Warburg-Christian)
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PDH Activity Assay Kit, Sufficient for 100 Colorimetric tests
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