Cellular Nicotinamide Adenine Dinucleotide Levels Research Articles

A molecular circuit linking the BCR to the NAD biosynthetic enzyme NAMPT is an actionable target in Richter syndrome

AbstractThis works defines, to the best of our knowledge, for the first time a molecular circuit connecting nicotinamide mononucleoside phosphoribosyl transferase (NAMPT) activity to the B-cell receptor (BCR) pathway. Using 4 distinct xenograft models derived from patients with Richter syndrome (RS-PDX), we show that BCR cross-linking results in transcriptional activation of the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme NAMPT, with increased protein expression, in turn, positively affecting global cellular NAD levels and sirtuins activity. NAMPT blockade, by using the novel OT-82 inhibitor in combination with either BTK or PI3K inhibitors (BTKi or PI3Ki), induces rapid and potent apoptotic responses in all 4 models, independently of their mutational profile and the expression of the other NAD biosynthetic enzymes, including nicotinate phosphoribosyltransferase. The connecting link in the circuit is represented by AKT that is both tyrosine- and serine-phosphorylated by PI3K and deacetylated by sirtuin 1 and 2 to obtain full kinase activation. Acetylation (ie, inhibition) of AKT after OT-82 administration was shown by 2-dimensional gel electrophoresis and immunoprecipitation. Consistently, pharmacological inhibition or silencing of sirtuin 1 and 2 impairs AKT activation and induces apoptosis of RS cells in combination with PI3Ki or BTKi. Lastly, treatment of RS-PDX mice with the combination of PI3Ki and OT-82 results in significant inhibition of tumor growth, with evidence of in vivo activation of apoptosis. Collectively, these data highlight a novel application for NAMPT inhibitors in combination with BTKi or PI3Ki in aggressive lymphomas.

Porcine placental extract increase the cellular NAD levels in human epidermal keratinocytes

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for numerous enzymes involved in energy metabolism. Because decreasing NAD levels is a common hallmark of the aging process in various tissues and organs, maintaining NAD levels has recently been of interest for the prevention of aging and age-related diseases. Although placental extract (PE) are known to possess several anti-aging effects, the NAD-boosting activity of PE remains unknown. In this study, we found that porcine PE (PPE) significantly increased intracellular NAD levels in normal human epidermal keratinocytes (NHEKs). PPE also attenuated the NAD depletion induced by FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT). Interestingly, only the fraction containing nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) restored NAD content in NHEKs in the absence of NAMPT activity. These results suggest that PPE increases intracellular NAD by providing NAD precursors such as NMN, NR, and NAM. Finally, we showed that the application of PPE to the stratum corneum of the reconstructed human epidermis significantly ameliorated FK866-induced NAD depletion, suggesting that topical PPE may be helpful for increasing skin NAD levels. This is the first study to report the novel biological activity of PE as an NAD booster in human epidermal cells.

CAMP-Mediated Autophagy Promotes Cell Survival via ROS-Induced Activation of PARP1: Implications for Treatment of Acute Lymphoblastic Leukemia.

PARP1 inhibitors augment the DNA damage-induced killing of ALL cells by limiting the opposing effects of cAMP-mediated autophagy, which involves ROS-induced PARP1 activation and depletion of cellular NAD levels.

Targeting Nicotinamide Adenine Dinucleotide (NAD) Glycohydrase Activity of CD38 Exerts Anti-Myeloma Effect Accompanying Intracellular NAD Elevation

Introduction. The development of novel agents has improved the outcomes of multiple myeloma (MM) patients. Especially, daratumumab, an anti-CD38 monoclonal antibody which exerts therapeutic effect against MM cells through direct cell damage, antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), has shown its high efficacy in clinical practice. CD38 is a transmembrane glycoprotein highly expressed in plasma cells. CD38 is also a major nicotinamide adenine dinucleotide (NAD) glycohydrase in mammalian tissues, which regulate cellular levels of NAD. However, the role of CD38 as a NAD glycohydrase (NADase) in survival of MM cells is not well understood. In the present study, we conducted CD38 enzyme activity inhibition on MM cells using a small molecule compound 78c, a specific inhibitor for NADase enzymatic activity of CD38, in order to study the role of CD38 NADase activity in MM cell survival and to examine whether CD38 enzyme inhibition could be a new therapeutic strategy of MM. Materials and methods. MM cell lines (NCI-H929, KMS-12BM, KMS-12PE, U266) were treated with CD38 NADase inhibitor, 78c, in vitro. Viability of MM cell lines and patient-derived MM cells were analyzed by flow cytometry after 7AAD staining. MM cell lines possessing CD38 positive and negative fraction were sorted according to the CD38 expression using CD38 Micro-Beads. CD38 low MM cell lines were treated with All-trans retinoic acid(ATRA)to increase surface CD38 expression. Intracellular NAD and NADH concentrations in MM cells were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Caspase inhibitor, Z-VAD-FMK, was used in combination with 78c to study the mechanism of 78c induced MM cell death. Results. 78c induced cell death in MM cell lines at low concentrations (IC50 10-20 μM). Addition of 78c to patient derived bone marrow cells showed cytotoxicity to MM cells, while toxicity to non-MM cells were limited. CD38 positive fraction of MM cell lines had better sensitivity to 78c compared to CD38 negative fraction. CD38 induction by ATRA in CD38 low MM cell lines showed increased sensitivity to 78c. These results proved that 78c efficacy correlates with surface CD38 expression. Comparison of intracellular NAD and NADH concentrations between CD38 positive and negative fractions of MM cell lines demonstrated a significant increase of NAD in the CD38 negative fraction compared to their positive counterparts, indicating that CD38 is indeed controlling the intracellular NAD concentration. Marked increase of NAD / NADH ratio was observed in 78c treated MM cell lines compared to control, proving that CD38 NADase inhibition affects intracellular NAD concentration in MM cells (Fig. 1). 78c treatment of MM cell lines significantly reduced the number of viable cells in the Annexin- / PI- region, however, addition of Z-VAD-FMK did not lead to recovery of viable cell numbers, indicating non-apototic cell death induction by CD38 NADase inhibition. Conclusions. CD38 is the major NADase in mammalian tissues, and involved in catabolism of NAD. CD38 NADase inhibitor, 78c, inhibited the growth of MM cells at low concentrations. 78c induced cell death was found to be highly specific to MM cells and its cytotoxic effect was associated with surface CD38 expression of MM cells. Increased amount of NAD in MM cells by 78c treatment suggests that NAD elevation is associated with MM cell death induced by CD38 NADase inhibition. Since, daratumumab has limited effect against CD38 NADase activity, modulation of intracellular NAD levels by CD38 NADase inhibition could provide a novel therapeutic strategy for MM (Fig. 2). Disclosures Matsuoka: Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Sorafenib-Induced Apoptosis in Hepatocellular Carcinoma Is Reversed by SIRT1.

Sorafenib is a multi-kinase inhibitor and one of the few systemic treatment options for patients with advanced hepatocellular carcinomas (HCCs). Resistance to sorafenib develops frequently and could be mediated by the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase sirtuin (SIRT)1. We aimed to test whether sorafenib efficacy is influenced by cellular NAD levels and NAD-dependent SIRT1 function. We analyzed sorafenib effects on apoptosis induction, NAD salvage, mitochondrial function, and related signaling pathways in HCC cell lines (HepG2, Hep3B, und HUH7) overexpressing SIRT1 or supplemented with the NAD metabolite nicotinamide mononucleotide (NMN) compared to controls. Treatment of HCC cell lines with sorafenib dose-dependently induced apoptosis and a significant decrease in cellular NAD concentrations. The SIRT1 protein was downregulated in HUH7 cells but not in Hep3B cells. After sorafenib treatment, mitochondrial respiration in permeabilized cells was lower, citrate synthase activity was attenuated, and cellular adenosine triphosphate (ATP) levels were decreased. Concomitant to increased phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), sorafenib treatment led to decreased activity of the mechanistic target of rapamycin (mTOR), indicative of energy deprivation. Transient overexpression of SIRT1, as well as NAD repletion by NMN, decreased sorafenib-induced apoptosis. We can, therefore, conclude that sorafenib influences the NAD/SIRT1/AMPK axis. Overexpression of SIRT1 could be an underlying mechanism of resistance to sorafenib treatment in HCC.

Keeping the balance in NAD metabolism.

Research over the last few decades has extended our understanding of nicotinamide adenine dinucleotide (NAD) from a vital redox carrier to an important signalling molecule that is involved in the regulation of a multitude of fundamental cellular processes. This includes DNA repair, cell cycle regulation, gene expression and calcium signalling, in which NAD is a substrate for several families of regulatory proteins, such as sirtuins and ADP-ribosyltransferases. At the molecular level, NAD-dependent signalling events differ from hydride transfer by cleavage of the dinucleotide into an ADP-ribosyl moiety and nicotinamide. Therefore, non-redox functions of NAD require continuous biosynthesis of the dinucleotide. Maintenance of cellular NAD levels is mainly achieved by nicotinamide salvage, yet a variety of other precursors can be used to sustain cellular NAD levels via different biosynthetic routes. Biosynthesis and consumption of NAD are compartmentalised at the subcellular level, and currently little is known about the generation and role of some of these subcellular NAD pools. Impaired biosynthesis or increased NAD consumption is deleterious and associated with ageing and several pathologies. Insults to neurons lead to depletion of axonal NAD and rapid degeneration, partial rescue can be achieved pharmacologically by administration of specific NAD precursors. Restoring NAD levels by stimulating biosynthesis or through supplementation with precursors also produces beneficial therapeutic effects in several disease models. In this review, we will briefly discuss the most recent achievements and the challenges ahead in this diverse research field.

Venetoclax in Combination with NAMPT Inhibitors Decreases Mitochondrial Bioenergetics through the Impaired AMPK/SIRT/PGC1α Signaling Pathway in CLL

Objective: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Altered mitochondrial metabolism has been shown to be involved in the pathogenesis of CLL. Nicotinamidephospho-ribosyltransferase (NAMPT) is a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage pathway. FK866 and GMX1778, chemical inhibitors of NAMPT, deplete cellular NAD and ATP levels and trigger apoptosis in CLL cells, suggesting NAMPT contributes to the prolonged survival of these cells. Venetoclax is an approved therapy for CLL and blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, leading to programmed cell death of CLL cells. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling axis senses the metabolic demands of cells and regulates mitochondrial function. Silent information regulator T1 (SIRT1) is a cytoplasmic enzyme that mediates NAD+-dependent deacetylation of target substrates. The importance of the AMPK/SIRT/PGC1α signaling pathway has to be explored yet in CLL. Venetoclax may trigger a switching off of AMPK and /or SIRT1 signaling leading to impaired PGC-1α expression/activity and diminished mitochondrial activity. The effects of these drugs on mitochondrial bioenergetics profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of Bcl-2, AMPK, SIRT1, and PGC1α were analyzed. We hypothesize that venetoclax, along with FK866 and GMX1778, will alter CLL mitochondrial bioenergetics profiles and function, cell viability, and ROS levels through the involvement of the AMPK/SIRT/PGC1α signaling pathway and enable lower doses and profound effects for combination therapies rather than single agent therapy.Methods: A high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria), a Clarke-type oxygen electrode is used to measure mitochondrial respiration rates of CLL cells at 370C. Freshly isolated CLL cells (10 mil./ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates, the following chemicals were added: oligomycin (2uM), FCCP: carbonyl cyanide p-trifluoromethoxy phenyhydrazone (2.5-12.5 uM), and antimycin A (2 uM). Oxygen consumption rate is expressed in pmol oxygen/s/mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by Novocyte flow cytometer.Results: Primary CLL cells treated with 1.25 nM of venetoclax for 24 or 48h showed significantly decreased basal respiration rates, maximal respiration rates, and spare respiratory capacity compared to DMSO vehicle control. These parameters were also significantly affected by 1nM of FK866 or 1 nM of GMX1778 except basal respiration rates. The combination treatment of venetoclax with FK866 or GMX1778 for 24 or 48h significantly decreased all these bioenergetics parameters compared to each individual drug. MMP was not affected by these drug treatments and cell viability corresponded to the effect of the mitochondrial bioenergetics profile. ROS levels after 24 hours in venetoclax treated CLL cells were significantly increased compared to DMSO, any single agent or in combination. However, after 48 hours of treatment, increased ROS levels were not statistically significant compared to DMSO. Protein levels of Bcl-2, P-AMPK, AMPK, SIRT1, and PGC1α were decreased by venetoclax alone or in combination with FK866 or GMX1778.Conclusion: Venetoclax, FK866 and GMX1778 affected bioenergetics profiles in CLL cells at doses as low as 1.25 nM, 1nM, and 1 nM, respectively. The combined effect of these drugs on the mitochondrial bioenergetics profiles and cell viability is more profound than each NAMPT inhibitor agent alone. Protein levels of Bcl-2, AMPK, SIRT1, and PGC1α in venetoclax treated samples were reduced compared to DMSO and each single agent NAMPT inhibitor. These novel data suggest the involvement of the AMPK/SIRT/PGC1α signaling pathway to target mitochondrial metabolism and provide rational therapeutic combinations that may lead to reduced toxicity and increased drug efficacy in CLL. DisclosuresBanerji:Roche: Other: Unrestricted grant received in the past; Janssen: Other: Unrestricted grant received in the past; Gilead: Other: Unrestricted grant received in the past; Abbvie: Other: Unrestricted grant received in the past; Teva: Other: Unrestricted grant received in the past.

Decreased NAD Activates STAT3 and Integrin Pathways to Drive Epithelial-Mesenchymal Transition

Nicotinamide adenine dinucleotide (NAD) plays an essential role in all aspects of human life. NAD levels decrease as humans age, and supplementation with NAD precursors plays a protective role against aging and associated disease. Less is known about the effects of decreased NAD on cellular processes, which is the basis for understanding the relationship between cellular NAD levels and aging-associated disease. In the present study, cellular NAD levels were decreased by overexpression of CD38, a NAD hydrolase, or by treating cells with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT). Quantitative proteomics revealed that declining NAD levels downregulated proteins associated with primary metabolism and suppressed cell growth in culture and nude mice. Decreased glutathione synthesis caused a 4-fold increase in cellular reactive oxygen species levels, and more importantly upregulated proteins related to movement and adhesion. In turn, this significantly changed cell morphology and caused cells to undergo epithelial to mesenchymal transition (EMT). Secretomic analysis also showed that decreased NAD triggered interleukin-6 and transforming growth factor beta (TGFβ) secretion, which activated integrin-β-catenin, TGFβ-MAPK, and inflammation signaling pathways to sustain the signaling required for EMT. We further revealed that decreased NAD inactivated sirtuin 1, resulting in increased signal transducer and activator of transcription 3 (STAT3) acetylation and phosphorylation, and STAT3 activation. Repletion of nicotinamide or nicotinic acid inactivated STAT3 and reversed EMT, as did STAT3 inhibition. Taken together, these results indicate that decreased NAD activates multiple signaling pathways to promote EMT and suggests that age-dependent decreases in NAD may contribute to tumor progression. Consequently, repletion of NAD precursors has potential benefits for inhibiting cancer progression.

ROS-Mediated 15-Hydroxyprostaglandin Dehydrogenase Degradation via Cysteine Oxidation Promotes NAD+-Mediated Epithelial-Mesenchymal Transition

Nicotinamide adenine dinucleotide (NAD) levels decrease with aging as a result of aging-associated CD38 upregulation. Here, we established a cell model with decreased cellular NAD levels by overexpressing CD38 or treating cells with FK866, an inhibitor of nicotinamide phosphoribosyltransferase. We revealed that decreased NAD triggered reactive oxygen species (ROS)-mediated degradation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which drove cells to undergo epithelial-mesenchymal transition (EMT). Moreover, we showed that oxidation of the Cys44 residue to sulfonic acid in 15-PGDH led to its degradation via non-canonical ubiquitination-proteasome and autophagy pathways. Mutation ofCys44 to alanine abolished ROS-induced 15-PGDH degradation. We demonstrated that 15-PGDH silencing promoted EMT, whereas supplementation with NAD precursors increased NAD and 15-PGDH stability, and reversed the EMT process. Taken together, these results suggest that declining NAD levels contribute to age-dependent increases in cancer incidence, and repletion of NAD precursors is beneficial for increasing 15-PGDH expression.

Effect of nicotinamide adenine dinucleotide on bupivacaine-induced neurotoxicity

Objective Investigating whether bupivacaine induces decline of intracellular nicotinamide adenine dinucleotide(NAD) level, and whether NAD level decreasing is involved in bupivacaine-induced neurotoxicity. Methods SH-SY5Y cell were treated with 1 mmol/L for indicated time points (30 min to 7 h), the intracellular NAD content and cell viability were detected. SH-SY5Y cells were treated with bupivacaine at concentrations varying from 1 mmol/L to 10 mmol/L for 30 min. The intracellular NAD content and cell viability were then detected. SH-SY5Y cells exposed to 5 mmol/L bupivacaine for 30 min, upon 30 min pre-, post-NAD treatment at different concentrations. The intracellular NAD levels and the cell viabilities in all groups were examined. Results The intracellular NAD level began to decline after 3 h bupivacaine treatment. The level was decreased to (27.8±8.47)% at 7 h time point of treatment. The intracellular NAD content of SH-SY5Y cells were decreased to (21.50±3.15)%, (25.73±7.22)%, and (16.07±13.93)% respectively after receiving 2, 5, 10 mmol/L bupivacaine treatment for 30 min. Thus, the content levels of NAD were significantly lower than untreated control(P 0.05). And cell viability were also decreased while concentrations of bupivacaine increasing[cell viability significantly declined to (49.44±8.55)%, (35.75±15.83)%, and (25.58±4.45)%, respectively, at the concentration of 2, 5, 10 mmol/L (P<0.05). The cellular NAD levels reached (85.87±11.82)%, (89.21±11.55)%, and (105.05±58.82)% in 2.5, 5, 10 mmol/L NAD pre-treatment groups respectively. The cellular NAD levels were significantly higher than the levels in bupivacaine groups(P<0.05). The cell viabilities were higher in cells receiving NAD pre-treatment or post-treatment than viability of cells were treated with 5 mmol/L bupivacaine alone. Thus, the pre-treatment group achieved better viability(P<0.05). Conclusions Intracellular NAD level decline was involved in bupivacaine induced neurotoxicity. Exogenous NAD increases cellular NAD level and attenuates the cytotoxicity resulting from bupivacaine. Key words: Nicotinamide adenine dinucleotide; Bupivacaine; Neurotoxicity

Inhibition of NAMPT decreases cell growth and enhances susceptibility to oxidative stress.

Nicotinamide adenine dinucleotide (NAD) is an essential molecule for living organisms and plays a vital role in aging and age-associated diseases. In eukaryotic cells, cellular NAD is mainly generated by the scavenge pathway in which nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the formation of nicotinamide mononucleotide. Inhibition of NAMPT is a therapeutic strategy for cancer treatment. To explore the effects of NAMPT inhibition on cellular processes, cells were treated with 10nM FK866, an NAMPT inhibitor, resulting in a decrease in the cellular NAD level, a lower growth rate, and enhanced susceptivity to oxidative stress as compared to the untreated cells. Quantitative proteomics revealed that 325proteins were downregulated in the FK866-treated cells, and were involved in diverse cellular processes including nucleobase-containing compound metabolic process, protein metabolic process, antioxidant and DNA repair processes. Downregulation of 4 selected proteins was confirmed by western blotting and quantitative PCR. Downregulation of antioxidant proteins GRX1 and catalase, and DNA-repair proteins PCNA and PARP1 contributed to the enhanced susceptibility of FK866-treated cells to oxidative stress. FK866 treatment also caused mitochondrial dysfunction through downregulation of mitochondrial ribosomal proteins. Taken together, these results demonstrate that FK866 treatment efficiently decreases the cellular NAD level and induces autonomous changes in proteostasis, leading to cell growth inhibition and increased susceptibility to oxidative stress.

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.

Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

Regulation of the Nampt-mediated NAD salvage pathway and its therapeutic implications in pancreatic cancer

Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor for the redox reactions in the metabolic pathways of cancer cells that have elevated aerobic glycolysis (Warburg effect). Cancer cells are reported to rely on NAD recycling and inhibition of the NAD salvage pathway causes metabolic collapse and cell death. However, the underlying regulatory mechanisms and clinical implications for the NAD salvage pathway in pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study showed that the expression of Nampt, the rate-limiting enzyme of the NAD salvage pathway, was significantly increased in PDAC cells and PDAC tissues. Additionally, inhibition of Nampt impaired tumor growth in vitro and tumorigenesis in vivo, which was accompanied by a decreased cellular NAD level and glycolytic activity. Mechanistically, the Nampt expression was independent of Kras and p16 status, but it was directly regulated by miR-206, which was inversely correlated with the expression of Nampt in PDAC tissues. Importantly, pharmacological inhibition of Nampt by its inhibitor, FK866, significantly enhanced the antitumor activity of gemcitabine in PDAC cells and in orthotopic xenograft mouse models. In conclusion, the present study revealed a novel regulatory mechanism for Nampt in PDAC and suggested that Nampt inhibition may override gemcitabine resistance by decreasing the NAD level and suppressing glycolytic activity, warranting further clinical investigation for pancreatic cancer treatment.

Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer's disease-relevant murine model.

BackgroundMitochondrial dysfunction is a hallmark of neurodegenerative diseases including Alzheimer’s disease (AD), with morphological and functional abnormalities limiting the electron transport chain and ATP production. A contributing factor of mitochondrial abnormalities is loss of nicotinamide adenine dinucleotide (NAD), an important cofactor in multiple metabolic reactions. Depletion of mitochondrial and consequently cellular NAD(H) levels by activated NAD glycohydrolases then culminates in bioenergetic failure and cell death. De Novo NAD+ synthesis from tryptophan requires a multi-step enzymatic reaction. Thus, an alternative strategy to maintain cellular NAD+ levels is to administer NAD+ precursors facilitating generation via a salvage pathway. We administered nicotinamide mononucleotide (NMN), an NAD+ precursor to APP(swe)/PS1(ΔE9) double transgenic (AD-Tg) mice to assess amelioration of mitochondrial respiratory deficits. In addition to mitochondrial respiratory function, we examined levels of full-length mutant APP, NAD+-dependent substrates (SIRT1 and CD38) in homogenates and fission/fusion proteins (DRP1, OPA1 and MFN2) in mitochondria isolated from brain. To examine changes in mitochondrial morphology, bigenic mice possessing a fluorescent protein targeted to neuronal mitochondria (CaMK2a-mito/eYFP), were administered NMN.MethodsMitochondrial oxygen consumption rates were examined in N2A neuroblastoma cells and non-synaptic brain mitochondria isolated from mice (3 months). Western blotting was utilized to assess APP, SIRT1, CD38, DRP1, OPA1 and MFN2 in brain of transgenic and non-transgenic mice (3–12 months). Mitochondrial morphology was assessed with confocal microscopy. One-way or two-way analysis of variance (ANOVA) and post-hoc Holm-Sidak method were used for statistical analyses of data. Student t-test was used for direct comparison of two groups.ResultsWe now demonstrate that mitochondrial respiratory function was restored in NMN-treated AD-Tg mice. Levels of SIRT1 and CD38 change with age and NMN treatment. Furthermore, we found a shift in dynamics from fission to fusion proteins in the NMN-treated mice.ConclusionsThis is the first study to directly examine amelioration of NAD+ catabolism and changes in mitochondrial morphological dynamics in brain utilizing the immediate precursor NMN as a potential therapeutic compound. This might lead to well-defined physiologic abnormalities that can serve an important role in the validation of promising agents such as NMN that target NAD+ catabolism preserving mitochondrial function.

Bioluminescent cell-based NAD(P)/NAD(P)H assays for rapid dinucleotide measurement and inhibitor screening.

Abstract The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z' value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels.

Overexpression of CD38 Decreases Cellular NAD Levels and Alters the Expression of Proteins Involved in Energy Metabolism and Antioxidant Defense

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells and mediates multiple cellular signaling pathways. In the present study, a 35% decrease of cellular NAD level is achieved by stable expression of the N-terminal truncated CD38, a NAD hydrolase. CD38-expressing (CD38(+)) cells have the lower growth rate and are more susceptive to oxidative stress than the wild type cells and empty vector-transfected (CD38(-)) cells. Quantitative proteomic analysis shows that 178 proteins are down-regulated in CD38(+) cells, which involve in diverse cellular processes including glycolysis, RNA processing and protein synthesis, antioxidant, and DNA repair. Down regulation of six selected proteins is confirmed by Western blotting. However, down-regulation of mRNA expressions of genes associated with glycolysis, antioxidant, and DNA repair is less significant than the corresponding change in protein expression, suggesting the low NAD level impairs the protein translational machinery in CD38(+) cells. Down-regulation of antioxidant protein and DNA-repair protein expression contributes to the susceptibility of CD38(+) cells to oxidative stress. Taken together, these results demonstrate that CD38(+) cells are a useful model to study effects of the cellular NAD levels on cellular processes and establish a new linker between cellular NAD levels and oxidative stress.

Is a deceptive role of IGF-1 in Sirt1–PARP1 interactions the primary step of postnatal regression of hypertrophic cardiomyopathy in infants of diabetic mothers?

Is a deceptive role of IGF-1 in Sirt1–PARP1 interactions the primary step of postnatal regression of hypertrophic cardiomyopathy in infants of diabetic mothers?

A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribosyltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimental evidence has indicated that Nampt might have proangiogenic activity and supports the growth of some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then we set up an HTS system after thorough optimizations of this method and validated that it is feasible and effective through a pilot screening on a small library. This HTS system should expedite the discovery of Nampt inhibitors as antitumor drug candidates.

Inhibition of the Kynurenine-NAD+Pathway Leads to Energy Failure and Exacerbates Apoptosis in Pneumococcal Meningitis

Pneumococcal meningitis causes neurological sequelae, including learning and memory deficits in up to half of the survivors. In both humans and in animal models of the disease, there is apoptotic cell death in the hippocampus, a brain region involved in learning and memory function. We previously demonstrated that in an infant rat model of pneumococcal meningitis, there is activation of the kynurenine (KYN) pathway in the hippocampus, and that there was a positive correlation between the concentration of 3-hydroxykynurenine and the extent of hippocampal apoptosis. To clarify the role of the KYN pathway in the pathogenesis of hippocampal apoptosis in pneumococcal meningitis, we specifically inhibited 2 key enzymes of the KYN pathway and assessed hippocampal apoptosis, KYN pathway metabolites, and nicotinamide adenine dinucleotide (NAD) concentrations by high-performance liquid chromatography. Pharmacological inhibition of kynurenine 3-hydroxylase and kynureninase led to decreased cellular NAD levels and increased apoptosis in the hippocampus. The cerebrospinal fluid levels of tumor necrosis factor and interleukin-1α and -β were not affected. Our data suggest that activation of the KYN pathway in pneumococcal meningitis is neuroprotective by compensating for an increased NAD demand caused by infection and inflammation;this mechanism may prevent energy failure and apoptosis in the hippocampus.