NAD Degradation Research Articles

Abstract P3197: Sarm1 Deficiency Protects Mice Against Diabetic Cardiomyopathy: Potential Mechanisms Via Ameliorating Lipotoxicity

Heart failure occurs at nearly twice the rate in diabetic patients as compared to normal patients. NAD levels are decreased in human and murine heart disease models including diabetic hearts. Strategies to raise NAD in these models have primarily focused on increasing NAD synthesis. Alternatively, inhibiting NAD consumption provides another viable path to increasing NAD levels in diabetic hearts. Sterile Alpha and Tir Motif Containing 1 (SARM1) is an NAD hydrolase that mediates axonal degeneration through NAD degradation and promotion of mitochondrial dysfunction. However, the role SARM1 plays in heart disease has not been investigated. We subjected male wild type (WT) and global SARM1 knockout mice (KO) to chronic diabetic stress induced by streptozotocin injections. We showed that 16-week diabetic stress promoted progressive decline in systolic and diastolic function as measured by longitudinal echocardiography. SARM1 deletion (diabetic KO) improved systolic and diastolic function of diabetic mice, despite similar glucose, and plasma aqueous and lipid metabolite levels as the diabetic wild-type mice. Diabetic KO hearts showed increased NAD levels, suggesting that SARM1 may be activated to promote NAD decline in diabetic hearts. Transcriptomic analysis identified 1948 differentially expressed genes in diabetic WT hearts, compared to non-diabetic WT hearts. Gene Module Network Analysis identified upregulation of fatty acid metabolic processes and suppression of genes involved in mitochondrial processes in diabetic hearts. We measured mitochondrial oxygen consumption rate and found suppressed mitochondrial respiration in diabetic WT hearts. SARM1 deficiency reversed the upregulated fatty acid metabolic genes, improved mitochondrial respiration, and elevated NAD levels in diabetic hearts. Genes such as CD36, HADHA, Acot2, and malonyl-CoA are all downregulated by SARM1 deficiency, indicating that SARM1 deficiency may relieve lipotoxicity by improving mitochondrial function and modifying metabolic transcriptional networks in diabetic hearts. Future analysis will shed light on how SARM1 contributes to lipotoxicity as a new pathogenic mechanism of diabetic cardiomyopathy.

Inhibition of phosphate transport by NAD+/NADH brush border membrane vesicles.

Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD+/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing Xenopus laevis oocytes. In BBMV, NAD+ required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in Xenopus oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A Ki value of 538 µM NAD+ in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD+/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD+/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time.

A Similarity-Based Method for Predicting Enzymatic Functions in Yeast Uncovers a New AMP Hydrolase

Despite decades of research and the availability of the full genomic sequence of the baker’s yeast Saccharomyces cerevisiae, still a large fraction of its genome is not functionally annotated. This hinders our ability to fully understand cellular activity and suggests that many additional processes await discovery. The recent years have shown an explosion of high-quality genomic and structural data from multiple organisms, ranging from bacteria to mammals. New computational methods now allow us to integrate these data and extract meaningful insights into the functional identity of uncharacterized proteins in yeast. Here, we created a database of sensitive sequence similarity predictions for all yeast proteins. We use this information to identify candidate enzymes for known biochemical reactions whose enzymes are unidentified, and show how this provides a powerful basis for experimental validation. Using one pathway as a test case we pair a new function for the previously uncharacterized enzyme Yhr202w, as an extra-cellular AMP hydrolase in the NAD degradation pathway. Yhr202w, which we now term Smn1 for Scavenger MonoNucleotidase 1, is a highly conserved protein that is similar to the human protein E5NT/CD73, which is associated with multiple cancers. Hence, our new methodology provides a paradigm, that can be adopted to other organisms, for uncovering new enzymatic functions of uncharacterized proteins.

Deep eutectic solvents as a stabilising medium for NAD coenzyme: unravelling the mechanism behind coenzyme stabilisation effect

DES choline chloride:urea effectively stabilises NAD coenzyme up to 50 days at room temperature. Consequently, this solvent could be used to minimise NAD degradation during storage, assays, and enzyme-catalysed oxidoreductive reactions.

Extracellular Hydrolysis of NAD+and its Secondary Metabolites by a new Aspergillus sp

In some organisms, the metabolism of extracellular nicotinamide adenine dinucleotide (NAD+) plays an important role in purine salvage and uptake. Since filamentous fungi are used to degrade NAD and produce beneficial secondary metabolites, various enzymes, fermented foods and organic acids. Many strains belonging to filamentous fungi were screened for the enzymes production that catalyzed hydrolytic dephosphorylation and deamination of NAD and its secondary metabolites. A fungal isolate which was found to be the most prominent strain was identified genetically as Aspergillus fumigatous. Extracts of this new strain catalyzes the hydrolysis the ester bond between the two nucleotides of NAD by pyrophosphatase activity, which catalyzes NAD to nicotinamide mononucleotide (NMN) and adenosine monophosphate (AMP), at that point NMN and AMP are hydrolyzed to nicotinamide riboside (NR) and adenosine separately by 5’–nucleotidase activity This research reports also existence of deaminase activity hydrolyzed adenosine to inosine. The study demonstrated major enzymatic pathways involved in NAD degradation and identification of intermediates formed by Aspergillus fumigatous. Separation and partial purification of these enzymes and some kinetic properties of them were achieved

Enhancement of NAD(H) pool for formation of oxidized biochemicals in Escherichia coli.

The NAD+/NADH ratio and the total NAD(H) play important roles for whole-cell biochemical redox transformations. After the carbon source is exhausted, the degradation of NAD(H) could contribute to a decline in the rate of a desired conversion. In this study, methods to slow the native rate of NAD(H) degradation were examined using whole-cell Escherichia coli with two model oxidative NAD+-dependent biotransformations. A high phosphate concentration (50mM) was observed to slow NAD(H) degradation. We also constructed E. coli strains with deletions in genes coding several enzymes involved in NAD+ degradation. In shake-flask experiments, the total NAD(H) concentration positively correlated with conversion of xylitol to L-xylulose by xylitol 4-dehydrogenase, and the greatest conversion (80%) was observed using MG1655 nadR nudC mazG/pZE12-xdh/pCS27-nox. Controlled 1-L batch processes comparing E. coli nadR nudC mazG with a wild-type background strain demonstrated a 30% increase in final L-xylulose concentration (5.6 vs. 7.9g/L) and a 25% increase in conversion (0.53 vs. 0.66g/g). MG1655 nadR nudC mazG was also examined for the conversion of galactitol to L-tagatose by galactitol 2-dehydrogenase. A batch process using 15g/L glycerol and 10g/L galactitol generated over 9.4g/L L-tagatose, corresponding to 90% conversion and a yield of 0.95g L-tagatose/g galactitol consumed. The results demonstrate the value of minimizing NAD(H) degradation as a means to improve NAD+-dependent biotransformations.

Increased Rate of NAD Metabolism Shortens Plant Longevity by Accelerating Developmental Senescence in Arabidopsis.

NAD is a well-known co-enzyme that mediates hundreds of redox reactions and is the basis of various processes regulating cell responses to different environmental and developmental cues. The regulatory mechanism that determines the amount of cellular NAD and the rate of NAD metabolism remains unclear. We created Arabidopsis thaliana plants overexpressing the NAD synthase (NADS) gene that participates in the final step of NAD biosynthesis. NADS overexpression enhanced the activity of NAD biosynthesis but not the amounts of NAD+, NADH, NADP+ or NADPH. However, the amounts of some intermediates were elevated, suggesting that NAD metabolism increased. The NAD redox state was greatly facilitated by an imbalance between NAD generation and degradation in response to bolting. Metabolite profiling and transcriptional analysis revealed that the drastic modulation of NAD redox homeostasis increased tricarboxylic acid flux, causing the ectopic generation of reactive oxygen species. Vascular bundles suffered from oxidative stress, leading to a malfunction in amino acid and organic acid transportation that caused early wilting of the flower stalk and shortened plant longevity, probably due to malnutrition. We concluded that the mechanism regulating the balance between NAD synthesis and degradation is important in the systemic plant response to developmental cues during the growth-phase transition.

Permeability transition pore-dependent and PARP-mediated depletion of neuronal pyridine nucleotides during anoxia and glucose deprivation.

Exposure of rat cortical neurons to combined oxygen and glucose deprivation results in loss of NAD(P)H autofluorescence that is only partially reversible following restoration of oxygen and glucose, suggesting catabolism of pyridine nucleotides. This study tested the hypothesis that metabolic inhibition caused by cyanide-induced chemical anoxia plus glucose deprivation promotes both release of mitochondrial NAD(H) in response to opening of the permeability transition pore (PTP) and NAD(P)(H) degradation through activation of poly (ADP-ribose) polymerase (PARP). The NAD(P)H autofluorescence of rat neonatal cortical neurons was monitored during and following acute (10-30min) exposure to the respiratory inhibitor, cyanide, in the absence and presence of glucose. Because nitric oxide-derived peroxynitrite is a known activator of PARP, we additionally assessed the effect of a nitric oxide generating agent on the NAD(P)H autofluorescence response to chemical anoxia plus glucose deprivation. Cyanide induced a rapid increase in autofluorescence, followed by a steady decline promoted by the presence of nitric oxide. This decline was primarily due to NAD(H) catabolism, as verified by measurements of total NAD(H) present in cellular extracts. Catabolism was partially blocked by an inhibitor of PARP, by a PTP inhibitor, and by either glucose or pyruvate as a source of reducing power. Overall, data suggest that metabolic, oxidative, and nitrosative stress during in vitro neuronal anoxia and glucose deprivation result in release of mitochondrial pyridine nucleotides in response to PTP opening and rapid, extensive NAD(H) degradation mediated by PARP activation. These events may contribute to the metabolic dysfunction that occurs in vivo during cerebral ischemia and reperfusion and therefore represent prime targets for neuroprotection.

Heterogeneous photocatalytic degradation of pesticides using decatungstate intercalated macroporous layered double hydroxides.

Decatungstate W10O32(4-) was efficiently intercalated between the layers of three-dimensionally ordered macroporous Mg2Al-layered double hydroxide. The structural and textural properties of as-prepared intercalated compound were characterized using different solid-state characterization techniques such as X-ray powder diffraction, FTIR and Raman spectroscopies and electronic microscopy. The photocatalytic properties of immobilized W10O32 (4-) within Mg2Al structure were investigated using 2-(1-naphthyl) acetamide (NAD) as a model of pesticide. The influence of different parameters such as amount of catalyst, pH and oxygen concentration were investigated. An optimal NAD degradation was obtained for a photocatalyst concentration of 60 mg l(-1). Under our experimental conditions, this heterogeneous photocatalyst induces photodegradation of 60 % of NAD after 17 h of irradiation at 365 nm and at pH 6.6. Interestingly, pesticide photodegradation leads to the mineralization of substrates to H2O and CO2 and the photocatalyst can be recycled and reused without any loss of activity over four cycles.

Targeting of NAD Metabolism in Pancreatic Cancer Cells: Potential Novel Therapy for Pancreatic Tumors

Here, we describe a novel interplay between NAD synthesis and degradation involved in pancreatic tumor growth. We used human pancreatic cancer cells, both in vitro (cell culture experiments) and in vivo (xenograft experiments), to demonstrate the role of NAD synthesis and degradation in tumor cell metabolism and growth. We demonstrated that pharmacologic and genetic targeting of Nampt, the key enzyme in the NAD salvage synthesis pathway, inhibits cell growth and survival of pancreatic cancer cells. These changes were accompanied by a reduction of NAD levels, glycolytic flux, lactate production, mitochondrial function, and levels of ATP. The massive reduction in overall metabolic activity induced by Nampt inhibition was accompanied by a dramatic decrease in pancreatic tumor growth. The results of the mechanistic experiments showed that neither the NAD-dependent enzymes PARP-1 nor SIRT1 play a significant role on the effect of Nampt inhibition on pancreatic cancer cells. However, we identified a role for the NAD degradation pathway mediated by the NADase CD38 on the sensitivity to Nampt inhibition. The responsiveness to Nampt inhibition is modulated by the expression of CD38; low levels of this enzyme decrease the sensitivity to Nampt inhibition. In contrast, its overexpression decreased cell growth in vitro and in vivo, and further increased the sensitivity to Nampt inhibition. Our study demonstrates that NAD metabolism is essential for pancreatic cancer cell survival and proliferation and that targeting NAD synthesis via the Nampt pathway could lead to novel therapeutic treatments for pancreatic cancer.

Mitochondrial Dysfunction and NAD+ Metabolism Alterations in the Pathophysiology of Acute Brain Injury

Mitochondrial dysfunction is commonly believed to be one of the major players in mechanisms of brain injury. For several decades, pathologic mitochondrial calcium overload and associated opening of the mitochondrial permeability transition (MPT) pore were considered a detrimental factor causing mitochondrial damage and bioenergetics failure. Mitochondrial and cellular bioenergetic metabolism depends on the enzymatic reactions that require NAD(+) or its reduced form NADH as cofactors. Recently, it was shown that NAD(+) also has an important function as a substrate for several NAD(+) glycohydrolases whose overactivation can contribute to cell death mechanisms. Furthermore, downstream metabolites of NAD(+) catabolism can also adversely affect cell viability. In contrast to the negative effects of NAD(+)-catabolizing enzymes, enzymes that constitute the NAD(+) biosynthesis pathway possess neuroprotective properties. In the first part of this review, we discuss the role of MPT in acute brain injury and its role in mitochondrial NAD(+) metabolism. Next, we focus on individual NAD(+) glycohydrolases, both cytosolic and mitochondrial, and their role in NAD(+) catabolism and brain damage. Finally, we discuss the potential effects of downstream products of NAD(+) degradation and associated enzymes as well as the role of NAD(+) resynthesis enzymes as potential therapeutic targets.

Photochemical Degradation of the Plant Growth Regulator 2‐(1‐Naphthyl) acetamide in Aqueous Solution Upon UV Irradiation

The photochemical degradation of 2-(1-naphthyl) acetamide (NAD) in aqueous solution using simulated sunlight excitation as well as UV light within the 254-300 nm range was investigated to obtain an insight into the transformation mechanism that could occur under environmental conditions. Several photoproducts were identified using HPLC/MS/MS techniques. The degradation quantum yield was found to be independent of the excitation wavelength, but showed a dependence of oxygen concentration. This increased by a factor of approximately 3 from aerated to oxygen-free solutions. There is a clear involvement of both triplet and singlet excited states in NAD photoreactivity. The participation of singlet oxygen as a significant route in NAD degradation was ruled out by comparison with the behavior using Rose Bengal as a photosensitizer. A mechanistic pathway implying hydroxylation process through NAD radical cation species as well as an oxidation reaction by molecular oxygen is proposed. The photochemical behavior of NAD appears to mainly involve the aromatic moieties without any participation of the amide side chain. Toxicity tests clearly show that the generated primary photoproducts are responsible for a significant increase in the toxicity. However, upon prolonged irradiation this toxicity tends to decrease.

Mitochondrial dysfunction and nicotinamide dinucleotide catabolism as mechanisms of cell death and promising targets for neuroprotection

Both acute and chronic neurodegenerative diseases are frequently associated with mitochondrial dysfunction as an essential component of mechanisms leading to brain damage. Although loss of mitochondrial functions resulting from prolonged activation of the mitochondrial permeability transition (MPT) pore has been shown to play a significant role in perturbation of cellular bioenergetics and in cell death, the detailed mechanisms are still elusive. Enzymatic reactions linked to glycolysis, the tricarboxylic acid cycle, and mitochondrial respiration are dependent on the reduced or oxidized form of nicotinamide dinucleotide [NAD(H)] as a cofactor. Loss of mitochondrial NAD(+) resulting from MPT pore opening, although transient, allows detrimental depletion of mitochondrial and cellular NAD(+) pools by activated NAD(+) glycohydrolases. Poly(ADP-ribose) polymerase (PARP) is considered to be a major NAD(+) degrading enzyme, particularly under conditions of extensive DNA damage. We propose that CD38, a main cellular NAD(+) level regulator, can significantly contribute to NAD(+) catabolism. We discuss NAD(+) catabolic and NAD(+) synthesis pathways and their role in different strategies to prevent cellular NAD(+) degradation in brain, particularly following an ischemic insult. These therapeutic approaches are based on utilizing endogenous intermediates of NAD(+) metabolism that feed into the NAD(+) salvage pathway and also inhibit CD38 activity.

Metabolism of NAD and N1-methylnicotinamide in growing and growth-arrested cells.

Nicotinamide is metabolized primarily into NAD and N1-methylnicotinamide in cultured cells of normal rat kidney. The metabolic pathways for the nicotinamide metabolites are independently regulated and are influenced by the growth stage of the cells. N1-Methylnicotinamide levels are 1.5--2-fold elevated in cells growth-arrested by treatment with histidinol, thymidine, or picolinic acid, or by serum starvation. This increase is due to a more rapid rate of synthesis rather than decrease in excretion. The rates of both synthesis and degradation of NAD are increased in serum-starved cells so that the NAD concentration is the same as it is in growing cells. NAD and N1- methylnicotinamide levels are not significantly increased when the intracellular nicotinamide concentration is increased 20-fold by addition of excess nicotinamide to the culture medium, demonstrating that the size of the nicotinamide pool does not limit synthesis of these compounds. In medium containing normal amounts of nicotinamide, the apparent first-order rate constant for the decay of NAD, radioactively labeled in the nicotinamide moiety, is about 4 h-1. Labeled N1-methylnicotinamide is not metabolized, but rather is excreted into the medium with a first-order rate constant of 3.9 h-1. The rate of loss of label from NAD, but not from N1-methylnicotinamide, is increased about twofold by addition of excess nicotinamide to the culture medium. This could be explained by a dilution of a labeled nicotinamide pool which is formed during NAD degradation and which is recycled into NAD but not into N1-methylnicotinamide. The results demonstrate a rapid turnover of NAD at the bond joining nicotinamide and ADP-ribose, in agreement with previous studies. In addition, the results show that nicotinamide is metabolized into N1-methylnicotinamide with what appears to be a carefully regulated synthetic mechanism. The existence of significant amounts of N1-methylnicotinamide in cultured cells raises the question of the physiological importance of this compound.

NAD degradation and regulation of CD38 expression by human monocytes/macrophages.

In recent years, evidence has accumulated that NAD+ serves as a precursor of metabolites that are involved in a number of regulatory processes. In this work we show that extracellularly added NAD+ was rapidly degraded by intact human monocytes to nicotinamide and ADP-ribose. Besides these main products, minor amounts of AMP, ADP and cADP-ribose were formed. Expression of CD38, which has been identified as NAD+-glycohydrolase (EC 3.2.2.6) degrading NAD+ into nicotinamide and ADP-ribose, was determined on freshly isolated human monocytes by flow cytometry and RT-PCR. Upon ligation with anti-CD38 mAb, CD38 underwent internalization, shedding and new expression. As monocytes possess an intracellular CD38 pool, it could serve as a source for newly expressed CD38. Differentiation of monocytes to macrophages resulted in down-regulation of surface expression of CD38. This decrease correlates with a reduction in NADase activity, indicating that the amount of functional active CD38 molecules decrease during differentiation. As CD38 mRNA was found to be diminished in macrophages, regulation of the gene product seems to occur at the level of transcription or mRNA stability.

NAD degradation and regulation of CD38 expression by human monocytes/macrophages

NAD degradation and regulation of CD38 expression by human monocytes/macrophages

Optimizing the energy status of skin cells during solar radiation

Ionizing- and ultraviolet-radiation cause cell damage or death by directly altering DNA and protein structures and by production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). These processes disrupt cellular energy metabolism at multiple levels. The formation of DNA strand breaks activates signaling pathways that consume NAD, which can lead to the depletion of cellular ATP. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteasome that degrades damaged nuclear proteins including histones. Identifying approaches to optimize these responses while maintaining the energy status of cells is likely to be very important in minimizing the deleterious effects of solar radiation on skin.

Rapid screening method for identification of cholera toxin-producing Vibrio cholerae O1 and O139.

A novel method of identifying cholera enterotoxin (CT)-producing Vibrio cholerae serogroups O1 and O139 was developed. The method uses degradation of NAD as a specific biochemical marker for the CT-producing strains. The substrate NAD at a concentration of 100 mumol/liter was markedly degraded when it was incubated at 37 degrees C for 2 h with the CT-producing stains at a final cell density equivalent to that of a twofold dilution of a McFarland no. 1 standard. NAD degradation was monitored by an enzyme-amplified color development assay. Subsequent tests conducted with a total of 119 strains of V. cholerae, including both clinical and environmental isolates, confirmed a significant correlation between NAD degradation and CT production for all V. cholerae strains belonging to serogroups O1 and O139. Since 2 of 11 non-O1, non-O139 V. cholerae strains not carrying the CT gene degraded NAD, serotyping of the strains prior to the test is recommended.

Modulatory role of GTP-binding proteins in the endogenous ADP-ribosylation of cytosolic proteins.

The endogenous ADP-ribosylation of cytosolic proteins and the pattern of NAD degradation were analyzed in subcellular fractions of rat liver in order to investigate the modulation of these reactions by GTP-binding (G) proteins. We could show that intracellular membranes from rat liver have a guanine nucleotide- and divalent cation-dependent pyrophosphatase activity able to rapidly degrade NAD to AMP. This enzymatic activity was investigated by two different approaches: the degradation of [32P]-NAD in the presence of intracellular membranes and the mono-ADP-ribosylation of cytosolic proteins. Divalent cations, preferentially Zn2+ and Mn2+, were required for the pyrophosphatase activity, since in the presence of the Zn2+ chelator TPEN (N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine) or EDTA, the NAD degradation was inhibited by about 50%. Accordingly, in the presence of TPEN the endogenous ADP-ribosylation of cytosolic proteins was enhanced, whereas Zn2+ caused a significant inhibition of this reaction. GDP beta S was able to strongly activate the mono-ADP-ribosylation of cytosolic proteins. This effect was abolished by GTP gamma S, suggesting that a G protein, or rather one of the subunits of a heterotrimeric G protein, is involved in the modulation of the pyrophosphatase and consequently, of endogenous ADP-ribosylation. We propose that a regulatory pathway involving a heterotrimeric G protein modulates enzymes affecting the NAD turnover and availability of NAD for endogenous mADPRTs.

4983513 Sulfhydryl compounds for suppressing the inhibitory effects of NAD degradation products on LD-L activity: Tsung-I Lin, Alice C Liu assigned to Beckman Instruments Inc

4983513 Sulfhydryl compounds for suppressing the inhibitory effects of NAD degradation products on LD-L activity: Tsung-I Lin, Alice C Liu assigned to Beckman Instruments Inc