Para-hydrogen induced polarization NMR and NAD(P)H-fluorescence lifetime imaging synergistically reveal enzymatic and thus metabolic changes in human tumor cells upon NAMPT inhibition: a proof-of-principle.

Proliferating cells have a sustained high demand for regeneration of electron acceptors as 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 label-free fluorescence lifetime imaging of Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H-FLIM) and signal-enhanced magnetic resonance (MR) spectroscopy synergize to study metabolic pathways in cells. RAMOS cells were analyzed by inhibiting Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ „Salvage pathway“ by FK866. FK866 treatment leads to NAD+ reduction followed by reduced 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. Additionally, we found a marked NADPH oxidase activity increase and reduced lactate dehydrogenase (LDH) activity. This change in LDH activity is confirmed by reduced flux of signal-enhanced pyruvate to lactate conversion measured in real time by MR. Our proof-of-principle study shows how spatially-resolved metabolic imaging techniques, i.e. NAD(P)H-FLIM, are complemented by real-time MR, paving the way towards a comprehensive spatio-temporal understanding of metabolic pathways in living cells.