Proliferating cells have a sustained high demand for regeneration of electron acceptors as nicotinamide dinucleotide (phosphate) (NAD(P)+/NAD(P)H) is involved in a number of critical redox reactions within cells. However, their analysis in living cells is still challenging. We propose that combining label-free NADH and NADPH fluorescence lifetime imaging (NAD(P)H-FLIM) and signal-enhanced nuclear magnetic resonance (NMR) spectroscopy allows new, deeper insights into changes in specific metabolic pathways in living cells. For proof of principle, NAD+-metabolism was perturbed by specific inhibition of the rate-limiting enzyme of the NAD+ “Salvage pathway” Nicotinamide phosphoribosyltransferase (NAMPT) by FK866 in RAMOS human lymphoma cells. FK866 treatment leads to NAD(H) reduction, followed by reduced RAMOS cell proliferation. The NAD(P)H-FLIM analysis revealed increased general NAD(P)H-dependent metabolic activity indicated by increased ratios of enzyme-bound to total NAD(P)H concentration upon NAMPT inhibition. More importantly, a marked reduction in lactate dehydrogenase (LDH) activity accompanied by NADPH oxidase activity increase is observed. Using signal-enhanced NMR spectroscopy a reduced flux of pyruvate to lactate catalyzed by LDH is detectable in real time in living cells. This strongly supports NAD(P)H-FLIM analysis and demonstrates that intervening in the NAD+ “Salvage pathway” can have specific and global consequences for cells. Our principle study shows how spatially-resolved metabolic imaging techniques, that is, NAD(P)H-FLIM, are complemented by real-time NMR, paving the way toward a comprehensive spatiotemporal understanding of metabolic pathways in living cells.
