Abstract
NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles.
Highlights
NAD is a key determinant of cellular energy metabolism
We have previously shown that the fluorescence lifetime of NADPH is identical to that of NADH in solution[19], demonstrating that fluorescence from the free, unbound pyridine nucleotides cannot be discriminated on the basis of their fluorescence lifetime
By combining the enzyme-bound NAD(P)H fluorescence lifetimes measured in the NADK þ and NADK À cells, the biochemically quantified [NADH] and [NADPH] values in each cell line[29] and a mathematical model in which NADH and NADPH were assumed to possess discrete and distinct fluorescence lifetimes when bound inside the cell, we found that tbound would describe the [NADPH]/[NADH] ratio by 1⁄2NADPH 1⁄4 tboundðnsÞ À 1:5 1⁄2NADH 4:4 À tboundðnsÞ
Summary
NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The nicotinamide adenine dinucleotide (NAD þ /NADH) and nicotinamide adenine dinucleotide phosphate (NADP þ /NADPH) redox couples are the major determinants of redox state in the cell The relative abundance of the two pyridine nucleotides and their redox balance mediates cell fate in a wide range of diseases, including cancer, diabetes and neurodegeneration Quantifying their behaviour is essential in understanding the role of metabolism in these diseases. Combined with computational and mathematical modelling, we have found that NAD(P)H fluorescence lifetime characteristics discriminate between NADH and NADPH This provides a unique approach to identify cells within complex tissues that are enriched in NADPH, raising questions about their metabolic roles and specialization. We have analysed the impact of altered metabolic state on NAD(P)H fluorescence decay characteristics, helping to place changes in lifetimes observed in transformed neoplastic cells on a firm biochemical footing
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