Lowered NAD Research Articles

Large changes in NAD levels associated with CD38 expression during HL-60 cell differentiation

NAD is an important cofactor involved in multiple metabolic reactions and as a substrate for several NAD-dependent signalling enzymes. One such enzyme is CD38 which, alongside synthesising Ca2+-releasing second messengers and acting as a cell surface receptor, has also been suggested to play a key role in NAD+ homeostasis. CD38 is well known as a negative prognostic marker in B-CLL but the role of its enzymatic activity has not been studied in depth to date. We have exploited the HL-60 cell line as a model of inducible CD38 expression, to investigate CD38-mediated regulation intracellular NAD+ levels and the consequences of changes in NAD+ levels on cell physiology. Intracellular NAD+ levels fell with increasing CD38 expression and this was reversed with the CD38 inhibitor, kuromanin confirming the key role of CD38 in NAD+ homeostasis. We also measured the consequences of CD38 expression during the differentiation on a number of functions linked to NAD+ and we show that some but not all NAD+-dependent processes are significantly affected by the lowered NAD+ levels. These data suggest that both functional roles of CD38 might be important in the pathogenesis of B-CLL.

Effect of Exercise on Cardiac Tissue Oxidative and Inflammatory Mediators in Chronic Kidney Disease

Background: Chronic renal failure (CRF) results in diminished physical activity and increased risk of cardiovascular disease (CVD). CVD risk factors are raised by sedentary life style and ameliorated by physical fitness in the general population. Accordingly, exercise improves hypertension, endothelial dysfunction, insulin resistance, dyslipidemia, inflammation and oxidative stress in high-risk populations. This study was designed to explore the effect of exercise on oxidative and inflammatory mediators in the left ventricle (LV) of CRF rats. Methods and Results: One week after 5/6 nephrectomy female rats were housed in either regular cages or cages equipped with running wheels for 4 weeks. Sham-operated rats housed in regular cages served as controls. Sedentary CRF rats exhibited azotemia, hypertension, anemia, oxidative stress, activation of NF-κB and upregulations of reactive oxygen species-generating enzyme, NAD(P)H oxidase, MCP-1, cyclooxygenase-2 (COX-2), and PAI-1 in LV. The CRF rats assigned to the exercise group ran 6.8 ± 0.7 km/day and 72 ± 8 min/day. Voluntary exercise reversed NF-κB activation and lowered NAD(P)H oxidase, PAI-1, MCP-1 and COX-2 abundance, increased LV mass by raising myofibrillar proteins and ameliorated anemia without affecting renal function or arterial pressure. Conclusions: CRF resulted in upregulation of prooxidant/proinflammatory pathways in LV. These changes were ameliorated by exercise, which indicates the potential cardiovascular benefit of exercise in renal insufficiency.

Ca2+ controls slow NAD(P)H oscillations in glucose‐stimulated mouse pancreatic islets

Exposure of pancreatic islets of Langerhans to physiological concentrations of glucose leads to secretion of insulin in an oscillatory pattern. The oscillations in insulin secretion are associated with oscillations in cytosolic Ca(2+) concentration ([Ca(2+)](c)). Evidence suggests that the oscillations in [Ca(2+)](c) and secretion are driven by oscillations in metabolism, but it is unclear whether metabolic oscillations are intrinsic to metabolism or require Ca(2+) feedback. To address this question we explored the interaction of Ca(2+) concentration and islet metabolism using simultaneous recordings of NAD(P)H autofluorescence and [Ca(2+)](c), in parallel with measurements of mitochondrial membrane potential (DeltaPsi(m)). All three parameters responded to 10 mm glucose with multiphasic dynamics culminating in slow oscillations with a period of approximately 5 min. This was observed in approximately 90% of islets examined from various mouse strains. NAD(P)H oscillations preceded those of [Ca(2+)](c), but their upstroke was often accelerated during the increase in [Ca(2+)](c), and Ca(2+) influx was a prerequisite for their generation. Prolonged elevations of [Ca(2+)](c) augmented NAD(P)H autofluorescence of islets in the presence of 3 mm glucose, but often lowered NAD(P)H autofluorescence of islets exposed to 10 mm glucose. Comparable rises in [Ca(2+)](c) depolarized DeltaPsi(m). The NAD(P)H lowering effect of an elevation of [Ca(2+)](c) was reversed during inhibition of mitochondrial electron transport. These findings reveal the existence of slow oscillations in NAD(P)H autofluorescence in intact pancreatic islets, and suggest that they are shaped by Ca(2+) concentration in a dynamic balance between activation of NADH-generating mitochondrial dehydrogenases and a Ca(2+)-induced decrease in NADH. We propose that a component of the latter reflects mitochondrial depolarization by Ca(2+), which reduces respiratory control and consequently accelerates oxidation of NADH.

Cloning, expression, and catalytic triad of recombinant arylformamidase

Arylformamidase (AFMID) is the second enzyme of the kynurenine pathway metabolizing tryptophan to nicotinic acid and nicotinamide adenine dinucleotide cofactors. Inhibition of AFMID by organophosphorus insecticides in developing chicken embryos is correlated with lowered NAD levels and severe teratogenesis. The cDNA sequence previously identified for mouse liver AFMID (AF399717) (MW 34229) was cloned and expressed in Escherichia coli. Residues identified as potential catalytic triad members (S162, D247, and H279) through sequence motif and homology modeling were mutated to alanine to probe their contributions to enzyme activity. The wild-type and mutant AFMIDs were expressed as amino terminal 6× His-tagged recombinant proteins to facilitate purification. Three chromatography steps isolated highly purified proteins for enzyme activity comparisons. Expressed AFMID showed high activity, 42 ± 1 μmol/min/mg protein, for its natural substrate, N-formyl- l-kynurenine. The same K m (0.18–0.19 mM) was observed for expressed and native cytosolic AFMID. The single mutants (S162A, D247A, and H279A) lost essentially all (>99%) activity. The predicted catalytic triad of S162, D247, and H279 is therefore confirmed by site-directed mutagenesis.

In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation.

MCF-7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP-treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increases in the enzyme activities involved in glutamate degradation and in the malate-aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6-bisphosphate and pyruvate accumulated in AMP-arrested cells. Based on the lowered NAD level in the AMP-treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P-ribose-PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented.

Critical time periods and the effect of tryptophan in malathion-induced developmental defects in [formula omitted] embryos

Xenopus laevis embryos were exposed to the organophosphorus insecticide malathion or its metabolite malaoxon during the first four days of development. The compounds produced the following defects in a dose dependent manner: reduced size, abnormal pigmentation, abnormal gut, enlargement of the atria and aorta, bent notochord and lowered NAD + levels. Notochords were bent downward between the third and sixth somites with concurrent compression of the somites. Anterior intestine diameter was increased with a concurrent reduction in the number of intestinal loops. When tryptophan was administered along with malathion, NAD + was measured at control levels or above, yet neither severity nor incidence of defects were reduced. The reduction of NAD + does not seem to be responsible for the defects seen in Xenopus as occurs for some defects in avian species. In experiments to determine critical time of exposure, the final 48 h, which follow organogenesis and are primarily devoted to growth and development, seem most critical.

Effects of Streptozotocin Exposure In Vitro on the Replication and Repair of DNA in Fetal Rat Pancreatic Islet Cells

It was recently proposed that the role for poly(ADP-ribose) synthetase during DNA repair was exerted via a depletion of cellular NAD in order to slow down energy-requiring processes in the cell, notably DNA replication. This would enable the cell to more efficiently repair damaged DNA. In the present study fetal rat pancreatic islet cells were exposed to 2.2 mM streptozotocin (SZ). Using [3H]thymidine labeling and autoradiographic techniques, it was found that cellular nuclear silver grain counts, an index of DNA repair synthesis, were doubled after SZ exposure. However, autoradiographically measured DNA replication remained unaffected. Cellular NAD + NADH contents were reduced by 60% in the SZ-treated islets. Estimates of the poly(ADP-ribose) synthetase activity showed that this was doubled in islets exposed to SZ. Immediately after the SZ treatment the islets exhibited a 35% reduction in insulin secretion in response to 16.7 mM glucose. Taken together, the present findings do not favor the suggested role for poly(ADP-ribose) synthetase during DNA repair. Rather we observed an increased activity of this enzyme, a lowered cellular NAD + NADH content, and an increased rate of DNA repair synthesis concomitant with an unchanged DNA replicative rate in the SZ-exposed islets.

Plasmodium berghei: Gluconeogenesis in the infected mouse liver studied by 13C nuclear magnetic resonance

Using 13C nuclear magnetic resonance, we have compared the gluconeogenic activity of perfused livers isolated from normal starved mice and mice highly parasitized with Plasmodium berghei, using [2- 13C]pyruvate as substrate. In both types of livers, 13C labeling of glucose carbons occurred in positions 1, 2, 5, and 6. The equal proportions of [1,6- 13C]- and [2,5- 13C]glucose in livers from malarial and normal mice suggests that pyruvate enters the gluconeogenic pathway directly and, to an equal extent, via the tricarboxylic acid cycle. The normalized signal heights indicated that at a given time after the addition of [2- 13C]pyruvate the degree of 13C labeling in glucose carbons was reduced in livers from malarial animals, when compared to livers from normal animals. During the course of the perfusion experiment, the [2- 13C]lactate resonance signal was always more intense from livers of malarial animals than from normal animals. A reduced activity of hepatic gluconeogenesis in malarial animals was further confirmed by a separate set of perfusion experiments which showed a 56% reduction of the measured rate of glucose production in livers from malarial animals, with respect to that of normal animals. A lowered NAD NADH ratio in livers from malarial animals would explain the increased proportion of lactate observed in the spectra and be related to a decreased gluconeogenic rate. A more reduced oxidoreduction level in the hepatocytes of a malarial animal would result from a defect in the oxidative phosphorylation activity of mitochondria.

Do organophosphate insecticides inhibit the conversion of tryptophan to NAD+ in ovo?

The hypothesis that organophosphate (OP) insecticides reduce the NAD+ levels of chick embryos by inhibiting kynurenine formamidase was tested. Fertile chicken eggs at 3 days of incubation were treated with a teratogenic dose of the organophosphate insecticide diazinon (DZN) in the presence or absence of exogenous L-tryptophan or nicotinamide, or one of the metabolic intermediates (L-kynurenine, 3-hydroxyanthranilic acid, quinolinic acid) between tryptophan and NAD+. By day 10 of development, DZN reduced the NAD+ content of the hind limbs of the embryos to less than 20% of normal and by day 15 it caused severe type I and type II teratogenic responses. The co-presence of tryptophan or one of its metabolites served to maintain the NAD+ levels of DZN-treated embryos close to or above normal and significantly alleviated the symptoms of type I teratisms. Tryptophan is virtually as effective as most of its metabolites in suppressing the effects of DZN on the NAD+ content and physical development of the embryos. This equivalence does not support the proposition that the inhibition of kynurenine formamidase causes the lowered NAD+ levels involved in OP-induced type I teratogenesis. It is consistent with the concept that the insecticide acts to decrease the availability of tryptophan to the embryo.

Relation of yolk sac membrane kynurenine formamidase inhibition to certain teratogenic effects of organophosphorus insecticides and of carbaryl and eserine in chicken embryos

One type of teratogenic effect induced in chicken embryos by a variety of organophosphorus (OP) and mcthylcarbamate (MC) insecticides is associated with lowered embryo NAD levels. This appears to result from inhibition of kynurenine formamidase, which impairs conversion of tryptophan to essential pyridine nucleotide cofactors. The yolk sac membrane and the embryonic liver contain two types of kynurenine formamidase, one sensitive and the other relatively insensitive to OP and MC inhibitors. The eserine-sensitive enzyme accounts for ~ 26 percent of the overall activity for hydrolysis of N 1N x- diformyl- l-kynurenine . In studies with twenty-one OP insecticides and two MC compounds injected at day 4 of incubation, the inhibition of the eserine-sensitive kynurenine formamidase at day 9 of incubation correlates well with the lowering of NAD levels at day 12 of incubation and the intensity of teratogenic signs. Teratogens of this type combine high potency for inhibiting the relevant kynurenine formamidase in the yolk sac membrane early in development, and possibly in the liver at later stages, with adequate in tiro stability to maintain the inhibition through critical stages of embryogenesis. Thus, kynurenine formamidase joins acetylcholinesterase in the list of OP- and MC-sensitive enzymes of critical physiological significance.

Kynurenine formamidase inhibition as a possible mechanism for certain teratogenic effects of organophosphorus and methylcarbamate insecticides in chicken embryos

At least two types of developmental anomalies are induced in chicken embryos by certain organophosphorus (OP) and methylcarbamate (MC) insecticides. One of them (Type I) leads to micromelia and abnormal feathering and another (Type II) involves arthrogryposis, wry neck and rumplessness. Type I but not type II teratogenesis is associated with a lowered embryo NAD level and is alleviated on restoring the NAD level by administration of intermediates in the tryptophan to NAD biosynthetic pathway. These and other observations with chicken embryos suggest but do not in themselves establish that impairment in the conversion of tryptophan to NAD, possibly by inhibition of kynurenine formamidase, leads to type I teratogenesis. This hypothesis is supported by finding that mouse liver kynurenine formamidase is extremely sensitive to in vivo inhibition by those OP and MC compounds which are the most potent NAD lowering agents and teratogens in the chicken embryo, i.e. crotonamide phosphates and pyrimidyl phosphorothionates such as dicrotophos and diazinon and MC compounds such as carbaryl. Teratological or other toxicological manifestations of kynurenine formamidase inhibition are probably restricted to species and developmental stages where reduced enzyme activity significantly impairs maintenance of normal levels of NAD or other essential biochemicals derived from kynurenine.

Niacin nucleotide synthesis and glycolytic activity in erythrocytes of patients suffering from pellagra

Erythrocytes of subjects with pellagra exhibit impaired synthesis of NAD from nicotinic acid in vitro. A detailed time course study of in vitro incorporation of 14C-nicotinic acid into NAD and its intermediates namely nicotinic acid mononucleotide (NaMN), nicotinic acid adenine dinucleotide (NaAD) was made in erythrocytes of patients with pellagra and normal subjects. The incorporation of 14C-nicotinic acid into the total nucleotides (NAD + NaAD + NaMN) was significantly reduced in the erythrocytes of pellagrins at 6 and 21 hours. Relatively more radioactivity was found in NaMN during these periods indicating thereby, that the conversion of NaMN to NaAd may be defective in pellagra. This was confirmed by label chasing experiment, in which NaMN of the intact cells was prelabeled with 14C-nicotinic acid, and its further conversion to NaAD was followed in vitro. The activity of nicotinate phosphoribosyl transferase, was normal in the erythrocytes of pellagrins. Concentration of ATP and glycolytic activity of the erythrocytes were investigated under conditions for NAD synthesis. A significant reduction in the basal levels of ATP was observed in pellagrins. As compared to normals, the rate of glycolytic activity, and the levels of ATP during NAD synthesis, was significantly lower in the erythrocytes of pellagrins. From the data obtained, it was concluded that the fall in ATP concentration found in the erythrocytes of pellagrins may not be the limiting factor for the optimal synthesis of NAD. Instead, the defect in lowered NAD synthesis, may be due to reduced activity of the enzyme NaMN adenyl transferase, which converts NaMN to NaAD.

The use of the oxidant “diamide” for studying the non-mitochondrial reducing capacity of Ehrlich ascites tumor cells

Diazenedicarboxylic acid bis(N,N-dimethylamide), (“diamide”) lowered non-mitochondrial NAD(P)H stores in Ehrlich ascites tumor cells in vitro by indirect reactions involving oxidation of glutathione and reduction of GSSG via glutathione reductase. The concentrations of diamide used did not alter the mitochondrial capacity to reduce NAD(P)H under anaerobic conditions. “Endogenous substrates” could be removed by multiple additions of diamide which indirectly inhibited NAD(P)H and GSH regeneration because of a lack of cellular reducing capacity. The regenerative power of the cells was restored by the addition of glucose. We conclude that diamide may prove to be a useful agent for studying the reducing capacity as well as the redox compartmentalization of cells in vitro.