Nicotinamide methylation and its relation to NAD synthesis in rat liver tissue culture. Biochemical basis for the physiological activities of 1-methylnicotinamide

The mode of [14C]nicotinamide conversion to NAD and 1-methylnicotinamide and the effects of exogenous 1-methylnicotinamide on this metabolic conversion were studied using rat liver slices incubated in a chemically defined culture medium. It was shown that at the physiological nicotinamide concentrations tested (11-500 microM), 1-methylnicotinamide is preferentially produced, rather than NAD. Upon increasing nicotinamide concentration to the levels that cause cytotoxicity (1-10 mM and higher), the rate of NAD synthesis dramatically increased and reached a level 6-fold higher than that of 1-methylnicotinamide. A dose-dependent inhibition (up to 60%) of NAD synthesis was seen by the exogenous addition of 1-methylnicotinamide; the degree of inhibition is affected also by the concentration of nicotinamide present as a precursor. A large depletion of intracellular ATP, associated with a marked accumulation of NAD, occurred in slices in response to the addition of high amounts of nicotinamide. However, the loss of ATP was overcome, when nicotinamide was given together with 1-methylnicotinamide. Finally, 1-methylnicotinamide per se was proven active in regulating cell growth by comparing the cytosolic activity of 1-methylnicotinamide oxidation of cultured RLC cells with that of rat liver. Thus, the previously observed growth stimulation of hepatic cells by 1-methylnicotinamide can reasonably been explained by its ATP-sparing effect due to the inhibition of NAD synthesis, a reaction which requires ATP.     

Nicotinamide methylation and its relation to NAD synthesis in rat liver tissue culture: Biochemical basis fro the physiological activities of 1-methylnicotinamide

The mode of [¹⁴C]lnicotinamide conversion to NAD and 1-methylnicotinamide and the effects of exogenous 1-methylnicotinamide on this metabolic conversion were studied using rat livers slices incubated in a chemically defined culture medium. It was shown that at the physiological nicotinamide concentrations tested (11–500 μM), 1-methylnicotinamide is preferentially produced, rather than NAD. Upon increasing nicotinamide concentration to the levels that cause cytotoxicity (1–10 mM and higher), the rate of NAD synthesis dramatically increased and reached a level 6-fold higher than that of 1-methylnicotinamide. A dose-dependent inhibition (up to 60%) of NAD synthesis was seen by the exogenous addition of 1-methylnicotinamide; the degree of inhibition is affected also by the concentrations of nicotinamide present as a precursor. A large depletion of intracellular ATP, associated with a marked accumulation of NAD, occurred in slices in response to the addition of high amounts of nicotinamide. However, loss of ATP was overcome, when nicotinamide was given together with 1-methylnicotinamide. Finally, 1-methylnicotinamide per se was proven active in regulating cell growth by comparing the cytosolic activity of 1-methylnicotinamide oxidation of cultured RLC cells with that of rat liver. Thus, the previously observed growth stimulation of hepatic cells by 1-methylnicotinamide can reasonably been explained by its ATP-sparing effect due to the inhibition of NAD synthesis, a reaction which requires ATP.     

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.     

Enhancement of DNA synthesis and cell proliferation by 1-methylnicotinamide in rat liver cells in culture: Implication for its in vivo role

1-Methylnicotinamide, a direct methylation product of nicotinamide, stimulates the DNA synthesis and proliferation of rat liver cells (RLC) in culture at concentrations higher than 20 μM. The effect of nicotinamide, which is a potent inhibitor of DNA synthesis and proliferation, is counteracted by 1-methylnicotinamide. The intracellular NAD concentration decreases within 2 h under 1-methylnicotinamide, whereas it increases in the presence of nicotinamide. The poly(ADP-ribose) synthesizing activity in the isolated nuclei remained unchanged. These results suggest a physiological role of 1-methylnicotinamide in the cell growth through a lowering of intracellular NAD level.     

Differential effects of kynurenine and tryptophan treatment on quinolinate immunoreactivity in rat lymphoid and non-lymphoid organs

Quinolinate is a tryptophan metabolite and an intermediary in nicotinamide adenine dinucleotide (NAD+) synthesis in hepatocytes. Kynurenine is an upstream metabolite in the same biochemical pathway. Under normal physiological conditions, kynurenine is thought to be produced primarily in the liver as an NAD+ precursor. However, during immune stimulation or inflammation, numerous extrahepatic tissues convert systemic tryptophan to kynurenine, and its concentration subsequently rises dramatically in blood. The fate and role of extrahepatic kynurenine are uncertain. In order to begin addressing this question, the present study was performed to determine which cell types can produce quinolinate from either systemic tryptophan or kynurenine. By using highly specific antibodies to protein-coupled quinolinate, we found that intraperitoneal injections of tryptophan led to increased quinolinate immunoreactivity primarily in hepatocytes, with moderate increases in tissue macrophages and splenic follicles. In contrast, intraperitoneal injections of kynurenine did not result in any significant increase in hepatocyte quinolinate immunoreactivity, but rather led to dramatic increases in immunoreactivity in tissue macrophages, splenic white pulp, and thymic medulla. These findings suggest that hepatocytes do not make significant use of extracellular kynurenine for quinolinate or NAD+ synthesis, and that, instead, extrahepatic kynurenine is preferentially metabolized by immune cells throughout the body. The possible significance of the preferential metabolism of kynurenine by immune cells during an immune response is discussed.     

Relationship between the ability of nicotinamide to maintain nicotinamide-adenine dinucleotide in rat liver cell culture and its effect on cytochrome P-450.

Rat hepatocytes cultured for 24 h lose 60% of their NAD content. Treatment with nicotinamide prevents the loss of NAD as well as the previously reported loss of cytochrome P-450, suggesting a possible causal relationship. However, isonicotinamide also prevents the loss of cytochrome P-450, but does not increase the concentration of NAD, demonstrating that the ability of nicotinamide to maintain cytochrome P-450 is not apparently related to its effect on the NAD content of cultured hepatocytes.     

Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.     

Pyridine nucleotide synthesis in 3T3 cells.

The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12-fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5-fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.     

Reactivation of NAD(H) biosynthetic pathway by exogenous NAD+ in Nil cells severely depleted of NAD(H)

The culture of Nil hamster fibroblasts in MEM lacking nicotinamide (NAm-MEM) leads to: (1) the rapid loss of intracellular total nicotinamide adenine dinucleotide (NAD(H)) content in these cells from a level of 150-200 pmoles/10(5) cells to less than 20 pmoles/10(5) cells; (2) the cessation of cell division and inhibition of DNA synthesis; and (3) a reduction of glucose consumption and lactic acid production. In most situations, following nicotinamide starvation, the restoration of intracellular NAD(H) follows rapidly the readdition of NAD+ (oxidized), nicotinamide mononucleotide (NMN), nicotinamide, or nicotinic acid. Resumption of cell division occurs after only a lag of about 24 hours. Nil cells subcultured for three consecutive times in the absence of nicotinamide (3(0) NAm- cells) exhibit different behavior. These severely starved cells are incapable of quickly restoring their intracellular NAD(H) content to normal levels when provided with any pyridine ring compound except NAD+. One-hour exposure of such cells to NAD+ allows utilization of nicotinamide to rapidly restore intracellular NAD(H). This short incubation with NAD+ does not result in any significant restoration of intracellular NAD(H) or lead to the accumulation of an intracellular pool of some precursor. This function of NAD+ as a stimulatory signal to the NAD(H)-biosynthetic pathway in severely starved Nil cells is a previously unreported role of NAD+, and does not require protein synthesis.     

Diabetic neuropathies. Metabolism of sorbitol in sciatic nerve tissue in streptozotocin diabetes

The increase of sorbitol and fructose levels caused by aldose reductase activation and sorbitol dehydrogenase inhibition were observed in sciatic nerve of streptozotocin-diabetic rats. Elevated polyol pathway activity has been implicated in the development of diabetic complications such as neuropathy. The regulation of polyol pathway enzymes is based on the changes of redox state of free nicotinamide nucleotides. The decrease of the NADP+/NADPH ratio in cytosolic compartment of sciatic nerve cells activated aldose reductase and the decrease of the NAD+/NADH ratio inhibited sorbitol dehydrogenase. Nicotinamide as a precursor of NAD+ biosynthesis increased the free NADP+/NADPH and NAD+/NADH ratios and inhibited the activity of polyol pathway. The sorbitol level decreased in sciatic nerve of nicotinamide-treated streptozotocin-diabetic rats as compared to non-treated ones. Thus, the data provide evidence for important role of nicotinamide, as an antidiabetic drug, in prevention or correction of diabetic neuropathy.     

Influence of streptozotocin upon the induction of tyrosine aminotransferase, the content of NAD and of 5-methyl cytosine in rat liver

The antibiotic, streptozotocin, has carcinostatic, carcinogenic, and diabetogenic properties. Moreover, it is capable of inducing the enzyme tyrosine aminotransferase in a permanent line of rat liver cells. In the present publication, the effects of streptozotocin upon the induction of tyrosine aminotransferase, NAD synthesis, and methylation of DNA in different organs were analyzed in vivo. If administered alone, streptozotocin slightly induced tyrosine aminotransferase. The induction of tyrosine aminotransferase caused by tryptophan or nicotinamide was inhibited by streptozotocin. Streptozotocin reduced the NAD content of the liver. NAD synthesis induced by tryptophan was reduced by streptozotocin, while that induced by nicotinamide was enhanced. DNA methylation in the form of 5-methyl cytosine was not influenced by streptozotocin.     

NAD metabolism and induction of tyrosine aminotransferase in the rat

The relationship between the NAD-metabolism and the induction of the tyrosine aminotransferase was studied. The content of NAD+ + NADH differs markedly from organ to organ. The highest values can be found in the liver. In intact animals tryptophan leads to an increase of NAD in liver and kidney, but not in brain and spleen. Nicotinamide, on the other hand, induces NAD synthesis in all the organs tested. In adrenalectomized animals, however, there is practically no rise of the NAD content after application of tryptophan contrary to the effect of nicotinamide. The enzyme tyrosine aminotransferase can be induced in intact animals by nicotinamide and tryptophan. This effect is much less pronounced in adrenalectomized animals. In adrenalectomized animals the induction of the tyrosine aminotransferase by tryptophan is markedly elevated by caffeine and theophylline. Under these conditions there is a significant increase of the NAD content as well. The tryptophan promoted induction of the tyrosine aminotransferase is influenced by inhibitors of the ADPR-transferase. The data presented give further evidence that the NAD adenoribosylation metabolism is involved in the induction of the tyrosine aminotransferase.     

The pathway of biosynthesis of nicotinamide–adenine dinucleotide in rat mammary gland

1. The pathway of NAD synthesis in mammary gland was examined by measuring the activities of some of the key enzymes in each of the tryptophan, nicotinic acid and nicotinamide pathways. 2. In the tryptophan pathway, 3-hydroxyanthranilate oxidase and quinolinate transphosphoribosylase activities were investigated. Neither of these enzymes was found in mammary gland. 3. In the nicotinic acid pathway, nicotinate mononucleotide pyrophosphorylase, NAD synthetase, nicotinamide deamidase and NMN deamidase were investigated. Both NAD synthetase and nicotinate mononucleotide pyrophosphorylase were present but were very inactive. Nicotinamide deamidase, if present, had a very low activity and NMN deamidase was absent. 4. In the nicotinamide pathway both enzymes, NMN pyrophosphorylase and NMN adenylyltransferase, were present and showed very high activity. The activity of the pyrophosphorylase in mammary gland is by far the highest yet found in any tissue. 5. The apparent K(m) values for the substrates of these enzymes in mammary gland were determined. 6. On the basis of these investigations it is proposed that the main, and probably only, pathway of synthesis of NAD in mammary tissue is from nicotinamide via NMN.     

Nicotinamide adenine dinucleotide metabolism in Candida albicans.

The functional pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis and their regulation were studied in the dimorphic fungus Candida albicans. The presence of a functional endogenous pathway of NAD biosynthesis from tryptophan was demonstrated. In addition, nicotinamide served as an efficient salvage precursor for NAD biosynthesis but nicotinate was not utilized. The pathway for nicotinamide utilization involved nicotinate and nicotinate nucleotides as intermediates, suggesting that the failure to utilize nicotinate involves a transport defect. The mechanisms that regulate NAD levels during exponential growth operated to maintain constant NAD levels when NAD biosynthesis occurred exclusively from endogenous or salvage pathways or from a combination of the two. The regulation also operated such that the salvage pathway was preferentially utilized.     

End-Product Regulation of the Tryptophan-Nicotinic Acid Pathway in Neurospora crassa

The regulation of the tryptophan-nicotinic acid pathway in Neurospora crassa was examined with mutants (nic-2, nic-3) which require nicotinamide for growth. The accumulation of N-acetylkynurenin and 3-hydroxyanthranilic acid by these mutants served to estimate the level of function of the early reactions in the pathway. In still cultures, maximal accumulation occurred with media containing growth-limiting amounts of nicotinamide; the accumulation of intermediates was almost negligible with nicotinamide in excess. Only nicotinamide and closely related compounds which also supported the growth of these mutants inhibited the accumulation of intermediates. The site of inhibition was assessed to be between tryptophan and kynurenin (or N-acetylkynurenin). The synthesis of N-acetylkynurenin was examined in washed germinated conidia suspended in buffer; the level of N-acetylkynurenin-synthesizing activity was inversely related to the concentration of nicotinamide in the germination medium. The addition of large amounts of nicotinamide to suspensions of germinated conidia did not affect their N-acetylkynurenin-synthesizing activity. Formamidase activity, kynurenin-acetylating activity, and gross tryptophan metabolism in germinated conidia was not influenced by the concentration of nicotinamide in the germination medium. The results obtained indicate that the site of inhibition by nicotinamide is the first step in the pathway, the tryptophan pyrrolase reaction. The data are interpreted as nicotinamide or a product thereof, such as nicotinamide adenine dinucleotide, acting as a repressor of the formation of tryptophan pyrrolase in N. crassa.     

Apparent maintenance of cytochrome P 450 by nicotinamide in primary cultures of rat hepatocytes.

The sole addition of a high, unphysiological, concentration of nicotinamide (25 mM) to a cell culture medium was found to maintain the cytochrome P 450 concentration of rat hepatocytes cultured for 24 hours at 71% of the level found in intact liver, whilst hepatocytes cultured without nicotinamide contained only 20% of their initial cytochrome P 450. Furthermore the P 450 concentration of hepatocytes cultured for 24 hours in the presence of 25 mM nicotinamide could be increased to the same level as found in intact rat liver by the inclusion of 1 mM nicotinamide into the medium used for cell isolation. Although the mechanism of action of nicotinamide is unknown this simple system for the maintenance of cytochrome P 450 in hepatocyte culture could provide the opportunity to study, under defined conditions $\text{in vitro}$, the factors that regulate cytochrome P 450 and hence determine hepatotoxicity and hepatocarcinogenesis.     

Microbial NAD Metabolism: Lessons from Comparative Genomics

Summary: NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life via horizontal gene transfer. Biochemical, genetic, and genomic analyses have advanced to the point at which the precursors and pathways utilized by a microorganism can be predicted. Challenges remain in dissecting regulation of pathways.     

Methylation of nicotinamide in rat liver cytosol and its correlation with hepatocellular proliferation

The changes in the activity of nicotinamide: S-adenosylmethionine methyltransferase (nicotinamide methylase) were studied in rat liver which was subjected to different rates of cellular proliferation. The cytosolic enzyme activity increased 3-4-fold in the first 24-48 h after partial hepatectomy and decreased again to the basal levels until 4 days post-operatively, whereas it remained unchanged in the livers of sham-operated animals. A single administration of thioacetamide at a dose of 50-250 mg/kg body weight, a treatment which induces hepatocellular proliferation as well, also enhanced the enzyme activity 2-3-fold 24 h after drug administration. This activity increase was associated with a marked lowering of intracellular NAD content of as much as 50% of the control levels. D-Galactosamine, a known hepatotoxic agent causing acute hepatitis in experimental animals and preventing DNA synthesis in regenerating liver, blocked the activity increase in regenerating rat liver. The rate of 1-methylnicotinamide synthesis, as measured by incubating liver slices in the culture medium supplemented with [14C]nicotinamide as a precursor, was found to be 2-4 times higher in the slices from regenerating liver and thioacetamide-treated rat liver than those from non-proliferating control liver. These results, together with our previous finding on the enhancement by 1-methylnicotinamide of the growth of cultured rat liver cells (Hoshino, J., Kühne, U. and Kr?ger, H. (1982) Biochem. Biophys. Res. Commun. 105, 1446-1452), support the view that nicotinamide methylase and its product, 1-methyl-nicotinamide, are involved in the control of hepatocellular DNA synthesis and proliferation.     

The control of nucleic acid and nicotinamide nucleotide synthesis in regenerating rat liver

1. The effect of injecting nicotinamide on the incorporation of [(14)C]orotate into the hepatic nucleic acids of rats after partial hepatectomy was investigated. 2. At 3h after partial hepatectomy the rapid incorporation of [(14)C]orotate into RNA, and at 20h after partial hepatectomy the incorporation of [(14)C]orotate into both RNA and DNA, were inhibited in a dose-dependent fashion by the previous injection of nicotinamide. 3. The injection of nicotinamide at various times before the injection of [(14)C]orotate at 20h after partial hepatectomy revealed an inhibition of the incorporation of orotate into RNA and DNA which was non-linear with respect to the duration of nicotinamide pretreatment. 4. The induction of a hepatic ATP depletion by ethionine demonstrated that the synthesis of hepatic NAD and NADP in partially hepatectomized rats was more susceptible to an ATP deficiency than in control rats. 5. The total hepatic activity of ribose phosphate pyrophosphokinase (EC 2.7.6.1) was assayed at various times after partial hepatectomy and found to be only marginally greater than the maximum rate of hepatic NAD synthesis induced in vivo by nicotinamide injection between 12 and 24h after partial hepatectomy. 6. It is suggested that a competition exists between NAD synthesis and purine and pyrimidine nucleotide synthesis for available ATP and particularly 5-phosphoribosyl 1-pyrophosphate. In regenerating liver the competition is normally in favour of the synthesis of nucleic acid precursors, at the expense of NAD synthesis. This situation may be reversed by the injection of nicotinamide with a subsequent inhibition of nucleic acid synthesis.     

Effect of tryptophan on isolated hepatocytes of rats

The addition of tryptophan to adult rat hepatocyte cultures stimulated DNA synthesis. The increase in DNA synthesis as measured by 3H-thymidine incorporation into DNA was observed on treatment of the cultures with tryptophan for 48 h but also as short as for 6 h in comparison with control cultures. An increase was also apparent at 30 h which was maintained for up to 48 h post treatment with tryptophan. The increase in DNA synthesis by tryptophan cannot be attributed to cell injury or to increased DNA degradation. Of the degradative enzymes added after harvesting the hepatocytes, only DNase decreased incorporation of 3H-thymidine. The observed effect was specific for tryptophan since treatment with kynurenine, isoleucine, methionine or serine failed to show a significant effect. Pretreatment of cultured hepatocytes with hydroxyurea prevented the tryptophan stimulated increase in DNA synthesis suggesting that the latter was due to replicative and not to reparative DNA synthesis. Experiments performed with the addition of diethylnitrosamine also alluded to tryptophan's role in replicative DNA synthesis. The mechanism of tryptophan-induced DNA synthesis is discussed.