Quantitative analysis of redox metabolism

Abstract

The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a significant role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging and in certain diseases, triggering intense interest in strategies to boost NAD levels, most notably through nicotinamide riboside (NR) and mononucleotide (NMN). A limitation in understanding NAD metabolism has been reliance on steady-state concentration measurements. Here, we established methods for NAD flux quantitation using stable isotope tracers combined with mathematical modeling. Cultured cells took nicotinamide (NAM) as the predominant NAD source. We showed that mitochondria directly import NAD and generate NAD from nicotinamide-containing nucleotides, but not from NAM. In vivo, NAD was made from tryptophan selectively in liver, which then excreted NAM. NAD fluxes varied widely across tissues, with high flux in small intestine and spleen and low flux in skeletal muscle. We also showed that intravenous, but not oral administration of NR or NMN delivered intact molecules to multiple tissues, with skeletal muscle displaying a preference for NR. In cell lines, newly synthesized NAD was consumed largely by PARPs and sirtuins. NAD kinase, which accounts for <10% of total NAD production, makes the anabolic and redox defense cofactor NADP(H). We further developed the quantitative tracing method to measure NADPH fluxes. While growing cells produce NADPH via the pentose phosphate pathway and folate metabolism which also make nucleotide precursors, we found that in differentiating adipocytes, a metabolic cycle involving malic enzyme make both NADPH and two-carbon units for fat synthesis. This study enables dissection of the production and consumption routes of redox cofactors across cells under different environmental conditions and murine tissues, and thus provides a novel window into redox metabolism.