Longevity and Nicotinamide riboside

GO term: nicotinamide riboside hydrolase activity

Ontology: Molecular Function (GO:0070635)

Definition: Catalysis of the reaction: nicotinamide riboside + H2O = nicotinamide + D-ribose.

Synonyms: N-ribosylnicotinamide hydrolase activity; nicotinamide ribonucleoside hydrolase activity

Click to expand...

Nicotinic Acid, Nicotinamide, and Nicotinamide Riboside:A Molecular Evaluation of NAD+ Precursor Vitamins in Human Nutrition.
Katrina L. Bogan and Charles Brenner
Annu. Rev. Nutr. 2008. 28:115–30
First published online as a Review in Advance on April 22, 2008
The Annual Review of Nutrition is online at nutr.annualreviews.org
This article’s doi: 10.1146/annurev.nutr.28.061807.155443

Key Words
sirtuins, poly(ADP-ribose) polymerase, cyclic ADP-ribose synthase, Wallerian degeneration, high-density lipoprotein cholesterol

Abstract
Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance re- verse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Be- cause current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and pro- pose areas for future research.

INTRODUCTION
Deficiency of niacin and/or tryptophan (Trp) causes pellagra, which is characterized by a darkly pigmented skin rash and the three D’s of dermatitis, diarrhea, and dementia. A century ago, pellagra was common among the rural poor in the southern United States and was thought to be an infectious disease. How- ever, in 1914, Joseph Goldberger tested the hypothesis that pellagra might be caused by a dietary deficiency and discovered that substituting corn-based diets with fresh milk, eggs, and meat cured and prevented the condition (29). Twenty-three years later, Conrad Elvehjem obtained a nicotinamide (Nam) fraction from de- proteinized liver and a sample of crystalline nicotinic acid (Na) and showed that these compounds have an antipellagragenic “antiblack tongue” activity on malnourished dogs (20). Subsequent biochemical studies identified Nam as a component of NAD+ and nicotinamide adenine dinucleotide phosphate (NADP) and showed that animals with pellagra have a significant decrease in muscle and liver NAD+ and NADP (5). Today pellagra occurs rarely in cases of extreme alcoholism and anorexia, or as a seasonal malady in underdeveloped parts of the world.

As schematized in Figure 1, the reason that a poor diet can produce a requirement for Na and Nam is that Trp, Na, and Nam are all NAD+ precursors (7). Trp is converted to NAD+ through an eight-step de novo pathway (Figure 2), so termed because the Nam base is essentially made from scratch. In contrast, Na and Nam are considered “salvageable precursors” that require only three steps and two steps, respectively, to rebuild NAD+. Nicotinamide riboside (NR) is an additional salvageable NAD+ precursor vitamin with a two-step pathway (14) and a three-step pathway (8) to form NAD+. As schematized in Figures 1 and 2, cells require ongoing NAD+ synthe- sis because NAD+ and NADP are not only coenzymes, which are recycled back and forth between oxidized (NAD+ and NADP) and reduced (NADH, NADPH) forms by hydride transfer enzymes, but are also substrates of NAD+ -consuming enzymes that break the glycosidic bond between the Nam moiety and the ADPribose moiety. NAD+- consuming enzymes transfer ADPribose and/or ADPribose polymers, form signaling com- pounds from NAD+ and NADP, and re- verse the acetyl modification of protein lysine residues. Each of these reactions con- sumes an NAD+ equivalent to a salvageable Nam product plus an ADPribosyl product (7).
NAD+-consuming enzymatic activities are induced, in part, by stresses such as DNA damage and inflammation. Many of these stresses are accompanied by specifically in- duced biosynthetic pathways, which appear to function to maintain NAD+ homeosta- sis. The term NAD+ homeostasis should be used cautiously, however, because it is not clear that cells are always in NAD+ home- ostasis. Mammalian NAD+ biosynthesis is not a closed, cell-autonomous system, and there appear to be situations in which cells actively increase and/or reduce the concentration of NAD+ and NAD+ metabolites to promote vital and/or regulatory functions, including cell death.

RECOMMENDED DAILY REQUIREMENTS OF THE NAD+ PRECURSORS
Despite the fact that the biosynthetic path- ways are not the same, in the literature of hu- man and animal nutrition, Nam and Na are collectively termed niacin and/or vitamin B3 . To protect against pellagra that can develop with Trp deficiency, recommended daily al- lowances (RDAs) of niacin are 16 and 14 mg per day for adult men and women, respec- tively (79). Because all plant, animal, and fun- gal inputs in our diet contain cellular NAD+ and NAD+ metabolites, foods provide NAD+ , NADH, NADP, and NADPH, which are con- sidered nutritional “niacin equivalents,” in ad- dition to Nam and Na. Whereas Nam and Na are the fully broken down NAD+ metabolites from animals and plants/fungi/bacteria respec- tively, NR and nicotinic acid riboside (NaR) can be considered partly broken down niacin equivalents. In the genetically tractable yeast system, all of the salvageable precursors (Na, Nam, NR, and NaR) support the growth of cells inactivated for de novo NAD+ synthesis (83). A single yeast cell deficient in de novo synthesis or undergoing a biological process that requires more than the minimum vital concentration of NAD+ must convert an available vitamin precursor to NAD+ in a cell-autonomous fashion. In contrast, humans exhibit the complexity of systemic NAD+ metabolism in which particular cells may uti- lize an NAD+ precursor to produce an ex- cess of NAD+ and export salvageable pre- cursors to other cells. Accordingly, dietary Trp is also classified as a niacin equivalent. However, because of the protein and other biosynthetic uses of Trp, 60 mg of Trp is con- sidered the equivalent of 1 mg of niacin (79). This physiological fact, that high levels of di- etary Trp result in circulation and excretion of Na (9, 10, 60, 70), has resulted in claims in textbooks and reviews that Na is derived from Trp. Whereas this is true in vertebrate organ- isms, there may not be a vertebrate intracellular pathway that is responsible.

[...]
NR is a newly discovered salvageable precursor of NAD+ that occurs in cow’s milk (14). Studies in Saccharomyces cerevisiae have shown that, like Na and Nam, NR is an NAD+ precursor that contributes to maintaining intracellular NAD+ concentration and improves NAD+ - dependent activities in the cell including Sir2-dependent gene silencing and longevity (8, 14). NR can either be converted to NAD+ by the Nrk pathway (14), which is induced by axotomy in dorsal root ganglion (DRG) neurons (71), or by the action of nucleoside phosphorylase and nicotinamide salvage (8). It has also been shown that the same two path- ways required for NR salvage in yeast cells can also be used for NaR salvage (83). In yeast cells, NR clearly qualifies as a vitamin by virtue of rescuing growth of strains deficient in de novo synthesis (14, 83), improving Sir2 functions (8), and utilizing a dedicated transporter (8a). Additionally, because cells deleted for the NR/NaR salvage enzymes have a significant deficiency in intracellular NAD+ when not supplemented with these compounds, it appears that NR and/or NaR are also normal metabolites (8).

Five lines of reasoning support designation of NR as an authentic NAD+ precursor vitamin in vertebrates. First, Haemophilus influenza, a flu-causing bacterium, which has no de novo pathway and cannot utilize Na or Nam, is strictly dependent on NR, NMN, or NAD+ for growth in the host bloodstream (22). Second, milk is a source of NR (14). Third, NR protects murine DRG neurons in an ex vivo axonopathy assay via transcriptional induction of the nicotinamide riboside kinase (NRK) 2 gene (71). Fourth, exogenously added NR and derivatives increase NAD+ accumulation in a dose-dependent fashion in human cell lines (94). Fifth, Candida glabrata, an opportunistic fungus that depends on NAD+ precursor vitamins for growth, utilizes NR during disseminated infection (51).

[...]
Sirtuin-dependent deacetylation of histones and other proteins re- sults in reprogrammed gene expression, mito- chondrial synthesis and function, cell survival, and longevity (91). Sirtuins have been recently reviewed as master switches of metabolism (18a).

[...]
NAD+ PRECURSORS IN FOODS
Consistent with Goldberger’s studies (29), niacin is abundant in meat, eggs, fish, dairy, some vegetables, and whole wheat. Notably, corn contains abundant Na and Nam, largely present in bound forms that are not bioavailable. Treatment with alkali is used to increase bioavailability, a practice that protected native and South American populations from deficiency. Untreated corn is considered “pellagragenic,” causing increased sensitivity to low dietary niacin concentrations in animal studies (46, 47). Milk, now known to be a natural source of NR (14), was shown to counteract the growth defect seen in corn-fed animals.

[...]
The use of NR as a precursor in mammalian cell types was first demonstrated in DRG neurons, which induce the NRK2 transcript when damaged by axotomy (71). The ubiquitous expression of Nrk1 in mammalian tissues (80) suggests utilization of NR and/or NaR (83) in a diverse array of cell types. However, Nrk2 is present in heart, brain, and skeletal muscle, and is notably absent in kidney, liver, lung, pan- creas, and placenta (48, 71). The fact that DRG neurons cannot be protected from damage-induced neuropathy by Na or Nam without concurrent gene expression of Na or Nam salvage genes suggests that NR is a uniquely useful precursor to the nervous system (71) when de novo synthesis of NAD+ from Trp is not sufficient.

ALTERATION OF NAD METABOLISM BY CALORIC RESTRICTION
Caloric restriction (CR) is the most effective intervention to extend the lifespan of multiple model organisms including mammals. CR is de- fined as a 20% reduction versus ad libitum feeding without compromising adequate nutrition (56). Although the mechanisms of CR remain elusive, it is thought that CR modulates fat and carbohydrate metabolism, attenuates oxidative damage, and activates a stress-induced hormetic response that mediates improved vitality and disease resistance (55). Among these three major mechanisms, modulation of fat and carbohydrate utilization is the most direct response to reduced dietary inputs, and hormesis is potentially the mechanism most influenced by the “signaling” aspect of CR.

In CR-treated mice, brain NAD+ levels are increased and Nam levels are decreased, and these changes accompany neuronal Sirt1 acti- vation, which reduces Alzheimer’s neuropathol- ogy (63). In fasted mice, NAD+ levels are increased in liver, which is accompanied by Sirt1 activation, PGC1α deacetylation, and increased mitochondrial biogenesis (67). The mechanisms by which lower food inputs in- crease NAD+ levels in brain and liver are com- pletely unknown. Two potential mechanisms that may account for this phenomenon are systemic mobilization of NAD+ precursors to the brain and liver and reduced NAD+ breakdown. Among the potential precursors that could mediate this phenomenon, Na and Trp seem unlikely because one would expect that increased food consumption would be required to increase their availability. Analysis of CR-induced systemic metabolites should permit the detection of either Nam or NR as candidate mediators of increased brain and liver NAD+ levels.

PROSPECTS FOR NR AS A SUPPLEMENT
The most fundamental use of NAD+ precursor molecules, Na and Nam, is in the prevention of pellagra. Like Na and Nam, NR is a natural product found in milk (14), which is incorporated into the intracellular NAD+ pool (94), and thus could be used as a general supplement, potentially for people who have adverse reactions to Na or Nam. More significantly, however, the specific utilization of NR by neurons may provide qualitative advantages over niacins in promoting function in the central and pe- ripheral nervous system.

NR may also find uses related to the pharmacological uses of Na or Nam, which are limited by the side effects of each. Because Gpr109A is specific for the acid and not the amide (85, 92), one would not expect NR to cause flushing. Similarly, the side effects associated with high-dose use of Nam in the prevention and treatment of diabetic disorders (65) raise substantial health and safety concerns (44). In light of the inhibitory effects of Nam on sirtuins and the protective roles of sirtuins in normal cellular metabolism (18a, 91), NR may represent an alternative supplement. Though uncertainties as to the mechanisms of action of therapeutic doses of Na and Nam exist, positive results with NR would clarify the mechanisms of action of Na and Nam. Because of the prevalence of PARP activation in neuropathies, inflammation, and neurodegeneration and the association of C. glabrata adherence with low NAD+, NR has great potential as a supplement or therapeutic agent that would elevate or maintain NAD+ in specific tissues. Future work will evaluate the pharmacokinetics, safety, and efficacy in animal and human systems to maintain health and to prevent disease.

Click to expand...

Nicotinamide Riboside Promotes Sir2 Silencing and Extends Lifespan via Nrk + and Urh1/Pnp1/Meu1 Pathways to NAD

Belenky, P., Racette, F. G., Bogan, K. L., McClure, J. M., Smith, J. S., & Brenner, C. (2007).
Cell, 129(3), 473–484. doi:10.1016/j.cell.2007.03.024

SUMMARY
Although NAD+ biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD+ precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD+ precursor that is converted to nicotinamide mononucleotide by specific nico- tinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD+ synthesis through two pathways, the Nrk1 pathway and the Urh1/ Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD+ metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD+ and increases Sir2 function.

INTRODUCTION
[...]
Calorie restriction (CR), the most powerful known intervention to extend lifespan in verte- brates, extends lifespan in wild-type yeast cells in a Sir2- and NAD+-dependent manner (Lin et al., 2000).
[...]
Two leading mechanisms from yeast molecular biology are that CR {Calorie restriction} promotes clearance of Sir2-inhibiting nicotinamide (Nam) (Anderson et al., 2003a) or a decrease in NADH (Lin et al., 2004), thereby increasing Sir2 function. It was also proposed that CR might increase the level of the Sir2 sub- strate, NAD+ (Guarente, 2000). It has now been shown that in livers of mice fasted for one day, NAD+ levels and Sirt1 protein levels are increased, leading to deacetylation of PGC-1a and a consequent gluconeogenic transcriptional program (Rodgers et al., 2005). Additionally, in a murine model of Alzheimer’s disease expressing the Swedish allele of human Ab precursor (Hsiao et al., 1996), CR almost completely prevents amyloid neuropathology coincident with increased Sirt1 protein expression in the neocortex, increased brain NAD+ levels, and reduced brain Nam (Qin et al., 2006).

Thus, a CR-mimetic small-molecule intervention to improve Sirt1 function would have considerable potential in metabolic and neuroprotective therapeutic areas. Indeed, evidence has recently been presented that high-dose resveratrol improves health, longevity (Baur et al., 2006), and muscle performance (Lagauge et al., 2006) in overfed mice through a mechanism that appears to involve Sirt1 activation. Although a nutritional approach of increasing Sir2 activity and longevity would be attractive, this has only been achieved by engineered gene overexpression in yeast (Anderson et al., 2002) because nicotinic acid fails to extend lifespan and Nam shortens lifespan (Bitterman et al., 2002; Gallo et al., 2004). Moreover, extra copies of NPT1 were reported to activate Sir2 and extend lifespan in a manner mimicking CR but without increasing NAD+ levels (Anderson et al., 2002).

[...]
Our investigation of NAD+ synthesis continues to indicate that not all of the metabolic steps for the molecule discovered 100 years ago as cozymase (Harden and Young, 1905) have been identified genetically, even though tryptophan, nicotinic acid, and Nam were identified as biosynthetic precursors long ago (Krehl et al., 1945). Accordingly, the lack of a complete set of genes controlling NAD+ metabolism may be hampering mechanistic analysis of Sir2-dependent and NAD+-dependent functions in all eukaryotic models under study. Recently, we discovered that nicotinamide riboside (NR), a natural product present in milk, is an NAD+ precursor that is converted to NAD+ through the action of conserved eukaryotic NR kinases including yeast and human Nrk1 and human Nrk2 (Bieganowski and Brenner, 2004). As shown in Figure 1A, the de novo pathway from tryptophan, the nicotinic-acid import/salvage pathway, and the yeast Nam salvage pathway all depend on the action of glutamine-dependent NAD+ synthetase Qns1 (Bieganowski et al., 2003). Thus, provision of extracellular NR defined a novel biosynthetic pathway to NAD+, a pathway that was shown to be Qns1 independent and Nrk1 dependent (Bieganowski and Brenner, 2004).

RESULTS
[...]
Given the four known pathways to produce NAD+ in yeast (Bieganowski and Brenner, 2004) and the Sirtuin-de- pendent degradative pathway to Nam (Tanner et al., 2000; Tanny and Moazed, 2001), we hypothesized that cells possess homeostatic mechanisms regulating NAD+ levels such that NR is not utilized when another precursor remains abundant. Because SDC medium was formulated to contain tryptophan and nicotinic acid but not Nam or NR (Wickerham, 1946), it was straightforward to determine whether depletion of tryptophan or nicotinic acid was limiting for utilization of exogenous NR.

[...]
Because nicotinic acid is not a necessary supplement for yeast cells with an intact de novo pathway, we elimi- nated the complication of nicotinic-acid-dependent changes in NAD+ concentration. Thus, we grew wild-type cells in SDC {synthetic dextrose complete} media without nicotinic acid and examined the effect on NAD+ levels of supplementation with 10 mM NR. As shown in Figure 3C, wild-type cells in nicotinic- acid-free media have a stable NAD+ level that is doubled by addition of 10 mM NR. Thus, NR is not only a compound that rescues the otherwise lethal qns1 deletion (Bieganow- ski and Brenner, 2004) or the phenotypes of Sir2-limited npt1 mutants (Figure 2) but also a vitamin that elevates intracellular NAD+ levels by 1 mM in wild-type cells, although its contribution to NAD+ accumulation depends on depletion of nicotinic acid.

[...]
Although yeast cells can double intracellular NAD+ levels with provision of either nicotinic acid or NR in the growth media, there are important differences between the salvage pathways of the two vitamins. As shown in Figure 2, nicotinic acid, which is almost universally used in yeast synthetic media though not required for S. cerevisiae growth, pro- duces an intracellular NAD+ level that drops by more than 2-fold over the course of growth in liquid culture. NR stably increases intracellular NAD+ levels and extends lifespan via two distinct metabolic pathways.

[...]
Vitamins are small molecules that are required in diets to prevent malnourishment. Metabolites are compounds that are naturally produced in particular biosynthetic pathways. Data presented in this study and in a previous study (Bieganowski and Brenner, 2004) establish the vitamin functions of NR in yeast. New data establish the role of NR salvage enzymes in maintaining NAD+ homeostasis in nonsupplemented cells. NR appears to be unique among NAD+ precursors in stably elevating NAD+ levels in yeast and may have advantages in human systems in which activation of Sirtuins is desired. Indeed, providing supplements to boost NAD+ synthesis may be a therapeutically important treatment modality for neurodegenerative conditions stemming from chemotherapy (Araki et al., 2004), physical nerve damage (Wang et al., 2005), Alzheimer’s disease (Qin et al., 2006), and multiple sclerosis (Kaneko et al., 2006).

In ex vivo experiments to protect dorsal-root-ganglion neurons from transection-induced axonopathy, the combination of Nam plus overexpression of Nam PRTase and the combination of nicotinic acid plus overexpression of nicotinic acid PRTase had neuro- protective activity. However, NR was the only nonphosphorylated, drug-like NAD precursor to protect DRG neurons from axonopathy without concurrent gene therapy (Sasaki et al., 2006). This appears to be because Nrk2 transcription is induced by nerve damage (Sasaki et al., 2006).

In addition to the potential value of increased NAD+ synthesis in neuroprotection, genetic and pharmacological data validate increasing Sirt1 activity in the effective me- tabolism of a high-fat diet (Lagauge et al., 2006). Because nicotinic acid causes flushing through a receptor mechanism unrelated to NAD+ synthesis (Benyo et al., 2005) and Nam inhibits Sirtuins (Bitterman et al., 2002), it will be important to test whether increasing net NAD+ synthesis with NR is as effective in promoting health of vertebrates as it is in extending lifespan of S. cerevisiae.{Saccharomyces cerevisiae is a species of yeast.}

Click to expand...

The NAD+/NADH Theory
It is important to note that there is an increasing body of scientific evidence suggesting that a host of favourable metabolic conditions are associated with a high ratio of NAD+ to NADH. If this is so, then the question of the very idea of NADH supplementation being in conflict with the latest research is not an unfair one. The first answer is simply that NADH supplementation in its own right is supported by a respectable amount of clinical research, therefore ensuring a following and a rightful place within the preventative health community. The second answer is related to the first in that the pharmacokinetics and mechanism of action for NADH supplementation are simply not clearly understood at this time. One proposed explanation that scientists have put forward is that endogenous NADH converts back into NAD+, which would explain its clinical credentials in the face of the emerging NAD+/NADH ratio theory.

Click to expand...

Introduction

p.495: In 1938, the pioneering vitamin-hunter Conrad Elvehjem and his coworkers put dogs on a synthetic diet supplemented with only the known B vitamins. When the dogs were near death and exhibited pellagra-like black tongue symptoms, the investigators fed the animals small-molecule fractions derived from liver. In this manner, nicotinic acid and nicotinamide, now collectively termed niacin, were identified as the “anti-black tongue factor” with essential nutritional activity (Elvehjem et al., 1938).

[...]
p.495: In 2000, it became clear that Sir2 and Sir2-related enzymes termed Sirtuins deacetylate {remove the acetyl group from} lysine residues with consumption of an equivalent of NAD and that this activity is required for Sir2 function as a transcriptional silencer (Imai et al., 2000).

[...]
p.495: NAD-dependent deacetylation reactions are required not only for alterations in gene expression but also for repression of ribosomal DNA recombination and extension of lifespan in response to calorie restriction (Lin et al., 2000, 2002). NAD is consumed by Sir2 to produce a mixture of 2and 3 O-acetylated ADPribose plus nicotinamide and the deacetylated polypeptide (Sauve et al., 2001).

[...]
p.495: In this study, we show that nicotinamide riboside, which was known to be an NAD precursor in bacteria such as Haemophilus influenza (Gingrich and Schlenk, 1944; Leder and Handler, 1951; Shifrine and Biberstein, 1960) that lack the enzymes of the de novo and PreissHandler pathways (Fleischmann et al., 1995), is an NAD precursor in a previously unknown but apparently conserved eukaryotic NAD biosynthetic pathway. [...] Finally, we utilize yeast mutants of defined genotype to hunt for vitamins in a pathway-specific manner and show that milk is a source of nicotinamide riboside.

[...]
p.496: As shown in Figure 2A, consistent with the scheme’s implicit assumptions about nicotinamide metabolism, nicotinamide does not rescue qns1 mutants even at 1 or 10 mM. However, apart from any intermediate or enzymatic transformation depicted in the scheme, nicotinamide riboside, [...] functions as a vitamin form of NAD at 10mM. [...] {Text under illustration}: (A) qns1 cells transformed with a plasmid carrying the QNS1 and URA3 genes were tested for growth on synthetic dextrose complete media and 5-fluoroorotic acid (5-FOA). NAD+ synthetase mutant qns1 is inviable without supplements or with 1 mM nicotinamide but is rescued by 10 mM nicotinamide riboside.

[...]
p.497: Second, anticancer agents such as tiazofurin (Cooney et al., 1983) and benzamide riboside (Krohn et al., 1992) have been shown to be metabolized intracellularly to NAD analogs tiazofurin adenine dinucleotide and benzamide adenine dinucleotide, which inhibit IMP dehydrogenase, the rate-limiting enzyme for guanine nucleotide biosynthesis. As these compounds can be considered analogs of nicotinamide riboside (Figure 3), generation of NAD analogs would necessarily involve phosphorylation of the 5 hydroxyl group and subsequent adenylylation of these intermediates.

[...]
{Text under illustration}: Figure5/p.499. Nicotinamide Riboside IsPresent in an Acid Whey Vitamin Fraction of Cow’s Milk
Yeast qns1 mutants grow when supplemented with whey in an NRK1-dependent manner, indicating that whey is a source of nicotinamide riboside.

[...]
p.499: We used the yeast qns1 mutant to screen for natural sources of nicotinamide riboside and, as shown in Figure 5, we found it in a vitamin fraction of cow’s milk. Unlike the original screen for vitamins in protein-depleted extracts of liver for reversal of black tongue in starving dogs (Elvehjem et al., 1938), this assay is pathway-specific in identifying NAD precursors. As shown in Figures 1 and 2, because of the qns1 deletion, nicotinic acid and nicotinamide do not score positively in this assay. Because the factor from milk requires nicotinamide riboside kinase for growth, the nutrient is clearly nicotinamide riboside and not NMN or NAD+.

[...]
p.499: However, animal genes for nicotinamidase have not been identified and there is no compelling evidence that nicotinamide and nicotinic acid are utilized as NAD precursors through the same route in mammals. The persistence of “niacin” as a mixture of nicotinamide and nicotinic acid may attest to the utility of utilizing multiple pathways to generate NAD and suggests that supplementation with nicotinamide riboside as third importable NAD precursor may be beneficial for certain conditions.

[...]
p.500: Though nicotinic acid treatment effects all of the key lipids in the desirable direction and has been shown to reduce mortality in target populations (Pasternak et al., 1996), its use is limited because of a side effect of heat and redness termed “flushing,” which is significantly effected by the nature of formulation (Capuzzi et al., 2000). Thus, it will be important to test whether nicotinamide riboside supplementation is a preferred route to improve lipid profiles in humans. Additionally, study of the expression and regulation of NAD biosynthetic enzymes is expected to reveal approaches to sensitize tumors to compounds such as tiazofurin, to protect normal tissues from the toxicity of compounds such as tiazofurin adenine dinucleotide, and to stratify patients for the most judicious use of tiazofurin chemotherapy.

p.500: To prepare nicotinamide riboside, 120 umol NMN (Sigma, concentration corrected by absorption) was treated with 1250 units of calf intestinal alkaline phosphatase (Sigma) for 1 hr at 37C in 1 ml 100 mM NaCl, 20 mM Tris [pH 8.0], 5 mM MgCl2. {This sound a bit complicated!}

{Maybe this could be a clue for easy preparing?} p.500: A whey vitamin fraction of commercial nonfat cow’s milk was prepared by adjusting the pH to 4 with HCl, stirring at 55C for 10 min, removal of denatured casein by centrifugation, and passage through a 5000 Da filter. In yeast media, nicotinamide riboside was used at 10 uM and whey vitamin fraction at 50% by volume.

Click to expand...

p.64:
Abstract

The etiology of multiple sclerosis (MS) is unknown but it manifests as a chronic inflammatory demyelinating disease in the central nervous system (CNS). During chronic CNS inflammation, nicotinamide adenine dinucleotide (NAD) concentrations are altered by (T helper) Th1-derived cytokines through the coordinated induction of both indoleamine 2,3-dioxygenase (IDO) and the ADP cyclase CD38 in pathogenic microglia and lymphocytes. While IDO activation may keep auto-reactive T cells in check, hyper-activation of IDO can leave neuronal CNS cells starving for extracellular sources of NAD. Existing data indicate that glia* may serve critical functions as an essential supplier of NAD to neurons during times of stress. Administration of pharmacological doses of non-tryptophan NAD precursors ameliorates pathogenesis in animal models of MS.

*Glia: Non-neuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons in the brain, and for neurons in other parts of the nervous system such as in the autonomic nervous system.


2.2. Biosynthesis of NAD

p.65: Twentieth century man is particularly susceptible to dietary induced NAD deficiency. The most devastating nutritional deficiency disease in the history of the United States of America was the NAD deficiency disease pellagra, an epidemic which killed over 120,000 people in the first two decades of the 1900s. NAD precursors are distinguished as one of the few known molecules identified as important enough to be forced into the public diet for improved health by the U.S. government. It should be realized however, that niacin requirements of individuals can vary depending on genetics and stress. [...] Recently, new NAD metabolic pathways have gained recognition for their important roles in MS. For example, the NAD precursor nicotinamide (NA) can ameliorate MS in the experimental autoimmune encephalomyelitis (EAE) animal model. Thus, we focus here on NAD biosynthesis to understand the potential role of NAD depletion in MS pathogenesis.

In all vertebrates, NAD can be synthesized by two pathways: de novo synthesis from tryptophan [3,4] and/or from vitamin precursors in the diet: NA, nicotinamide (NAM), and nicotinamide riboside (NAMR). [...] For the de novo pathway to be completed it is necessary to have adequate riboflavin (vitamin B2), pyridoxyl phosphate (vitamin B6), and ascorbate (vitamin C).

p.66: NAD can also be synthesized from three different essential non-amino acid precursors in the diet: nicotinic acid (NA), nicotinamide (NAM), and nicotinamide riboside (NAMR). Yeast with an inactivated de novo pathway may use any of the salvageable precursors (NA, NAM, NAMR, or nicotinic acid riboside) to support life. Nicotinic acid riboside has only been examined in yeast. These non-amino acid dietary NAD precursors are collectively termed vitamin B3 and require two (NAM, NAMR) or three (NA) steps respectively to generate NAD.

p.66: It should be realized that not every cell is capable of converting each precursor to NAD at all times although NAD is essential to survival. Because tissue and cell-type specific enzyme expression differences exist in metazoans, the precursors are differentially utilized in the gut, brain, blood and other organs [1] (Table 1). The de novo pathway is clearly active in liver, neuronal and immune cells. The pathway from NA is expressed in the liver, kidney, heart and intestine. While, vertebrates do not possess the nicotinamidase enzyme that converts NAM to NA, intestinal bacteria in the vertebrate gut use nicotinamidase to convert NAM to NA, which in turn is used to synthesize NAD via the Preiss and Handler salvage pathway. The NAMR salvage pathway is expressed in neurons and in cardiac and skeletal muscle.

Collectively, experimental observations support the idea that by increasing NAD, we may be able to prevent or reduce the effects of MS pathogenesis. Mammals elevate their NAD during autoimmune encephalomyelitis, while tryptophan levels are decreased in the cerebrospinal fluid (CSF) and sera of MS patients. Caloric restriction (CR) raises NAD levels and provides protection against EAE-mediated pathogenesis. CR reduces inflammation, demyelination, and neurodegeneration, but does not suppress immune function. CR increases both the expression of the rate-limiting enzyme controlling NAD biosynthesis and lifespan in a SIRT1 dependent manner. Consequently, the body may synthesize NAD to help control autoimmune encephalomyelitis.


2.3. Changes in NAD Levels in Animal Models of MS and NAD Precursors

p.67: Under conditions of NAD deficiency, neurons are exceptionally vulnerable to the degeneration characteristic of MS. The EAE model of MS indicates that endogenous levels of NAD are elevated in the CNS. This presumably occurs due to the combined increases in lymphocyte infiltration, hematopoietic cell proliferation, and IDO activity in professional APCs commonly observed in MS.

While net increases in NAD in the CNS are observed in EAE models, we predict that these increases are largely restricted to the immune system and come at the risk of NAD deficiency in neurons. Extracellular levels of the precursor used for de novo biosynthesis (tryptophan) are significantly decreased in the serum and CSF of MS patients. Fortunately, this loss of available NAD precursor can be partially rescued either by administrating pharmacological doses of nicotinamide or by enforced caloric restriction. Both approaches lead to increased NAD levels and profoundly ameliorate EAE pathogenesis. By contrast, administration of tryptophan is known to increase lymphoproliferation. Thus high dose tryptophan administration may be expected to exacerbate immune-mediated demyelinating pathogenesis, actually promoting neurodegeneration due to hyper activation of the immune system (discussed in greater detail in section 8.1.1).

{Inserting one line here from section 8.1.1} p.82: Complementation through tryptophan administration however, is likely not to be advisable in large part because tryptophan deprivation is a basic mechanism that is used to limit inflammation. Also, tryptophan in high doses is the most toxic of all amino acids [180], it is mitogenic for T cells [30,31], and tryptophan may be involved in causing deadly eosinophilia myalgia syndrome.

{So, if I understand this correctly, depletion of tryptophan is a sign of inflammation in the body, and taking tryptophan could make things worse???}


2.4. Glia as Suppliers of NAD to Neurons

[...]
p.68: Given the divisions of labor reserved for glia, it makes sense that glia are likely more efficient at NAD biosynthesis than neurons and consequently serve critical functions as providers of NAD to neurons. Collectively experimental data supports the notion that glial may be required for the essential delivery of NAD to neurons starting from tryptophan, nicotinic acid/niacin, or nicotinamide/niacinamide, but not nicotinamide riboside.


3.3. Etiology of MS

p.69: Whether infectious agents, environmental toxins, mutagenesis, or nutrient deficiencies cause MS is uncertain. Inheritance studies of MS have found genetic links affecting disease susceptibility, but have not revealed any single genetic mutation. Given the high frequency of MS in the population, it seems most likely that more than one factor is involved. For example, geographical epidemiology and animal models clearly reveal important roles for vitamin D signaling, while deficiencies of vitamin B12 or sadenosylmethionine can directly cause demyelination. Meanwhile recurrent reactivation of a latent herpes infection resembles relapsing-remitting MS and is commonly found in the CNS, where its potential role in MS pathogenesis is difficult to resolve owing to its nearly ubiquitous presence.


8. POTENTIAL NAD-RELATED PHARMACOTHERAPEUTIC STRATEGIES
FOR TREATING MS

8.1. NAD Precursor Complementation

8.1.2 Nicotinic Acid

[...]
p.83: With the statins as the most commercially successful class of pharmaceuticals in the world, yet nicotinic acid still outperforms them in spite of unparalleled investments to develop something better, this is truly remarkable. Thus, nicotinic acid acid is the gold standard for correcting dyslipidemia on the whole. [...] Nicotinic acid appears to be preferred over nicotinamide as a NAD precursor primarily when it is applied at the high concentrations. Nicotinic acid is the more preferred substrate in evolution based on experimental radioactive labeling and comparative genomic studies examining to date (presentation by Mathias Ziegler at PARP 2008 meeting, Tucson, Arizona, U.S.A.). In glia, nicotinic acid provides greater levels of NAD biosynthesis per mole than nicotinamide or tryptophan by 200 and 500 fold respectively. This also appears to be true for glia. However, neurons are distinctively inefficient in their ability to convert dietary NAD precursors to NAD [27]. As described above this may be another biosynthetic job for glia like that of cholesterol biosynthesis. {Reading p.68, if I've understood correctly, Nicotinamide Riboside is an exception}

p.85: Most curiously, while nicotinic acid is well known for providing tremendous relief in animal models of atherosclerosis, it can actually increase the levels of a biomarker known to be directly correlated with increased risk of atheroslerosis (homocysteine) in rat studies as well as lowering of pyridoxyl phosphate (vitamin B6) and cobalamin (vitamin B12). Increases in plasma homocysteine have also been observed in patients treated with high doses of niacin.

[...]
Interestingly, the additional application of pyridoxyl phosphate or methionine would correct the elevation in homocysteine while still exerting the positive effects on lipid profile. These observations suggest that once the body has enough NAD to perform most of its desired chemistry, then one the next co-factors to become rate-limiting for performing of endogenous biochemistry is pyridoxyl phosphate (vitamin B6) or cobalamin (vitamin B12). This makes sense given that NAD is perhaps followed by pyridoxyl phosphate in the shear total number of known reactions requiring these co-factors respectively. Furthermore, it is well known that deficiencies of cobalamin lead to demyelination and such deficiencies have on occasion been observed in MS. Thus, in order to maintain optimal NAD function it seems ideal to include increased administration of pyridoxyl phosphate and cobalamin.

8.1.3. Nicotinamide

p.85: Nicotinamide is incapable of exerting the profound benefits correcting lipodystrophy. Sometimes nicotinamide is referred to as no-flush niacin, however this can be misleading as it suggests that it may be useful in correcting bad lipid profiles. More basic research has been performed using nicotinamide than nicotinic acid for many years. Still, there unique aspects of nicotinamide activated signal transduction continue to be realized.

[...]
Nicotinamide when administered daily causes a dramatic protection against demyelination and neurological signs in MOG-induced EAE. This is accompanied by a persistent elevation in whole body NAD levels, where untreated mice suffered severe depletion of NAD levels. It is essential that the nicotinamide be administered with regularity in order to maintain the elevated levels of NAD. At the highest nicotinamide concentrations it prevented death and was capable of delaying onset of behavioral deficits from 8 days to 26 days. No adverse reactions were noted upon administration of nicotinamide, which is on the whole a highly safe molecule even when administered at high doses.

8.1.4. Nicotinamide Riboside

[...]
p.86: Thus there is a long history of clinical use for high doses of nicotinic acid and nicotinamide. By contrast, nothing is known regarding the utility of nicotinamide riboside in the clinic.

Of potentially great clinical therapeutic significance nicotinamide riboside is distinguished as the only NAD precursor capable of providing NAD directly to neurons and delaying Wallerian degeneration assay. [...] More clinical research is needed given the tremendous potential therapeutic benefit of nicotinamide riboside therapy for treating neurodegenerative diseases. [...] Perhaps, the effect is more immediately beneficial to the neuron since glia are not apparently required for synthesis of NAD from nicotinamide riboside.


8.3. SIRT1 Activation

p.87: Activation of SIRT1 using resveratrol/SRT501, or nicotinamide riboside/SRT647 has been shown to minimize the clinical signs and inflammatory response in EAE, while treatment with the SIRT1 inhibitor Sirtinol would prevent these beneficial effects. Here we briefly review the current state of Sirtuin knowledge with emphasis on potential targeting in the context of MS.

[...]
While we cannot change our genetics, we can change some of our epigenetics. Most epigenetic modifications are heritable and not alterable either. However, as the defining class III histone deacetylases, the Sirtuins can be targeted by pharmacologically modifying NAD levels. Functionally, the Sirtuins are extremely highly conserved evolution. They serve pivotal roles exerting control of metabolic functions in response to nutrient availability as observed in all examined organisms ranging from man to bacteria!

[...]
The classic SIRT1 activator resveratrol is now known to also increase SIRT3 expression. Accordingly it stands to reason that the ability of high dose nicotinamide to provide NAD and subsequently ATP may involve the NAD mediated activation of SIRT3 leading to sustained levels of ATP. Given that nicotinamide when applied at high concentrations is protective against Wallerian degeneration and EAE, greater attention should be directed at considering nicotinamide in the context of clinical MS. [...] It should be mentioned that man actually empirically discovered resveratrol's benefits in the form of many plant-based medicines over 5,000 years ago.

[...]
This ability of resveratrol to promote neurite outgrowth may be of particular significance in the context of recovery from MS.


10. LACK OF DIETARY VARIETY CAUSING NAD DEFICIENCIES AND POTENTIAL CONNECTIONS TO MS ETIOLOGY TODAY

[...]
p.96: Humans are particularly vulnerable to NAD deficiency. This became particularly evident with introduction of the first rice and flour foods to the general population in the 1870's, which immediately resulted in epidemics of the NAD deficiency disease known as pellagra. Between 1900 and 1940, at least 100,000 individuals in the southern United States died of pellagra ([334] and excellent review [335]). In 1907, pellagra was the leading cause of death in mental hospitals [5]. Joseph Goldberger noticed the disease was particular to people on a limited diet while those ingesting cow's milk were spared.

[...]
p.97: Pellagra is often considered to be a disease that no longer exists in the developed countries other than in cases of tuberculosis drug-induced pellagras, chemotherapy-induced pellagra, or in connection with alcoholism. However, analysis of western niacin status suggests inadequate niacin status occurs with a fairly high frequency at approximately 15% of the population. The most consistent neuropathologic abnormality in pellagra is reported to be central chromatolysis of neurons, where chromatolysis is defined as the loss of Nissl body staining as a result of neuronal death from injury, fatigue, or depletion of energy supplies.

p.97: Pellagra can result from either a passive process evoked by dietary insufficiency or even active drug induced processes including tuberculosis drugs and excessive alcohol consumption. By extension, pellagric pathology can also arise from immune system mediated NAD depletion. However, NAD deficiency can also arise from activation of NAD depleting pathways. NAD depleting pathways can be activated by poor diet or autoimmune dys-regulation.

[...] The modern western diet remains disproportionately high in refined sugars and corn. The annual sugar consumption per individual has increased by over 10 fold in the western world since in the early 1800s. All of these dietary changes correlated directly with increased refining. How well this may historically correlate with the incidence of MS is unknown. Significantly however, glucose actively promotes NAD depletion through PARP-1 activation. Nicotinamide can both inhibit PARP-1 enzyme activity and supply NAD to the cell. Nicotinamide protects against diabetes in animal models and continues to warrant active consideration in clinical studies (reviewed [142]). Limitations in NAD likely persist as a major rate-limiting factor controlling age-related disease onset. Extremely high doses of nicotinic acid of over 1000 fold higher than the RDA are commonly used to correct dyslipidemia that often arises from excessive high fat diets. The minimal amount of NAD precursor needed to stave off disease is likely to depend on dietary habits and immune function.

[...]
With corn so prevalent in the diet and the often very simplistic diets that lacked variety, including mostly corn meal and molasses, people in the southern United States of America soon suffered from pellagra. In 1912 it was shown that a maize only diet caused a multitude of health problems. More recently it was shown that corn only diets lead to severe defects in brain serotonin levels .A rather homogenous modern diet deficient in tryptophan may contribute more than we realize to MS pathogenesis a result of insufficient dietary NAD precursor.

Click to expand...

nicklebleu said:

I was wondering, if whey protein isolates should be introduced into an optimal diet.

From these article it seems that cowderived whey could be beneficial, as long as casein has been removed.
Mercola has been going on about whey for some time, but bearing in mind that he also sells it and has some comercial interest in it.

The main problem would be to source a well manufactured product and that might be quite difficult as the run-of-the-mill products from the weightlifter food shops probably wouldn't qualify.

Click to expand...

ChromaDex Gains Rights to Make NR

IRVINE, Calif.—ChromaDex Corp. gained exclusive worldwide rights to a novel manufacturing process for nicotinamide riboside (NR) developed by Cornell University, allowing the company to launch novel, proprietary ingredient that could be used in cardiovascular health, glucose control, anti-aging and cognitive function products.

"Despite its potential to deliver significant health benefits, nicotinamide riboside, or NR, is currently not available to the market as a dietary ingredient," said Frank L. Jaksch Jr., CEO of ChromaDex. "The technology we have licensed from Cornell provides a cost-effective, commercially viable manufacturing process for NR, thereby solving the key barrier to entry."

NR, a recently discovered vitamin found naturally in milk, is a more potent version of the more commonly known niacin (vitamin B3). Like niacin, NR is a precursor to nicotinamide adenine dinucleotide (NAD), which is elevated by a calorie-restricted diet. According to ChromaDex, increasing cellular NAD has demonstrated cell-protective and positive metabolic effects. The company also said lab tests have shown promise for its ability to improve cardiovascular health, glucose levels and cognitive function, and have demonstrated evidence of anti-aging effects.

"We chose NR as a largely unevaluated molecule for the purpose of enhancing cellular NAD levels, and our published scientific work has verified that NR is perhaps the most potent NAD enhancing agent ever identified," said Anthony Sauve, associate professor of pharmacology at Weill Cornell Medical College.

To facilitate commercialization of NR, Sauve developed an efficient synthesis of this compound, and this synthesis enables large quantities of NR to be produced for a variety of potential uses.

Click to expand...

Why Billionaire Frost, Honig Are Buying ChromaDex

(PRWEB) August 31, 2012

ChromaDex announces date to release human clinical efficacy data on pterostilbene.

As the authority source for photochemical reference materials, ChromaDex has the benefit of understanding market demand for ingredients far before many of its end markets do. The legacy lab business was able to sense, for example, growing demand for stevia sweetener many years prior to commercialization based on the spike in stevia extract orders. It was able to leverage the same intellectual capital advantage to develop nictoinamide riboside (NR), a vitamin precursor of coenzyme NAD. Gould Partners believes that this vitamin will result in even greater positive awareness of the legacy business and make the firm an established consultant for the pharmaceutical, beverage & food, and dietary supplement industries.

NR: THE "NO FLUSH" NIACIN ALTERNATIVE

Research has shown that NR can be converted to NAD in a process that extends the lifespan of yeast cells via increasing NAD-dependent Sir2 enzyme activity and NAD levels. ChromaDex recently licensed exclusive patent rights to the technology behind engineered yeast fermentation of NR. NAD has already shown to yield positive metabolic effects; improve cardiovascular health, cognitive function, glucose levels and insulin activity; and protect against neurodegeneration, the effects of aging, and weight gain on a high-lipid diet. The product is thus diversified across a variety of high-growth unmet needs. Moreover, ChromaDex offers several other products, like pTeroPure, that target similar markets and provide multiple cross-selling opportunities.

NAD precursor vitamins nicotinamide and nicotinic acid, collectively called "niacin or vitamin B3" are widely applied in energy drinks, milk, and meat, among other household goods. Abbott Labs' NIASPAN is the FDA-aproved leader in niacin, which has shown to boost HDL ("good") cholesterol, lower bad cholesterol, and slow the buildup of clogging plaque. But the product has also commonly resulted in a flushing so severe that Abbott dedicates an entire page of its NIASPAN website to helping consumers deal with the side effect. The issue has been further compounded by a recent study that found it failed to prevent heart attacks.

ChromaDex's NR has shown no "flushing" side effects with similar positive effects at lower dosing. Since the company has additional manufacturing patents from Cornell University, the path to commercialization will be relatively short. This will likely pronounce stock reactions to data announcements.

WHY BILLIONAIRE PHILLIP FROST, HONIG ARE BUYING

In light of the impressive product portfolio, it should be of no surprise that the firm's largest shareholder is Billionaire healthcare investor Phillip Frost. He now owns roughly one-fifth of the business and has leveraged his position at Opko Health to extend the firm's distribution network to Latin America and emerging markets. Frost is also the Chairman of the world's largest generic producer and Israel's largest public company Teva Pharmaceutical. The speculation over the firm's largest shareholder further extending products across top patient populations will protect investors from any downside momentum in the stock price. At the same time, should any connections materialize, the stock has more room to appreciate - making for favorable risk/reward.

Phillip Frost is not the only wealthy investor backing ChromaDex. Corporate finance specialist Barry Honig has parlayed large sums of money into small-cap stocks and generated strong returns time and time again. Growing revenues form $3M to north of $125M, he actively led InterCLICK as Chairman to its $270M buyout from Yahoo! He was the mastermind behind SendTec, Neviant, and Seisint - transforming operations from one level to the next. He is now actively accumulating shares of ChromaDex with the most recent purchase being 8/15/12. Honig actually shares an office with Frost who has also been adding to his leading stake. The product line - from NR to pTeroPure and CG3 - and lab testing businesses have already established sustainable competitive advantages to drive the growth story going forward. If these investors (1) continue to not only put their money where their mouths are while (2) adding to the underlying fundamentals, ChromaDex will have even more impressive days ahead. Investors are encouraged to buy shares before the major data announcement on pTeroPure, so as to maximize upside potential.

Disclaimer: The distributor of this research report, Gould Partners, is not a licensed investment adviser or broker dealer. We are a consultant to a third-party representing ChromaDex and have received three thousand five hundred dollars for independent research. Investors are cautioned to perform their own due diligence as information contained within this report has been derived from public sources and cannot be guaranteed by us to be fully accurate. Always discuss investments with a licensed professional before making any financial decision. Statements made herein are often "forward-looking statements" as defined under Section 27A of the Securities Act of 1933, Section 21E of the Securities Act of 1934, and the Private Securities Litigation Reform Act of 1995. Since these statements are uncertain, actual results may be materially different from those expected.

Click to expand...