Littérature scientifique sur le sujet « NAD, NAMPT, NMNAT2 »

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.

Metabolic dysregulation underlies malignant phenotypes attributed to cancer stem cells, such as unlimited proliferation and differentiation blockade. Here, we demonstrate that NAD+ metabolism enables acute myeloid leukemia (AML) to evade apoptosis, another hallmark of cancer stem cells. We integrated whole-genome CRISPR screening and pan-cancer genetic dependency mapping to identify NAMPT and NMNAT1 as AML dependencies governing NAD+ biosynthesis. While both NAMPT and NMNAT1 were required for AML, the presence of NAD+ precursors bypassed the dependence of AML on NAMPT but not NMNAT1, pointing to NMNAT1 as a gatekeeper of NAD+ biosynthesis. Deletion of NMNAT1 reduced nuclear NAD+, activated p53, and increased venetoclax sensitivity. Conversely, increased NAD+ biosynthesis promoted venetoclax resistance. Unlike leukemia stem cells (LSCs) in both murine and human AML xenograft models, NMNAT1 was dispensable for hematopoietic stem cells and hematopoiesis. Our findings identify NMNAT1 as a previously unidentified therapeutic target that maintains NAD+ for AML progression and chemoresistance.

The homeostasis of NAD+ anabolism is indispensable for maintaining the NAD+ pool. In mammals, the mainly synthetic pathway of NAD+ is the salvage synthesis, a reaction catalyzed by nicotinamide mononucleotide adenylyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNATs) successively, converting nicotinamide (NAM) to nicotinamide mononucleotide (NMN) and NMN to NAD+, respectively. However, the relationship between NAD+ anabolism disturbance and diabetic nephropathy (DN) remains elusive. Here our study found that the disruption of NAD+ anabolism homeostasis caused an elevation in both oxidative stress and fibronectin expression, along with a decrease in Sirt1 and an increase in both NF-κB P65 expression and acetylation, culminating in extracellular matrix deposition and globular fibrosis in DN. More importantly, through constitutively overexpressing NMNAT1 or NAMPT in human mesangial cells, we revealed NAD+ levels altered inversely with NMN levels in the context of DN and, further, their changes affect Sirt1/NF-κB P65, thus playing a crucial role in the pathogenesis of DN. Accordingly, FK866, a NAMPT inhibitor, and quercetin, a Sirt1 agonist, have favorable effects on the maintenance of NAD+ homeostasis and renal function in db/db mice. Collectively, our findings suggest that NMN accumulation may provide a causal link between NAD+ anabolism disturbance and diabetic nephropathy (DN) as well as a promising therapeutic target for DN treatment.

Abstract Objectives We demonstrated that ethanol decreased cellular NAD+ levels not only by consuming it but by repressing the expression of genes involved in the NAD+ salvage pathway, consequently altering cellular bioenergetics in macrophages. Therefore, we explored whether nicotinamide riboside (NR), an NAD+ precursor, could restore cellular NAD + and reverse changes in glycolytic capacities triggered by ethanol alone or ethanol with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway. Methods RAW 264.7 macrophages were treated with 80 mM ethanol with or without 1 mM of NR and 50 nM FK866 for 72 h. Expression of genes involved in the NAD+ salvage pathway and glycolysis and cellular NAD+ levels were measured. Parameters related to glycolysis were determined using a Seahorse XFe24 Extracellular Flux analyzer. Sirtuin 1 (SIRT1) was inhibited by its inhibitor sirtinol or siRNA-mediated knockdown or activated by resveratrol to evaluate its contribution to the effects of ethanol on the NAD+ salvage pathway. Results NR significantly increased, but NAMPT inhibition decreased cellular NAD+ content in control and ethanol-treated cells. However, ethanol decreased, and NAMPT inhibition further lowered NAD+ content. Decreases in cellular NAD+ and mRNA levels of genes in the NAD+ salvage pathway, such as Nmrk1, Nampt, Nmnat1, and Nmnat3, by ethanol and FK866 were attenuated by NR. Interestingly, both NR and FK866 diminished increases in glycolytic capacity, glycolytic reserve, and non-glycolytic acidification caused by ethanol. Ethanol increased the expression and activity of hypoxia-inducible factor 1α (HIF1α) and glycolytic genes, and pyruvate dehydrogenase (PDH) phosphorylation, all of which were attenuated by NR and FK866, with the latter being more potent. Ethanol and FK866, to a much greater extent, repressed Sirt1 expression. SIRT1 inhibition reduced Nampt expression, but SIRT1 activation and NR led to the opposite effects in ethanol-exposed macrophages. Conclusions NR attenuates decreases in cellular NAD + content and glycolytic capacities by ethanol alone or ethanol with NAMPT inhibition at least partly through the activation of the SIRT1 pathway in macrophages. Funding Sources University of Connecticut College of Agriculture, Health and Natural Resources & USDA multistate Hatch W4002.

Acute myeloid leukemia (AML) is primarily a disease of older adults with poor treatment outcomes. Despite years of intensive research, the standard induction therapy for AML has remained largely unchanged for decades. Thus, the development of new and efficacious therapeutic targets for AML is urgently needed. Leukemia cells exhibit multiple metabolic aberrations that may be therapeutically targeted. Here, we show that nicotinamide adenine dinucleotide (NAD+) promotes leukemogenesis and causes chemotherapy treatment resistance through fueling energetic metabolism, and pinpoints nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) is a novel therapeutic target for AML. To identify novel genes essential for AML, we performed a whole genome CRISPR dropout screen by using MOLM13 cell line and identified 1,951 essential genes (Fig. A). By searching druggable targets among these genes, we narrowed down to 345 genes, among which we found two genes, NMNAT1 (nicotinamide nucleotide adenylyltransferase 1) and NAMPT (nicotinamide phosphoribosyltransferase), both involved in key steps in NAD+ biosynthesis. We comprehensively analyzed dependency scores for all genes involved in the NAD+ biosynthetic pathways (de novo synthesis pathway, the Preiss-Handler pathway and the salvage pathway) across a broad panel of cancer cell lines from the Dependency Map database (https://depmap.org/portal/). The results showed that NMNAT1 and NAMPT are both strongly selective and uniquely required for hematological malignancies compared to other cancers (Fig. B). Since little success has been achieved for NAMPT inhibitors in clinical trials, our attention was drawn to NMNAT1, which encodes a nuclear localized enzyme catalyzing the final step in NAD+ biosynthesis. We confirmed that deletion of NMNAT1 in AML cells significantly reduced nuclear NAD+ level and cell viability over time while sparing normal hematopoietic progenitor cells, suggesting that NMNAT1 is targetable to AML. Overexpression of wild-type Nmnat1 but not the enzymatically inactive forms rescued NMNAT1-KO AML, indicating that the catalytic activity of NMNAT1 is required for AML. To study the role of NAD+ in AML, we first measured NAD+ levels in leukemic and normal cells, and found higher NAD+ levels in leukemia-initiating cells from a murine MLL-AF9-induced AML model compared to normal cells. Supplementation of NAD+ metabolites (NMN, NAM and NR) increased AML proliferation, enhanced glycolysis (lactate production) and oxidative phosphorylation (ATP production), resulting in chemotherapy resistance (Fig. C). Deletion of NMNAT1 sensitized AML cell to chemotherapy treatment. To study the role of NMNAT1 in leukemogenesis in vivo, we genetically deleted NMNAT1 in murine or human leukemia cells, transplanted them into recipient mice, and found that deletion of NMNAT1 reduced leukemic burden and extended leukemia-free survival (Fig. D). Finally, to reveal the molecular mechanisms underlying NMNAT1 KO-mediated cell death (increased levels of gamma-H2AX), RNA-seq and functional assay of NAD+ dependent enzymes were performed. We found that the reduction of nuclear NAD+ resulting from NMNAT1 deletion upregulated genes involved in DNA repair pathway, which may be linked to impaired PARPs and Sirtuins activity. Our findings reveal the important function of NAD+ in leukemogenesis and chemoresistance, and identify NMANT1 as a novel therapeutic target for AML. Figure Disclosures No relevant conflicts of interest to declare.

Sprint interval training (SIT) is reported to improve blood glucose control and may be a useful public health tool. The sirtuins and associated genes are emerging as key players in blood glucose control. This study investigated the interplay between the sirtuin/NAD system and individual variation in insulin sensitivity responses after SIT in young healthy individuals. Before and after 4 weeks of SIT, body mass and fat percentage were measured and oral glucose tolerance tests performed in 20 young healthy participants (7 females). Blood gene expression profiles (all 7 mammalian sirtuin genes and 15 enzymes involved in conversion of tryptophan, bioavailable vitamin B3, and metabolic precursors to NAD). NAD/NADP was measured in whole blood. Significant reductions in body weight and body fat post-SIT were associated with altered lipid profiles, NAD/NADP, and regulation of components of the sirtuin/NAD system (NAMPT, NMNAT1, CD38, and ABCA1). Variable improvements in measured metabolic health parameters were evident and attributed to different responses in males and females, together with marked inter-individual variation in responses of the sirtuin/NAD system to SIT.

Mitochondrial sirtuins have diverse role specifically in aging, metabolism and cancer. In cancer, these sirtuins play dichotomous role as tumor suppressor and promoter. Previous studies have reported the involvement of sirtuins in different cancers. However, till now no study has been published with respect to mitochondrial sirtuins and glioma risks. Present study was purposed to figure out the expression level of mitochondrial sirtuins (SIRT3, SIRT4, SIRT5) and related genes (GDH, OGG1-2α, SOD1, SOD2, HIF1α and PARP1) in 153 glioma tissue samples and 200 brain tissue samples from epilepsy patients (taken as controls). To understand the role of selected situins in gliomagenesis, DNA damage was measured using the comet assay and oncometabolic role (oxidative stress level, ATP level and NAD level) was measured using the ELISA and quantitative PCR. Results analysis showed significant down-regulation of SIRT4 (p = 0.0337), SIRT5 (p<0.0001), GDH (p = 0.0305), OGG1-2α (p = 0.0001), SOD1 (p<0.0001) and SOD2 (p<0.0001) in glioma patients compared to controls. In case of SIRT3 (p = 0.0322), HIF1α (p = 0.0385) and PARP1 (p = 0.0203), significant up-regulation was observed. ROC curve analysis and cox regression analysis showed the good diagnostic and prognostic value of mitochondrial sirtuins in glioma patients. Oncometabolic rate assessment analysis showed significant increased ATP level (p<0.0001), NAD+ level [(NMNAT1 (p<0.0001), NMNAT3 (p<0.0001) and NAMPT (p<0.04)] and glutathione level (p<0.0001) in glioma patients compared to controls. Significant increased level of damage ((p<0.04) and decrease level of antioxidant enzymes include superoxide dismutase (SOD, p<0.0001), catalase (CAT, p<0.0001) and glutathione peroxidase (GPx, p<0.0001) was observed in patients compared to controls. Present study data suggest that variation in expression pattern of mitochondrial sirtuins and increased metabolic rate may have diagnostic and prognostic significance in glioma patients.

BackgroundThe activation of NAD+-dependent deacetylase, Sirt1, by the administration of nicotinamide mononucleotide (NMN) ameliorates various aging-related diseases.MethodsDiabetic db/db mice were treated with NMN transiently for 2 weeks and observed for effects on diabetic nephropathy (DN).ResultsAt 14 weeks after the treatment period, NMN attenuated the increases in urinary albumin excretion in db/db mice without ameliorating hemoglobin A1c levels. Short-term NMN treatment mitigated mesangium expansion and foot process effacement, while ameliorating decreased Sirt1 expression and increased claudin-1 expression in the kidneys of db/db mice. This treatment also improved the decrease in the expression of H3K9me2 and DNMT1. Short-term NMN treatment also increased kidney concentrations of NAD+ and the expression of Sirt1 and nicotinamide phosphoribosyltransferase (Nampt), and it maintained nicotinamide mononucleotide adenyltransferase1 (Nmnat1) expression in the kidneys. In addition, survival rates improved after NMN treatment.Conclusions:Short-term NMN treatment in early-stage DN has remote renal protective effects through the upregulation of Sirt1 and activation of the NAD+ salvage pathway, both of which indicate NMN legacy effects on DN.

Obesity has become a serious problem in public health worldwide, causing numerous metabolic diseases. Once the differentiation to mature adipocytes is disrupted, adipocyte hypertrophy and ectopic lipid accumulation leads to the inflammation in adipose tissue and systemic metabolic disorders. Intracellular metabolic state is known to change during cell differentiation and it affects the cell fate or the differentiation through epigenetic mechanism. Although the mechanism of preadipocyte differentiation has been well established, it is unknown how metabolic state changes and how it affects the differentiation in predipocyte differentiation. Nicotinamide adenine dinucleotide (NAD+) plays crucial roles in energy metabolism as a coenzyme in multiple redox reactions in major catabolic pathways and as a substrate of sirtuins or poly(ADP-ribose)polymerases. NAD+ is mainly synthesized from salvage pathway mediated by two enzymes, Nampt and Nmnat. The manipulation to NAD+ metabolism causes metabolic change in each tissue and changes in systemic metabolism. However, the role of NAD+ and Nampt in adipocyte differentiation remains unknown. In this study, we employed liquid chromatography-mass spectrometry (LC-MS)- and gas chromatography-mass spectrometry (GC-MS)-based targeted metabolomics to elucidate the metabolic reprogramming events that occur during 3T3-L1 preadipocyte differentiation. We found that the tricarboxylic acid (TCA) cycle was enhanced, which correlated with upregulated NAD+ synthesis. Additionally, increased alpha-ketoglutarate (αKG) contributed to histone H3K9 demethylation in the promoter region of PPARγ, leading to its transcriptional activation. Thus, we concluded that NAD+-centered metabolic reprogramming is necessary for the differentiation of 3T3-L1 preadipocytes.

L'interesse per la modulazione del metaboloma della nicotinamide adenina dinucleotide (NAD) sta guadagnando interesse a causa del suo potenziale terapeutico in diverse patologie umane. La soppressione della via di recupero della nicotinamide da parte degli inibitori della nicotinamide fosforibosil transferasi (NAMPT), tuttavia, ha dato risultati inconcludenti nei pazienti neoplastici perché diverse vie metaboliche aggirano il blocco enzimatico convergendo direttamente sulla nicotinamide mononucleotide adenil transferasi (NMNAT) per la sintesi di NAD. Sfortunatamente, inibitori NMNAT-specifici non sono stati identificati. Qui, il nostro gruppo, riporta l'identificazione di Vacor come substrato metabolizzato dall'azione consecutiva di NAMPT e NMNAT2 nell'analogo del NAD, Vacor adenina dinucleotide (VAD). Ciò porta all'inibizione di entrambi gli enzimi, nonché delle deidrogenasi NAD-dipendenti: ciò causa una rapida deplezione del NAD, blocco della glicolisi, a cui conseguono mancanza di energia e morte per necrosi delle cellule cancerose che esprimono NMNAT2. Al contrario, la mancanza di espressione NMNAT2 conferisce completa resistenza a Vacor. Sorprendentemente, Vacor stimola la formazione di VAD e la soppressione della crescita in xenotrapianti di neuroblastoma e melanoma NMNAT2-positivi. I nostri dati mostrano un primo tentativo di sfruttare l'intero percorso di recupero della nicotinamide come strategia antimetabolica nel trattamento delle neoplasie. // Interest in the modulation of nicotinamide adenine dinucleotide (NAD) metabolome is gaining great momentum because of its therapeutic potential in different human disorders. Suppression of nicotinamide salvage by nicotinamide phosphoribosyl transferase (NAMPT) inhibitors, however, gave inconclusive results in neoplastic patients because several metabolic routes circumvent the enzymatic block converging directly on nicotinamide mononucleotide adenylyl transferases (NMNATs) for NAD synthesis. Unfortunately, NMNAT inhibitors have not been identified. Here, we report the identification of Vacor as a substrate metabolized by the consecutive action of NAMPT and NMNAT2 into the NAD analog Vacor adenine dinucleotide (VAD). This leads to inhibition of both enzymes, as well as NAD-dependent dehydrogenases, thereby causing unprecedented rapid NAD depletion, glycolytic block, energy failure, and necrotic death of NMNAT2-proficient cancer cells. Conversely, lack of NMNAT2 expression confers complete resistance to Vacor. Remarkably, Vacor prompts VAD formation and growth suppression in NMNAT2-positive neuroblastoma and melanoma xenografts. Our data show the first evidence of harnessing the entire nicotinamide salvage pathway for antimetabolic strategies.

NAD (Nicotinamide Adenine Dinucleotide) is a pyridine involved in numerous biological processes, namely nutrient catabolism sustaining cellular energy metabolism. During aging, NAD levels decrease and a global proteostasis deregulation is observed. As many age-related diseases are neurodegenerative and characterized by the accumulation of protein aggregates, we hypothesized that NAD could prevent or ameliorate protein aggregation. To study the role of NAD metabolism in proteostasis, we used SH-SY5Y cells exposed to chemicals that modulate the levels of protein aggregation and NAD metabolism. Cells were stained with the ProteoStat® kit to detect protein aggregates and analysed by flow cytometry and fluorescence microscopy. Cell viability was measured with propidium iodide by flow cytometry and metabolic state was measured using the colorimetric resazurin assay. SH-SY5Y cells showed increased protein aggregation levels in the presence of the proteasome inhibitor MG132 over time. MG132 induced more aggregation than treatment of cells with a NAD metabolism inhibitor, although supplementation with NAD appeared to decrease protein aggregates. Fluorescence microscopy analysis corroborated the flow cytometry result. In all tested conditions cell viability was not altered, in contrast with metabolic state that was altered by chemical treatments. Supplementation with NAD appeared to decrease protein aggregates, and further studies are warranted to elucidate the role in proteostasis of the different NAD precursors and associated pathways.
NAD (Nicotinamida Adenina Dinucleótido) é uma piridina envolvida em vários processos biológicos, nomeadamente, catabolismo de nutrientes que sustentam a produção de energia na célula. Durante o envelhecimento, os níveis de NAD diminuem e uma desregulação global da proteostase é observada. Como muitas doenças ligadas ao envelhecimento são neurodegenerativas, onde ocorre acumulação de agregados de proteínas, colocamos a hipótese que o NAD poderá prevenir ou melhorar a agregação proteica. Para estudar o papel do metabolismo do NAD na proteostase, expusemos células SH-SY5Y a químicos que modelam os níveis de agregação proteica e de NAD. As células foram marcadas com o kit ProteoStat® para detetar os agregados proteicos e analisadas por citometria de fluxo e microscopia confocal. A viabilidade celular foi medida com iodeto de propídio por citometria de fluxo e o estado metabólico foi medido usando o ensaio colorimétrico da resazurina. Células SH-SY5Y apresentaram um aumento de agregados proteicos na presença do inibidor de proteossoma MG132 ao longo do tempo. MG132 induziu mais agregação do que o tratamento com um inibidor do metabolismo de NAD, no entanto a suplementação com NAD pareceu diminuir esses agregados. A microscopia confocal corroborou os resultados de citometria de fluxo. Em todas as condições testadas, a viabilidade celular não foi alterada, em contraste com o estado metabólico que foi alterado pelos tratamentos. Suplementação com NAD pareceu diminuir a agregação proteica e estudos futuros serão necessários para elucidar o papel na proteostase dos diferentes precursores de NAD e vias metabólicas associadas.
Mestrado em Biomedicina Molecular