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 NADdependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-NЈ-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-NЈ-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.

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The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly-and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.Several enzymes that transform NAD, used as a bona fide substrate, into metabolites displaying pleiotypic properties have been recently identified (1-4). Among them, ADP-ribose (ADPR) 2 transferases such as poly(ADP-ribose) polymerase (PARP)-1, -2, -3, -10, tankyrases, v-PARP, and others members of the same family are involved in processes such as nuclear homeostasis, cell differentiation, and death (5, 6). Mono(ADPribose) transferases (MART) are NAD-consuming enzymes present on cell membranes (but probably also in cytosol and organelles) that are still poorly understood (7). Additional NAD-consuming enzymes are CD-38/CD157, two ectoenzymes synthesizing ADPR, and the intracellular Ca 2ϩ -mobilizing compound cyclic ADPR (8), and sirtuins, a family of proteins encompassing seven members (Sirt1-7) involved in numerous processes such as chromatin homeostasis, transcription, cell death, and lifespan extension (9).Because of the constitutive activity of the NAD-consuming enzymes, eukaryotic cells evolved a rescue pathway leading to NAD resynthesis from nicotinamide (Nam). NAD rescue occurs in parallel to NAD neosynthesis from tryptophan or nicotinic acid (the so-called "kynurenine" and "Priess-Handler" pathways, respectively) (10, 11). Recently, work from the Brenner laboratory (12) identified nicotinamide riboside as an additional NAD precursor. The biochemical route of NAD rescue is composed by two enzymatic steps, the first operated by nicotinamide phosphoribosyltransferase (Nampt), forming nicotinamide mononucleotide from Nam and phosphoribosyl pyrophosphate, and the second driv...

Arginase 2, inducible-and endothelial-nitric-oxide synthase (iNOS and eNOS), indoleamine 2,3-dioxygenase (IDO) and TGF-beta, might impair immune functions in prostate cancer (PCA) patients. However, their expression was not comparatively analysed in PCA and benign prostatic hyperplasia (BPH). We evaluated the expression of these genes in PCA and BPH tissues. Seventy-six patients (42 BPH, 34 PCA) were enrolled. Arginase 2, eNOS and iNOS gene expression was similar in BPH and PCA tissues. TGF-beta1 gene expression was higher in BPH than in PCA tissues (p=0.035). IDO gene expression was more frequently detectable (p=0.00007) and quantitatively higher (p=0.00001) in PCA tissues than in BPH. IDO protein, expressed in endothelial cells from both BPH and PCA, was detectable in tumour cells in PCA showing evidence of high specific gene expression. In these patients, IDO gene expression correlated with kynurenine/tryptophan ratio in sera. Thus high expression of IDO gene is specifically detectable in PCA. 1 High expression of indoleamine 2,3-dioxygenase gene in prostate cancerChantal FEDER-MENGUS 1 * and Stephen WYLER 1 * , Tvrtko HUDOLIN 2 , Robin RUSZAT 1 , Lukas BUBENDORF 3 , Alberto CHIARUGI 4 , Maria PITTELLI 4 , Walter P. Seventy-six patients, 42 BPH and 34 PCA, were enrolled. Expression of arginase 2, eNOS and iNOS genes was similar in BPH and PCA tissues. TGF-β1 gene expression was significantly higher in BPH than in PCA tissues (p=0.035). In contrast IDO gene expression was more frequently detectable in PCA as compared to BPH (p=0.00007). Its extent was also significantly higher in PCA than in BPH (p=0.00001).Immunohistochemistry revealed IDO protein in endothelial cells from both BPH and PCA. However, in PCA showing evidence of high specific gene expression, IDO was detectable in tumor cells as well. In patients bearing these tumors, IDO gene expression correlated with kynurenine/tryptophan ratio in sera.These data indicate that while genes encoding Arginase 2, iNOS, eNOS and TGF-β immunosuppressive factors are expressed in PCA and BPH, high expression of IDO gene is only detectable in PCA.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a NAD-consuming enzyme with an emerging key role in epigenetic regulation of gene transcription. Although PARP-1 expression is characteristically restricted to the nucleus, a few studies report the mitochondrial localization of the enzyme and its ability to regulate organelle functioning. Here, we show that, despite exclusive nuclear localization of PARP-1, mitochondrial homeostasis is compromised in cell lines exposed to PARP-1 pharmacological inhibitors or small interfering RNA. PARP-1 suppression reduces integrity of mitochondrial DNA (mtDNA), as well as expression of mitochondria-encoded respiratory complex subunits COX-1, COX-2, and ND-2. Accordingly, PARP-1 localizes at promoters of nuclear genes encoding both the mtDNA repair proteins UNG1, MYH1, and APE1 and the mtDNA transcription factors TFB1M and TFB2M. It is noteworthy that poly(ADPribosyl)ation is required for nuclear gene expression of these mitochondrial proteins. Consistent with these findings, PARP-1 suppression impairs mitochondrial ATP production. Our results indicate that PARP-1 plays a central role in mitochondrial homeostasis by epigenetically regulating nuclear genes involved in mtDNA repair and transcription. These data might have important implications in pharmacology of PARP-1 inhibitors as well as clinical oncology and aging.