Pharmacological Effects of Exogenous NAD on Mitochondrial Bioenergetics, DNA Repair, and Apoptosis

• DOI: 10.1124/mol.111.073916
• Publication Date: 2011-09-15T10:02:49Z
• Authors: Maria Pittelli; Roberta Felici; Vanessa Pitozzi; Lisa Giovannelli; Elisabetta Bigagli; Francesca Cialdai; Giovanni Romano; Flavio Moroni; Alberto Chiarugi
• Keywords: 0301 basic medicine; DNA Repair; Apoptosis; Hep G2 Cells; NAD; Membrane Potentials; Mitochondria; Rats; POLY(ADP-RIBOSE) POLYMERASE-1 ACTIVITY; NICOTINAMIDE ADENINE-DINUCLEOTIDES; B-DRIVEN TRANSCRIPTION; CELL-DEATH; Mice; 03 medical and health sciences; Animals; Humans; Energy Metabolism; HeLa Cells

During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N'-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N'-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.