NAD Biosynthesis Research Articles

Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis

Nicotinate phosphoribosyltransferase (NaPRT, EC 2.4.2.11) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD+. Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD+ levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared.Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihydroxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.

Liquid chromatography/time-of-flight mass spectrometry for the analysis of plant samples: A method for simultaneous screening of common cofactors or nucleotides and application to an engineered plant line

Intense efforts are currently devoted to improve plant metabolomic analyses so as to describe more accurately the whole picture of metabolic pathways. Analyses based on liquid chromatography/time-of-flight mass spectrometry (LC-TOF) are now widely distributed among plant science laboratories. However, the use of reliable, sensitive LC-TOF methods to identify and quantify micromolar or inframicromolar key metabolites is often impeded by the sensitivity of the technique to sample preparation or chromatographic conditions. Typically, the sample matrix has a substantial influence on ionization efficiency and therefore, on the detectability of such compounds. Here, we describe a new method to analyze simultaneously 23 nucleotides and cofactors from plant extracts, taking advantage of solid-phase extraction (SPE) prior to injection. The influence of common m/z fragments in several metabolites and adducts is considered. We applied this method to characterise metabolic intermediates of NAD biosynthesis in Arabidopsis thaliana, using a wild-type and an engineered transgenic plant line that produces bacterial quinolinate phosphoribosyl transferase (nadc). We show that sample pre-purification with SPE is strictly required not only for compound quantification and identification but also to allow ionization of matrix-sensitive compounds (e.g. nicotinamide) or alleviate fragmentation of others (e.g. NAD). When exogenous substrate quinolinate was infiltrated into Arabidopsis leaves to increase the natural content in downstream metabolites, a clear correlation between intermediates of NAD biosynthesis was seen, showing the accuracy of our method for quantification in biological samples. Nadc plants only showed very modest changes in NAD-related metabolites and furthermore, they were associated with slightly lower photosynthetic performance and ATP production.

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells: FROM ENTRY OF EXTRACELLULAR PRECURSORS TO MITOCHONDRIAL NAD GENERATION

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule.

An integrated approach to characterize genetic interaction networks in yeast metabolism

Intense experimental and theoretical efforts have been made to globally map genetic interactions, yet we still do not understand how gene-gene interactions arise from the operation of biomolecular networks. To bridge the gap between empirical and computational studies, we: i) quantitatively measure genetic interactions between ~185,000 metabolic gene pairs in Saccharomyces cerevisiae, ii) superpose the data on a detailed systems biology model of metabolism, and iii) introduce a machine-learning method to reconcile empirical interaction data with model predictions. We systematically investigate the relative impacts of functional modularity and metabolic flux coupling on the distribution of negative and positive genetic interactions. We also provide a mechanistic explanation for the link between the degree of genetic interaction, pleiotropy, and gene dispensability. Last, we demonstrate the feasibility of automated metabolic model refinement by correcting misannotations in NAD biosynthesis and confirming them by in vivo experiments.

Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (Helianthus tuberosus L.) heterotrophic tissues

Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K (m) and V (max) values of 82 ± 1.05 μM and 4.20 ± 0.20 nmol min(-1) mg(-1) protein, respectively. The enzyme's pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells.

Activity of the cancer metabolism inhibitor MPC-9528 in xenograft models: Comparison of different dosing schedules.

10529 Background: MPC‐9528 is a potent, selective, orally bioavailable inhibitor of nicotinamide phosphoribosyltransferase (Nampt) that catalyzes the rate-limiting step in NAD biosynthesis from nic...

NMNAT Expression and its Relation to NAD Metabolism

Nicotinamide mononucleotide adenylyltransferease (NMNAT), a rate-limiting enzyme present in all organisms, reversibly catalyzes the important step in the biosynthesis of NAD from ATP and NMN. NAD and NADP are used reversibly in anabolic and catabolic reactions. NAD is necessary for cell survival in oxidative stress and DNA damage. Based on their localization, three different NMNAT's have been recognized, NMNAT-1 (homohexamer) in the nucleus (chromosome 1 p32-35), NMNAT-2 (homodimer) in the cytoplasm (chromosome 1q25) and NMNAT-3 (homotetramer) in the mitochondria. NMNAT also catalyzes the metabolic conversion of potent antitumor prodrugs like tiazofurin and benzamide riboside to their active forms which are analogs of NAD. NAD synthase-NMNAT acts as a chaperone to protect against neurodegeneration, injury-induced axonal degeneration and also correlates with DNA synthesis during cell cycle. Since its activity is rather low in tumor cells it can be exploited as a source for therapeutic targeting. Steps involved in NAD synthesis are being utilized as targets for chemoprevention, radiosensitization and therapy of wide range of diseases, such as cancer, multiple sclerosis, neurodegeneration and Huntington's disease.

NAMPT in Regulated NAD Biosynthesis and its Pivotal Role in Human Metabolism

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first reversible step in NAD biosynthesis and nicotinamide (NAM) salvage. The enzyme is designed for efficient capture of nicotinamide by coupling of ATP hydrolysis to assist in extraordinary NAM binding affinity and formation of nicotinamide mononucleotide (NMN). NAMPT provides the mechanism to replenish the NAD pool in human metabolism. In addition to its role in redox biochemistry, NAD fuels the sirtuins (SIRTs) to regulate transcription factors involved in pathways linked to inflammation, diabetes and lifespan. NAMPT-mediated lifespan expansion has caused a focus on the catalytic mechanism, regulation and inhibition of NAMPT. Structural, mechanistic and inhibitor design all contribute to a developing but yet incomplete story of NAMPT function. Although the first generation of NAMPT inhibitors has entered clinical trials, disappointing outcomes suggest more powerful and specific inhibitors will be needed. Understanding the ATP-linked mechanism of NAMPT and the catalytic site machinery may permit the design of improved NAMPT inhibitors as more efficient drugs against cancer.

Abstract LB-393: The cancer metabolism inhibitor MPC-9528 induces tumor regression in xenograft models with multiple dosing schedules by causing rapid and sustained reduction in tumor NAD

Abstract Introduction: MPC-9528 is a potent, selective, orally bioavailable inhibitor of nicotinamide phosphoribosyltransferase (Nampt), which catalyzes the rate-limiting step for NAD biosynthesis from nicotinamide. Inhibition of Nampt by MPC-9528 results in cell death as a consequence of NAD depletion and inhibition of ATP synthesis. MPC-9528 is a potent tumoricidal agent of cancer cell lines of diverse origin and shows anti-tumor activity in multiple xenograft models. Here we explore the determinants of efficacy in xenograft models by comparing the effects of MPC-9528 oral dosing schedules in terms of both tumor NAD depletion and survival. Methods: HT1080 human fibrosarcoma cells were implanted subcutaneously into athymic mice (nu/nu) for tumor studies. Dosing was initiated when median tumor volume was >100 mm3. MPC-9528 was dosed orally, either once weekly, once daily, or twice daily, for a total of two or three weeks. For pharmacodynamic (PD) studies, animals were dosed with MPC-9528 and tumors were collected at various times post-dosing for NAD determination by LC-MS/MS. Results: MPC-9528 (75 mg/kg) dosed once weekly for three weeks in a HT1080 xenograft model resulted in 75% tumor regression on study Day 23, 8 days after the last dose. The ED50 for anti-tumor activity with this schedule was 44 mg/kg and doses at or below 35 mg/kg showed no anti-tumor activity. All doses were equally well-tolerated with <10% change in median body weight. In the same model, MPC-9528 dosed once daily at 10 mg/kg or twice daily at 3 mg/kg for 14 days showed 91% and 92% tumor regression, respectively, by the end of dosing on study Day 15. Two animals in each of the MPC-9528 dose groups showed complete tumor regression. MPC-9528 (4 mg/kg) dosed twice daily resulted in complete tumor regression in 9 out of 10 animals. The plasma concentration versus time profiles of MPC-9528 dosed at 3, 10 or 75 mg/kg indicated that anti-tumor activity was not dependent on maximal concentration of the compound in blood or the total absorbed dose but rather on maintenance of compound concentration above a threshold efficacious concentration. PD studies revealed a rapid and sustained reduction of NAD in tumors from mice dosed with MPC-9528 and that the magnitude and kinetics of NAD change depended on the dose and schedule. Conclusions: MPC-9528 administration results in regression of tumors when plasma concentrations of compound are maintained above a threshold. MPC-9528 shows comparable efficacy when given intermittently, on a weekly schedule, or continuously on a once, or twice daily schedule. This unique attribute of MPC-9528 reflects its mechanism of action as a cell metabolism inhibitor that functions by inhibiting NAD synthesis in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-393. doi:10.1158/1538-7445.AM2011-LB-393

The NAD+/NADH redox state in astrocytes: Independent control of the NAD+ and NADH content

The intracellular redox state is established by several redox pairs, such as NAD(+) /NADH and NADP(+) /NADPH and glutathione. This redox state is a crucial determinant of cellular metabolism and function. Astrocytes are an important cell population contributing to brain metabolism and brain energy supply, so a careful control of these redox pairs is essential for proper brain function. Despite this, little is known about control of the NAD(+) and NADH content within the brain or in astrocytes. Therefore, we here analyzed the NAD(+) and NADH content of mouse tissue and cultured cortical astrocytes. The NAD(+) /NADH ratio increased in most tissues during development from newborn to adult mice. The basal redox ratio of cultured astrocytes was about 3.8 and similar to the redox ratio of the cortex of newborn mice. Although the NADH content of these cells was highly sensitive to the concentration of energy substrates and to modulation of energy metabolism, the NAD(+) content was surprisingly constant under these conditions. In contrast, application of nicotine amide or nicotinamide mononucleotide, which are precursors for NAD(+) biosynthesis, slowly increased NAD(+) content while leaving NADH levels unaffected. Finally, inhibiting the NAD(+) -degrading enzyme poly-(ADP-ribose)-polymerase increased NAD(+) content slightly without affecting NADH levels, whereas inhibition of sirtuins had no effect. These results indicate that, in addition to converting NAD(+) to NADH and vice versa during redox reactions, the content of both partners of this redox pair is additionally controlled by other mechanisms.

Structural and Kinetic Characterization of NAD Synthetase from H. sapiens and M. tuberculosis

NAD synthetase catalyzes the ATP‐dependent NAD formation from nicotinic acid adenine dinucleotide in NAD biosynthesis. This enzyme is essential for survival of Mycobacterium tuberculosis but not of humans. In this work, we are determining structural and mechanistic differences between NAD synthetase from M. tuberculosis (NAD synthetaseTB) and from Homo sapiens (NAD synthetaseHS) using x‐ray crystallography and kinetic analysis. NAD synthetaseTB is homooctameric with a 40 Å long inter‐subunit ammonia tunnel that allows transport of ammonia from the glutaminase active site to the synthetase active site. Structural complexes of NAD synthetaseTB with ligands have been solved within a 2.6–3.0 Å resolution, revealing key amino acid residues responsible for substrate binding and catalysis. In addition, NAD synthetaseHS expressed in Sf9 insect cells was co‐crystallized with an intermediate analog for structural comparison. This structure solved at a 3.0 Å resolution using molecular replacement and kinetic characterization are here reported. The crystal structures and kinetic parameters of both NAD synthetaseHS and NAD synthetaseTB can help clarify the basis of substrate recognition and specificity, leading to the design of high affinity inhibitors specifically targeting NAD synthetaseTB.

Hepatic FoxOs Regulate Lipid Metabolism via Modulation of Expression of the Nicotinamide Phosphoribosyltransferase Gene

FoxO transcription factors have been implicated in lipid metabolism; however, the underlying mechanisms are not well understood. Here, in an effort to elucidate such mechanisms, we examined the phenotypic consequences of liver-specific deletion of three members of the FoxO family: FoxO1, FoxO3, and FoxO4. These liver-specific triply null mice, designated LTKO, exhibited elevated triglycerides in the liver on regular chow diet. More remarkably, LTKO mice developed severe hepatic steatosis following placement on a high fat diet. Further analyses revealed that hepatic NAD(+) levels and Sirt1 activity were decreased in the liver of the LTKO mice relative to controls. At the mechanistic level, expression profile analyses showed that LTKO livers had significantly down-regulated expression of the nicotinamide phosphoribosyltransferase (Nampt) gene encoding the rate-limiting enzyme in the salvage pathway of NAD(+) biosynthesis. Luciferase reporter assays and chromatin immunoprecipitation analyses demonstrated that Nampt is a transcriptional target gene of FoxOs. Significantly, overexpression of Nampt gene reduced, whereas knockdown increased, hepatic triglyceride levels in vitro and in vivo. Thus, FoxOs control the Nampt gene expression and the NAD(+) signaling in the regulation of hepatic triglyceride homeostasis.

Niacin (Vitamin B3, Nicotinic Acid) Decreases Apolipoprotein B (ApoB) and VLDL Secretion from Mouse Hepatocytes

Niacin reduces plasma atherogenic apolipoprotein B (apoB)‐containing lipoproteins. To further understand if part of the mechanism of the VLDL‐lowering effect is on secretion of apoB‐containing lipoproteins, we isolated primary hepatocytes from Apobec1−/ − mice, which, like human liver, express apoB100 only. Niacin was well tolerated by hepatocytes showing no hepatotoxicity up to 100 mM. Metabolic labeling and pulse‐chase studies showed that niacin increased intracellular apoB100 degradation by 30%, decreased VLDL secretion from hepatocytes by 40%. These effects were found depend on medium supplementation of nicotinamide, the amide of niacin, which supplies sufficient NAD biosynthesis. The nicotinamide supplementation resulted in a dose‐dependent increase of apoB100 secretion and recovery, suggesting that adequate cellular NAD level is important for maximum VLDL secretion and a critical condition for niacin‐induced apoB100 degradation. Intraperitoneal niacin administration to Apobec1−/− mice caused a 40% decrease in the hepatic VLDL‐triglyceride production rate in vivo. Taken together, these data suggest that niacin decreases apoB100 and VLDL secretion from hepatocytes. The molecular mechanism responsible for these effects of niacin on the assembly and secretion steps of apoB100‐containing lipoprotein is under further investigation. Supported by grants from the NIH (HL58541) and Merck & Co., Inc.

Liver X Receptor (LXR) activation negatively regulates visfatin expression in macrophages

Adipose tissue macrophages (ATM) are the major source of visfatin, a visceral fat adipokine upregulated during obesity. Also known to play a role in B cell differentiation (pre-B cell colony-enhancing factor (PBEF)) and NAD biosynthesis (nicotinamide phosphoribosyl transferase (NAMPT)), visfatin has been suggested to play a role in inflammation.Liver X Receptor (LXR) and Peroxisome Proliferator-Activated Receptor (PPAR)γ are nuclear receptors expressed in macrophages controlling the inflammatory response. Recently, we reported visfatin as a PPARγ target gene in human macrophages. In this study, we examined whether LXR regulates macrophage visfatin expression. Synthetic LXR ligands decreased visfatin gene expression in a LXR-dependent manner in human and murine macrophages. The decrease of visfatin mRNA was paralleled by a decrease of protein secretion. Consequently, a modest and transient decrease of NAD+ concentration was observed. Interestingly, LXR activation decreased the PPARγ-induced visfatin gene and protein secretion in human macrophages.Our results identify visfatin as a gene oppositely regulated by the LXR and PPARγ pathways in human macrophages.

Comparative genomics of NAD(P) biosynthesis and novel antibiotic drug targets

NAD(P) is an indispensable cofactor for all organisms and its biosynthetic pathways are proposed as promising novel antibiotics targets against pathogens such as Mycobacterium tuberculosis. Six NAD(P) biosynthetic pathways were reconstructed by comparative genomics: de novo pathway (Asp), de novo pathway (Try), NmR pathway I (RNK-dependent), NmR pathway II (RNK-independent), Niacin salvage, and Niacin recycling. Three enzymes pivotal to the key reactions of NAD(P) biosynthesis are shared by almost all organisms, that is, NMN/NaMN adenylyltransferase (NMN/NaMNAT), NAD synthetase (NADS), and NAD kinase (NADK). They might serve as ideal broad spectrum antibiotic targets. Studies in M. tuberculosis have in part tested such hypothesis. Three regulatory factors NadR, NiaR, and NrtR, which regulate NAD biosynthesis, have been identified. M. tuberculosis NAD(P) metabolism and regulation thereof, potential drug targets and drug development are summarized in this paper.

Acetylation of p53 Protein Is Crucial for Its Function In Myeloid Cells.

AbstractAbstract 1480Severe congenital neutropenia (CN) is a heterogeneous disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 × 109/L. G-CSF treatment increases blood neutrophil counts in more than 90% of individuals with CN. CN is also considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop Acute Myelocytic Leukemia (AML) or Myelodysplastic Syndrome (MDS). Surprisingly no mutations in genes, which typically occur in primary AML/MDS patients, were detected in CN patients who developed leukemia. But studies in CN patients reveal a high association of G-CSF receptor mutation and the incidence of leukemia, indicating the dysregulation of certain factors downstream of G-CSF receptor signalling. Recently, we reported that Nicotinamide Phosphoribosyltransferase (NAMPT), a protein involved in biosynthesis of NAD+, was significantly increased in CN patients treated with G-CSF as compared to healthy individuals. Elevated NAMPT/NAD+ levels correlated with increased levels of SIRT1, a NAD+-dependent deacetylase which is involved in the deacetylation of histone and non-histone proteins e.g. p53. The acetylation of tumor-suppressor p53 is considered necessary for its transcriptional activation, while SIRT1-mediated deacetylation of p53 has been shown to attenuate the transcriptional activity of p53. Therefore, we asked if deacetylation-dependent inactivation of p53 might play a role in leukemic transformation in CN patients. In this study we demonstrate that the presence of NAMPT or NAD+ enhances the SIRT1-mediated deacetylation of p53 in both the 293T cell line and the promyelocytic leukemia NB4 cell line. Treatment with exogenous recombinant NAMPT also leads to a decrease in the acetylation levels of endogenous p53 in CD34+ cells. The cyclin-dependent kinase inhibitor 1A (p21, Cip1) protein is a well-known target of p53 and is involved in cell cycle arrest. We have shown that over-expression of NAMPT leads to down-regulation of p21 mRNA, and specific knockdown of SIRT1 leads to up-regulation of p21 mRNA. The presence of NAMPT also decreases the mRNA levels of p21 in both NB4 and CD34+ cells. The compound FK866 specifically inhibits NAMPT and has recently entered clinical trials as a potential chemotherapeutic agent. In a recent preclinical in vitro study FK866 has been shown to elicit massive cell death in numerous leukemia/lymphoma cell lines, but the underlying molecular mechanism remains unknown. We tested if inhibition of NAMPT using FK866 enhances the tumor-supressing role of p53 by increasing its acetylation levels. We have demonstrated that the treatment of NB4 cells with FK866 increases the acetylation of endogenous p53, and this increased acetylation is in part due to decreased interaction of p53 with SIRT1. In addition, the mRNA levels of p21 down-regulated in CD34+ and NB4 cells on treatment with NAMPT were up-regulated on use of FK866. Knockdown of p53 using specific shRNA against p53 inhibits the expression of p21 and treatment with FK866 under p53 knockdown does not induce the expression of p21 when compared to control cells. In functional studies we show that over-expression of NAMPT leads to increased proliferation of both 293T and NB4 cells. Treatment with FK866 leads to increased death of NB4 cells compared to the cells in which p53 has been silenced, due to the lack of p53 available to be acetylated. Taken together, our conclusion is that NAMPT/NAD+ activated SIRT1 mediates deacetylation of p53 leading to down-regulation of the downstream target gene p21. This inhibition of the tumor-suppressor functions of p53 might be involved in the leukemic transformation seen in CN. Disclosures:No relevant conflicts of interest to declare.

Porcine NAMPT gene: search for polymorphism, mapping and association studies

NAMPT encodes an enzyme catalysing the rate-limiting step in NAD biosynthesis. The extracellular form of the enzyme is known as adipokine visfatin. We detected SNP AM999341:g.669T>C (referred to as 669T>C) in intron 9 and SNP FN392209:g.358A>G (referred to as 358A>G) in the promoter of the gene. RH mapping linked the gene to microsatellite SW944. Linkage analysis placed the gene on the current USDA – USMARC linkage map at position 92 cM on SSC9. Association analyses were performed in a wild boar × Meishan F2 family (W × M), with 45 traits recorded (growth and fattening, fat deposition, muscling, meat quality, stress resistance and other traits), and in a commercial Landrace × Chinese-European (LCE) synthetic population with records for 15 traits (growth, fat deposition, muscling, intramuscular fat, meat colour and backfat fatty acid content). In the W × M, SNP 669T>C was associated with muscling, fat deposition, growth and fattening, meat quality and other traits and in the LCE with muscling, meat quality and backfat fatty acid composition. In the W × M, SNP 358A>G was associated with muscling, fat deposition, growth and other traits. After correction for multiple testing, the NAMPT haplotypes were associated in the W × M with, in descending order, muscling (q = 0.0056), growth (q = 0.0056), fat deposition (q = 0.0109), fat-to-meat ratio (q = 0.0135), cooling losses (q = 0.0568) and longissimus pHU (q = 0.0695). The SNPs are hypothesized to be in linkage disequilibrium with a causative mutation affecting energy metabolism as a whole rather than fat metabolism alone.

Regulation of the Expression of Genes Involved in NAD De Novo Biosynthesis in Corynebacterium glutamicum

Three genes, nadA, nadB, and nadC, involved in NAD de novo biosynthesis are broadly conserved in the genomes of numerous bacterial species. In the genome of Corynebacterium glutamicum, nadA and nadC but not nadB are annotated. The nadA and nadC genes are located in a gene cluster containing two other genes, designated ndnR and nadS herein. ndnR encodes a member of the Nudix-related transcriptional regulator (NrtR) family. nadS encodes a homologue of cysteine desulfurase involved in Fe-S cluster assembly. The gene cluster ndnR-nadA-nadC-nadS is genetically characterized herein. Mutant strains deficient in nadA, nadC, or nadS required exogenous nicotinate for growth, and the nicotinate auxotrophy was complemented by introduction of the corresponding gene in trans, indicating that each of these genes is essential for growth in the absence of an exogenous source of NAD biosynthesis. The results of reverse transcriptase PCR analyses and ndnR promoter-lacZ expression analyses revealed that the expression of ndnR, nadA, nadC, and nadS genes was markedly and coordinately repressed by nicotinate. The expression of these genes was enhanced by the disruption of ndnR, resulting in the loss of the nicotinate-responsive regulation of gene expression. These results suggest that NdnR acts as a transcriptional repressor of NAD de novo biosynthesis genes and plays an essential role in the regulation of the response to nicotinate.

Visceral adipose tissue visfatin in nonalcoholic fatty liver disease

Background. Visfatin is a novel adipocytokine predominantly expressed and secreted by visceral adipose tissue. It is realized for its multiple functions of central importance in NAD biosynthesis, innate immunity and inflammation. Its phosphoribosyl transferase activity regulates cellular energetics and NAD dependent enzymes such as SIRTUINS. Although its expression in various tissues and circulating levels are documented, visceral visfatin levels in Nonalcoholic fatty liver disease (NAFLD) patients have not been reported.Objective. The aim of the present study was to assess visceral adipose tissue visfatin levels in NAFLD.Materials and methods. A total of 115 patients undergoing diagnostic laparoscopy were recruited in the study and categorized into two groups based on standard criteria for NAFLD. Visceral adipose tissue TNF-α, IL-6 and visfatin levels were measured by ELISA. Blood glucose, lipids, liver enzymes and non esterified fatty acids (NEFA) were estimated using standard procedures. Formalin fixed, Hematoxylene Eosin stained liver biopsy specimens were examined for the presence of steatosis and the degree of steatosis was ascertained as per Brunt’s classification.Results. The visceral visfatin level declined significantly (P < 0.001) in all groups of NAFLD as compared to non NAFLD group, while plasma NEFA level increased with progressive steatosis (P < 0.02). Significant increase in TNF a was observed in all groups of NAFLD, while IL-6 increased in NASH only.Conclusion. A significant decline in visceral adipose tissue visfatin level was found to be associated with degree of steatosis in NAFLD patients.

On the catalytic role of the active site residue E121 of E. colil-aspartate oxidase

l-aspartate oxidase (LASPO) is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD +. The enzyme oxidizes l-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor. In accordance with its catalytic activities, LASPO displays strong primary and tertiary structure similarity with the flavin containing subunit of the proteins belonging to the succinate dehydrogenase/fumarate reductase family. The similarity extends to the active site residues, with LASPO differing from the other enzymes of the family only for the presence of a conserved glutamate (E121), which is substituted by apolar amino acids in the other enzymes. Three complementary approaches have been used to define the role of E121 in LASPO: characterization of mutants (E121A, E121Q, E121D and E121K), investigation of the catalytic activities of WT and mutants towards substrates and substrate analogues and molecular docking studies. All mutants retain fumarate reductase activity. On the contrary, all mutants lack l-aspartate oxidase activity, although retaining the ability to bind l-aspartate (except for E121K). These results and investigations on the oxidase activity towards substrate analogues suggest that the roles of E121 in catalysis include orienting l-aspartate in a productive binding mode and favouring proton abstraction from C2 by an active site base. Molecular docking studies of the substrate ( l-aspartate), inhibitor ( d-aspartate) and product (imino aspartate) in the active site of LASPO confirm that (a) the substrate/product energetically favoured orientation in the active site supports the conclusions reported above, (b) E121 interacts favourably with the charged amino group of the substrate and (c) different ligands might assume different orientations in the active site of the enzyme.