Detection and pharmacological modulation of nicotinamide mononucleotide (NMN) in vitro and in vivo.
The emerging key role of NAD-consuming enzymes in cell biology has renewed the interest in NAD resynthesis through the rescue pathways. The first step of the nicotinamide-dependent NAD-rescue pathway is operated by nicotinamide phosphoribosyl transferase (NaPRT) forming nicotinamide mononucleotide (NMN). Because of the difficulties in measuring NMN, numerous open questions exist about the pathophysiological relevance of NaPRT and NMN itself. Here, we describe a new method of fluorimetric NMN detection upon derivatization of its alkylpyridinium group with acetophenone. By adopting this method, we analyzed the kinetics of nicotinamide-dependent NAD recycling in HeLa and U937 cells. Measurement of NMN contents in subcellular fractions revealed that the nucleotide is highly enriched in mitochondria, suggesting intramitochondrial NAD synthesis. NMN increases in cells undergoing hyperactivation of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1, or exposed to gallotannin, a putative inhibitor of NMN-adenylyl transferases. Evidence that the inhibitor of NAD resynthesis FK866 selectively inhibits NaPRT having no effect on NMNAT activity is also provided. Importantly, NMN reduces NAD and ATP depletion in cells undergoing PARP-1 hyperactivation, significantly delaying cell death. Finally, we show that a single injection of FK866 in the mouse induces long-lasting (up to 16 h) but mild (approximately 20%) reduction of NMN contents in different organs, suggesting slow rate of basal NAD consumption in vivo. Data provide new information on the biochemistry and pharmacology of NAD biosynthesis, allowing a better understanding of pyridine nucleotide metabolism.
Department of Preclinical and Clinical Pharmacology, University of Florence, Italy.
Laura
L
Formentini
Flavio
F
Moroni
Alberto
A
Chiarugi
D000098:Acetophenones / Q000494:pharmacology; D000178:Acrylamides / Q000494:pharmacology*; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D016207:Cytokines / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051379:Mice; D009243:NAD / Q000096:biosynthesis*; D009536:Niacinamide / Q000494:pharmacology; D009537:Nicotinamide Mononucleotide / Q000378:metabolism*; D054409:Nicotinamide Phosphoribosyltransferase / Q000037:antagonists & inhibitors*; D009928:Organ Specificity; D010880:Piperidines / Q000494:pharmacology*; D020298:U937 Cells
D000098:Acetophenones; D000178:Acrylamides; D016207:Cytokines; D004791:Enzyme Inhibitors; D005561:Formates; C480543:N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide; D010880:Piperidines; D009243:NAD; C030544:formic acid; D009537:Nicotinamide Mononucleotide; D009536:Niacinamide; D054409:Nicotinamide Phosphoribosyltransferase; C085455:nicotinamide phosphoribosyltransferase, human; C038699:acetophenone