Separating NADH and NADPH fluorescence in live cells and tissues using FLIM

TY - JOUR

T1 - Separating NADH and NADPH fluorescence in live cells and tissues using FLIM

AU - Blacker, Thomas S

AU - Mann, Zoe F

AU - Gale, Jonathan E

AU - Ziegler, Mathias

AU - Bain, Angus J

AU - Szabadkai, Gyorgy

AU - Duchen, Michael R

PY - 2014/5/29

Y1 - 2014/5/29

N2 - 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.

AB - 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.

KW - Animals

KW - Cochlea/chemistry

KW - Energy Metabolism

KW - Fluorescence

KW - Glycolysis

KW - HEK293 Cells

KW - Humans

KW - NAD/analysis

KW - NADP/analysis

KW - Optical Imaging/methods

KW - Oxidation-Reduction

KW - Rats

U2 - 10.1038/ncomms4936

DO - 10.1038/ncomms4936

M3 - Article

C2 - 24874098

SN - 2041-1723

VL - 5

SP - 3936

JO - Nature Communications

JF - Nature Communications

ER -