Pharmacological Nicotinamide: Mechanisms Centered Around SIRT1 Activity

C H A P T E R

29 Pharmacological Nicotinamide: Mechanisms Centered Around SIRT1 Activity Eun Seong Hwang Department of Life Science, University of Seoul, Seoul, Republic of Korea

Nicotinamide (NAM) along with nicotinic acid (a.k.a. niacin) and nicotinamide riboside are members of the vitamin B3 family. They are a major source of nicotinamide adenine dinucleotide (NAD+), a key factor in metabolism and energy homeostasis. In 1938 Elvehjem et al. reported that pellagra (then called “black tongue disease”), a major manifestation of vitamin B3 deficiency, could be cured by NAM.1 This was the first report on the therapeutic effectiveness of NAM. During the last 50 years NAM at gram doses (in most cases 1–3 g a day), which exceeds the recommended daily allowance of 20 mg of niacin as recommended by the US Food and Drug Administration, has shown pharmacologic or therapeutic effectiveness in various diseases and conditions. NAM is currently used as a treatment for diseases such as bullous pemphigoid and skin cancers. Studies have also reported the usefulness of NAM in protecting neurons and vascular cells from ischemic reperfusion and similar oxidative stresses and preventing the onset of diabetes mellitus and immune suppression caused by UV radiation in patients with cancer. Effects against HIV, uremic pruritus, and inflammatory diseases have also been reported. Furthermore, NAM has been increasingly incorporated in many topical agents for skin protection and cosmetic purposes. These therapeutic effects are summarized in Table 29.1. NAM administration at gram-level doses (referred herein as pharmacological doses) has so far been shown to be safe and, encouraged by this, NAM is commercially available as tablets or in powder form. It is now necessary to understand the mechanisms underlying the diverse effects of NAM and make its administration even more secure to facilitate the development of broader therapeutic strategies. NAM is converted to NAD+ once it is taken up in cells, and the increase in cellular NAD+ levels appears to be the major mechanism underlying its effectiveness. NAM administration can affect energy metabolism by increasing the NAD+ redox level. In addition, NAM and NAD+ modulate the activities of many enzymes involved in stress responses and metabolism. Among these are two important proteins, SIRT1 and PARP-1, which play critical roles in cellular defense as well as organismal health and longevity. Importantly, the cellular NAD+ level and its redox state are not static. It is subject to change in association with diet, pathophysiological cues, cellular stresses, circadian rhythm, and aging process.44–47 This suggests that the NAD+ level is a prime target for manipulation in efforts to intervene in disease progression or to promote health and longevity. Recent studies indicate that this may be feasible. The administration of high doses of NAM has been shown to ameliorate some disease conditions in humans and enhance health and longevity in animals.48, 49 In this chapter current understanding of the mechanisms underlying the pharmacological effectiveness of NAM is introduced.

29.1 BIOCHEMISTRY AND FUNCTIONS OF NAD+ AND NAM The pharmacological effects of NAM are triggered via several different routes. One of them, the supply of NAD+ to cells, plays a dominant role. In many cases under test conditions the effect of NAM was abolished by treatment with FK866, a potent inhibitor of nicotinamide phosphoribosyl transferase (NAMPT),50 which catalyzes the first and ratelimiting step in the pathway of NAD+ production from NAM (salvage pathway of cellular NAD+ synthesis) (Fig. 29.1). Furthermore, nicotinic acid (niacin) and nicotinamide riboside, other NAD+ precursors, are similarly effective under

Pharmacoepigenetics https://doi.org/10.1016/B978-0-12-813939-4.00029-2

781

© 2019 Elsevier Inc. All rights reserved.

TABLE 29.1

Some Examples of Proposed Therapeutic Effectiveness of High-dose Nicotinamide

Pharmacological effects

Examples of clinical effectsa

Antiinflammatory and immunomodulatory and antiskin aging

• Treats and alleviates osteoarthritis and inflammatory conditions in patients • Oral and topical applications in humans have been shown effective against acne, rosacea, melasma, psoariasis, and bullous pemphigoid; Prophylaxis against nonmelanoma skin cancer • Phase III trial reduced the rate of new actinic keratosis and nonmelanoma skin cancers • Effective (in combination with tetracycline) against lupus erythematosus-associated skin disease in dogs and cats • Protects rats against NO-mediated vascular failure in endotoxic shock

Antidiabetic

• Prevents the onset and progression of diabetes • Preserves β-cell function in recent onset type 1 diabetes patients for up to 2 years after diagnosis

Neuroprotective

• Attenuates focal ischemic brain injury in rats • Treats dementia and neurological diseases and protects against cerebral ischemia, multiple sclerosis, and Alzheimer disease in animals • Improved cognitive function in subjects with mild to moderate AD in phase II trial

Antidepressive

• Enhances recovery from behavioral deficits, mental disorders, and discomforts ranging from schizophrenia to ADHD; has been shown effective at treating various types of depression in humans

Anti-HIV and AIDS

• Lessens HIV production and alleviates AIDS • Suppressed AIDS progression and improved survival in an AIDS cohort study

Renal protective

• Retards diabetic nephropathy development and ameliorates hyperphosphatemia in hemodialysis patients

a

References to the examples are listed in Table 29.2.

4-PY

Nucleus 2-PY

NADPH

NAM +

NADP

NADH

MeNAM NAMPT

PRPP

NAM

Microdomain

NAM

NMN

NNMAT-1

NAMPT

NAD+ Sirtuins NAM

eNAMPT

ARTs

PRPP

NNMAT-1 SIRT1

NAM

NAD+

acetyl-

NMN QNS1

SIRT1-target factor on promotger

NAAD

NRK1,2

NMA1,2

NAMN NPT1

NR NA

Conversion of NAM to NAD and other metabolites in the cytosol and nucleus. NAM transported to the cytosol is converted to NAD+ through the savage pathway (blue box). NAM is combined with PRPP to produce NMN which in turn is adenylated to become NAD+ through the activities of NAMT and NMNAT-1. A small fraction of NAD+ is reduced to NADH or phosphorylated to NADP+, which can be reduced to NADPH. NAD+ is also degraded to NAM and ADP-ribose by the enzymes SIRT1, PARP-1, and other ADP-ribosyl transferases (ARTs). NAM inhibits the activities of these NAD+-consuming enzymes. A limited amount of NAM is removed through the conversion to methyl-NAM (MeNAM), which is further metabolized to 2-PY and 4-PY; however, these conversions are inefficient. Meanwhile, in the nucleus (brown circle), NMNAT-1 was found to colocalize with SIRT1 on target DNA and facilitate NAD+ supply to SIRT1.51 NAMPT was also found to activate SIRT1 in target gene expression51 and is expected to localize near SIRT1 forming a microdomain (beige circle) in which the local concentration of NAM is lowered. This hypothetical microdomain creates a condition where SIRT1 is activated on its target promoter, even in the presence of high cellular levels of NAM. In addition, cells secrete eNAMPT, which converts NAM in the media or in body fluids to NMN. This lowers the cellular levels of NAM, but the production of NAD+ may be maintained by the influx of NMN instead of NAM and thus may facilitate SIRT1 activation. Meanwhile, nicotinic acid (NA) and nicotinamide riboside (NR) are converted to NAD+ via nicotinic acid mononucleotide (NAMN) and nicotinic acid adenine dinucleotide (NAAD) (for NA) and via NMN (for NR). 2-PY, N-methyl-2-pyridone-5-carboxamide; 4-PY, N-methyl-4-pyridone-5-carboxamide; Acetyl-●, acetylated transcription factor, a SIRT1 target; ARTs, mono-ADP-ribosyl transferases; eNAMPT, extracellular NAMPT; NMA1, NMA2, nicotinate mononucleotide adenyl transferase; NMNAT-1, NMN adenyl transferase; NPT1, nicotinate phosphoribosyl transferase 1, 2; NRK1, NRK2, NR kinase 1, 2; PRPP, phosphoribosyl pyrophosphate; QNS1, NAD synthetase. Reprinted and modified by permission from Springer Nature. Hwang ES, Song SB. Nicotinamide is an inhibitor of SIRT1 in vitro, but can be a stimulator in cells. Cell Mol Life Sci 2017; 74(18):3347-3362. © Springer International Publishing 2017.

FIG. 29.1

+

29.1 BIOCHEMISTRY AND FUNCTIONS OF NAD+ AND NAM

783

test conditions in disease as well as in health48, 49 (Fig. 29.1). To understand the mechanisms underlying the diverse effects of NAM it is important to understand the association between NAM and NAD+ as well as their biochemical properties. The absence of a dietary supply of NAM causes pellagra,52 a disease caused by NAD+ deficiency. Therefore, NAM is a major source of NAD+ in animal cells. A minor portion of the NAD+ pool is filled through de novo synthesis from tryptophan. NAD+, known to be present at the 0.3-mM level in animal cells at normal physiology,53 is utilized and functions largely in two different ways. First, NAD+ constitutes a key cellular redox with its reduced form NADH functioning in signaling and bioenergetics. The NAD+ redox state is closely associated with reactive oxygen species (ROS) generation while modulating the level of mitochondrial oxidative phosphorylation,54 and therefore is related to the levels of cellular oxidative stress and the onset of senescence. Second, NAD+ is a substrate for many hydrolysis enzymes in nonredox reactions and is cleaved to generate NAM, which is converted back to NAD+ through the salvage pathway. Meanwhile, NAM can function as an inhibitor of such reactions. Therefore, a NAD+-NAM cycle exists in cells (Fig. 29.1), and this plays important roles in the regulation of these nonredox reactions.

29.1.1 NAD+ as a Coenzyme in Redox Reactions: A Key Determinant of the Levels of ATP and ROS NAD+ is a coenzyme for a variety of dehydrogenases that mediate redox reactions. Typically, it accepts a highenergy electron from glyceraldehyde 3-phosphate to become NADH during glycolysis. Therefore, an increase in the cellular NAD+ level can facilitate glycolytic flux. Meanwhile, high cellular NAD+ levels can cause a decrease in the NADH/NAD+ ratio in mitochondria, thereby lowering the flow of electrons through the electron transport chain and causing a decline in ATP production. Concurrently, because electrons are prone to leak from the chain and thereby lead to the formation of superoxide radicals, mitochondrial ROS generation increases parallel with the level of electron flux. Therefore, the NAD+ level modulates the ROS level in addition to ATP production. Although cells are equipped with ROS-scavenging enzymes, such as superoxide dismutases, these enzymes apparently are not sufficiently effective at maintaining low ROS levels, particularly as their proliferation approaches the stage of replicative senescence. On the other hand, enhanced NAD+/NADH lowers ROS levels at the point of generation.9 Therefore, enhancing the cellular NAD+ level can be an excellent antioxidative strategy (Fig. 29.2). Indeed, an increase in the NAD+ level in human cells by the administration of 5 mM NAM has been shown to cause a >30% decline in the level of mitochondrial superoxide. Furthermore, through this antioxidative effect, NAM alleviated oxidative damage that may accumulate as cells continue to proliferate and thereby promoted cellular longevity.55, 56

29.1.2 NAD+ as a Substrate for Enzymes in Nonredox Reactions A class of hydrolases cleave NAD+ into ADP-ribose and NAM.57 The ADP-ribose moiety is transferred to either substrate proteins or a poly(ADP-ribose) chain, or linked to the acetyl group that is detached from the substrate, or converted to cyclic ADP-ribose58 (Fig. 29.3). Of these, two families of proteins, poly(ADP-ribose) polymers (PARPs) and sirtuins, are better known for their roles in cells and organisms. Most of the effects of NAM or NAD+ in health promotion or disease alleviation are attributed to changes in the activities of these proteins. The roles of other ADP-ribosyl transferases (ARTs) are also being recognized, albeit rather slowly. 29.1.2.1 PARPs PARP family proteins bind to damaged parts of DNA and trigger DNA repair responses.59 PARP-1, the best known and most abundant member of the family, binds to single-strand breaks of DNA and is activated to cleave NAD+ to make polymers of ADP-ribose (poly(ADP-ribose)) (Fig. 29.3B), which signal DNA repair proteins.60 PARP activity is conceivably important as a guardian of genome integrity because there is a strong correlation between cellular PARP activity and the life span of mammals.61 However, PARP proteins can impose certain adverse effects on cells and the body depending on the manner in which they are activated and exert activities. PARPs under severe oxidative stresses, such as those imposed by ischemic reperfusion in which sudden exposure to a large amount of oxygen triggers oxidative damage to cellular components, are activated to a great extent and thus exhaust the cellular NAD+ pool, thereby inducing an energy crisis.62 In addition, PARP-1 induces the mitochondrial permeability transition pore (mPTP), through which small molecules diffuse across mitochondrial membranes bringing about deleterious changes, such as Ca+2 efflux, ROS accumulation, and dissipation of membrane potential (ΔΨm),63 and thereby causes the apoptotic death of vulnerable cells like neurons64, 65 (Fig. 29.2B). Meanwhile, PARP-1 enhances NF-κB-mediated transcription of the genes encoding for proinflammatory cytokines, chemokines, and inflammatory mediators. This activity can occasionally exacerbate inflammation and underlies PARP-1-mediated induction and aggravation of inflammatory diseases in animal models.6, 66, 67

FIG. 29.2 Nicotinamide-mediated downregulation of mitochondrial ROS generation. (A) The mitochondrial production of ROS is linked to oxidative phosphorylation. High NADH/NAD+ ratios push the transfer of electrons to the electron transport chain. (Yellow, blue, and pink ovals depict complexes I, III, and IV, respectively, in the mitochondrial inner membrane. The green cylinder is ATP synthase. Black arrows indicate electron flow in the chain.) At high NADH/NAD+ ratios more electrons pass through the chain and produce a high proton gradient leading to a high level of ATP production. At high ΔΨm some electrons are pushed in the reverse direction and leak out from complex I and complex III (red lines). These electrons attach to free O2 forming superoxide radicals (O2
), which are removed by getting converted to H2O as a result of the activities of ROS scavengers, SOD, thioredoxin reductase (TRXR), and catalase. However, this is apparently not effective enough, as some superoxide radicals are left to become hydroxyl radicals. On the other hand, a low level of NADH leads to decreased ROS generation while driving ΔΨm at reduced rates. NAM treatment leads to low NADH/NAD+ ratios in mitochondria and thereby attenuates ROS generation, functioning as an efficient antioxidative strategy as shown in Ref. 9. (B) ROS are also generated through mPTP. mPTP is traditionally viewed as a VDAC-ANT complex, which is located at “contact sites” between the inner and outer membranes of mitochondria through interaction with creatine kinase (Crk). Recent studies have reported phosphate carrier (PiC), F0F1 ATP synthase, not VDAC, as the essential pore component. Acetylated CypD binds to ANT and PiC and induces mPTP opening. NAM induces activation of SIRT3, which deacetylates CypD and drives its dissociation from ANT and PiC, thereby inducing mPTP closure. mPTP opening is also induced by high levels of Ca2+ and ROS and inorganic phosphate, as well as by dissipation of ΔΨm and low levels of ATP.

O H H

O NH2

O–

+ N

O P O

O P O

O

NH2

N N O

O–

N

Nicotinamide

PARP

N O

OH OH

O P O

NH2

NAD+ NH2

O–

O O

O

Rib OH OH N

N N

O P O

Rib

N

N

P

Ad

P

N

O

O–

Ad

NH2

P

OH OH

NAD+

NADH

PARP

Ad

P

P

P

Rib

Rib Rib P

(A)

Acceptor protein

NAD+

Ad P

OH OH

Rib

Rib Rib

Rib

P

Nicotinamide

Ad P

Rib

Acceptor protein

(B)

NAD+ Ac

NAD+

O

Substrate protein

O NH2

CD38

NH2

N

N

Nicotinamide

Nicotinamide

H 2N N

Sirtuins OH O O OH

O O O P O P O OH OH

N

N

O

N

O

O

HO

(C)

O O

O

P

OH

OH

OH

OH OH

Ac

Substrate protein

P

O

N

O-acetyl-ADP-ribose HO

(D)

O

N

HN

N

OH

N

Cyclic ADP-ribose TRENDS in Cell Biology

NAD+ in redox reactions is reduced to NADH, whereas in nonredox reactions it is cleaved to NAM and ADP-ribose. (A) NAD+ is reduced to NADH constituting NAD+/NADH redox. (B–D) It is also a cosubstrate for NAD+-consuming enzymes (nonredox enzymes), such as poly-ADP-ribose polymerases (PARPs) (B), sirtuins (C), and CD38/157 ectoenzymes (D). NAD+ is commonly cleaved by these enzymes to NAM and ADP-ribose. NAM functions as an inhibitor of the reactions of these enzymes. Therefore, NAM and NAD+ form a reciprocal regulatory circuit. Reproduced from Imai S, Guarente L. NAD+ and sirtuins in aging and disease. Trends Cell Biol 2014; 24(8):464-471. © 2014 Elsevier Ltd.

FIG. 29.3

29.2 REGULATION OF SIRT1 BY NAM

785

29.1.2.2 Sirtuins Sirtuins are a family of proteins that possess dual enzymatic activities (deacetylase and ADP-ribosyl transferase) (Fig. 29.3C) and regulate the activity of target proteins through deacetylation.68 All seven members of the human sirtuin family (SIRT 1–7), characterized by different localization and substrate specificity, largely play roles in metabolic homeostasis and stress resistance as well as metabolism in diverse eukaryotes.69, 70 As a result of their dependence on a high NAD+/NADH ratio and the capability to modulate the NAD+ level, sirtuin activities are closely associated with the cellular state of energy and redox.71 The action mechanisms of SIRT1 and SIRT3 are better known and their roles better appreciated than those of other member proteins. In addition, collaboration between these two proteins in certain cellular target events (e.g., enhancing mitochondrial quality and activity) has also been demonstrated in recent studies. After a brief summary of the activities of these proteins the way in which they, particularly SIRT1, are regulated by NAM and possible factors that affect this regulation will be introduced. 29.1.2.3 SIRT1 SIRT1 plays a key role in the maintenance of health, development, and longevity as well as in the prevention of many aging-associated disorders. The targets of SIRT1 are transcription factors and coregulators involved in diverse cellular activities including energy homeostasis, differentiation, cell growth, survival, apoptosis, autophagy, inflammation, and DNA damage responses. This is the reason the modulation of SIRT1 activity rather than PARP-1 activity may be more important in the diverse pharmacological effects of NAM. SIRT1, in general, exerts effects by modulating the activities of multiple targets. For example, SIRT1 suppresses inflammation by inducing inhibitory deacetylation of the RelA/p65 subunit of NF-κB72, 73 and stimulatory deacetylation of FoxO3, a transcription factor that induces antioxidant genes.74, 75. Active FoxO3 also suppresses the nuclear translocation of RelA/p65 and thereby inhibits NF-κB signaling.76 SIRT1 also mediates mitochondrial quality maintenance by facilitating mitochondrial biogenesis and degradation. Mitochondrial degradation is achieved by activating peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α),77 a key transactivator for the expression of mitochondrial protein genes.78 The degradation of old mitochondria is mediated by autophagy,79 in which SIRT1 plays a triggering role by deacetylating the critical components in autophagosome formation.80, 81 Further activities of SIRT1 will be discussed with regard to the specific therapeutic effects of NAM. 29.1.2.4 SIRT3 SIRT3, a mitochondrial sirtuin, appears to play a role in mitochondrial integrity and functional efficiency. It deacetylates mitochondrial proteins that are involved in fatty acid metabolism and oxidative phosphorylation. This is believed to enhance the efficiency of oxidative phosphorylation and ATP production.82, 83 Furthermore, SIRT3mediated deacetylation of cyclophilin D (CypD) leads to mPTP closure, thereby attenuating mitochondria depolarization and ROS generation and preserving mitochondrial integrity84 (Fig. 29.2B). It appears that high NAD+ levels enhance mitochondrial quality, especially through the concurrent activation of SIRT1 and SIRT3. In addition to NAM, treatments involving nicotinamide mononucleotide (NMN) or NAM riboside (other NAD+ precursors) have been shown to commonly increase the mRNA of proteins functioning in oxidative phosphorylation and enhance the oxidative performance of mitochondria in a manner dependent on SIRT1 and SIRT3.69, 85

29.2 REGULATION OF SIRT1 BY NAM Because sirtuins (and other nonredox enzymes) commonly cleave NAD+ and produce NAM,57 it is conceivable that NAM behaves as an inhibitor of sirtuins. Indeed, NAM has been shown to bind to SIRT1 and inhibit its activity in a noncompetitive manner.86, 87 In fact, NAM has frequently been used as an inhibitor of SIRT1.88 However, the fact that NAM exerts such diverse cell-beneficial effects yet also inhibits SIRT1 appears contradictory. However, as will be shown, many of these effects can be explained by sirtuin activation rather than inhibition, although the latter also appears to exert positive effects in certain conditions. The existing variables that affect the direction and readiness of the effect of NAM administration need to be better understood.

29.2.1 Dual Effects of NAM on SIRT1 Activity The inhibitory capacity of NAM was first observed on Sir2p, a yeast sirtuin. Sir2p activity was abolished by treatment with 5 mM NAM.89 The overexpression of PNC1, a yeast nicotinamidase that converts NAM to nicotinic acid, reactivated Sir2p,90 whereas the deletion of PNC1 had the opposite effect.91 NAM inhibits human SIRT1 in vitro with an IC50 ranging

786

29. PHARMACOLOGICAL NICOTINAMIDE: MECHANISMS CENTERED AROUND SIRT1 ACTIVITY

from 50 to 180 μM.92–94 Experimentally, millimolar doses of NAM have been successfully used to inhibit SIRT1 in such a way as to prove a cellular event was being mediated by SIRT1 activation. For example, treatment with resveratrol, a potent activator of SIRT1 or SIRT1 overexpression, attenuated TNFα-induced expression of molecules functioning during the onset and maintenance of the reaction to inflammation. This effect was abolished by pretreatment of 20 mM NAM for 1 h, which resulted in an increase in the acetylation level of NF-κB.95 In addition, NAM treatment attenuated resveratrol-mediated suppression of morphological deterioration and increase in cell viability under oxidative stress while increasing the level of acetylation of p53, a major mediator of stress-induced apoptosis.96 The stimulatory effect of NAM on SIRT1 has also been demonstrated. NAM and SIRT1 activators have been found to commonly induce autophagy activation and mitochondrial changes suggestive of mitophagy, such as mitochondrial fragmentation and a decrease in mitochondrial content, when used for treatment in human cells.38, 56, 80, 81 Importantly, a rapid increase in NAD+ levels and the NAD+/NADH ratio accompanied the increase in SIRT1 activity in NAM-treated cells. Together, these findings demonstrate that NAM has dual effects on SIRT1.

29.2.2 Pharmacokinetics of NAM: Interconversion of NAD+ and NAM in Cells NAM is rapidly absorbed from the gastrointestinal tract, and peak concentrations in blood are reached within 1 h of oral ingestion in humans. It is also rapidly cleared from the circulation indicating it is readily absorbed in cells. Moreover, immediately after administration of a large dose of NAM, SIRT1 activity can be inhibited in cells. However, over time NAM is converted into NAD+, and the concentration of NAM decreases while that of NAD+ increases. This predicted pattern of change in the levels of NAM and NAD+ has indeed been demonstrated in both animal tissues and cultured cells. Intraperitoneal administration of NAM at a dose of 500 mg/kg in mice (estimated concentration close to 1 mM) caused linear and rapid increase in NAD+ level in the liver (close to 12-fold levels in 12 h). This level then decreased but remained several times higher than the basal level after 24 h.97 The patterns were similar in other tissues tested, but the extent of increase was smaller. Furthermore, NAM treatment at 10 mM in human fibroblasts caused an increase in NAD+ levels in a similar pattern98, 99 (Fig. 29.4). The NAD+/NADH ratio also increased. The NAD+ level increased nearly twofold in rat liver100 and 1.6-fold in human fibroblasts and hepatoma cells after approximately 5 h.56, 99 The KM of SIRT1 for NAD+ is known to be in the 34–171 μM range.101–103 A level twice as high as the basal level would reach 360–540 μM,46, 53, 104 which falls well above KM and would allow maximum velocity for SIRT1. Therefore, NAD+ levels, when considered alone, easily become stimulatory to SIRT1 after treatment with NAM at millimolar doses. Changes in the levels of NAM itself upon administration have not been addressed as extensively; however, an immediate and steep increase followed by a gradual and lengthy decrease has been reported.105 After intraperitoneal injection in mice the levels of radiolabeled NAM rapidly increased in the tissues tested during the initial 10 min and then decreased while radioactive NAD+ emerged and increased linearly (Fig. 29.5). There appeared to be a large variation in the kinetics of the change in NAM in different organs: in skeletal muscle the increased level of NAM declined the quickest, whereas in

FIG. 29.4 Rapid increase of NAD+ level in human fibroblasts treated with NAM. Upon treatment with 10 mM NAM the cellular NAD+ level increased rapidly during the initial 12 h and decreased thereafter, but remained near the twofold level for 6 days. Reproduced from Hwang ES, Song SB. Nicotinamide is an inhibitor of SIRT1 in vitro, but can be a stimulator in cells. Cell Mol Life Sci. 2017; 74(18):3347–3362. © Springer International Publishing 2017.

787

29.2 REGULATION OF SIRT1 BY NAM

Heart

Kidney 80 nam

nam

nad

80 Radioactivity

Radioactivity

100

60 40 20 0

60 40 20 0

0

60 min

0

60 min

Spleen 14

nam

Skeletal muscle 3

nad

12

nam

nad

2.5

10

Radioactivity

Radioactivity

nad

8 6 4 2

2 1.5 1 0.5

min

0

0 0

60 min

0

60 min

Observed changes in the levels of NAM and NAD+ in tissues administered a high-dose of NAM. Representative changes in the levels of NAM and NAD+ in mice after NAM administration are as adopted from data reported by Collins and Chaykin.105 The y axis of the graphs represents the radioactivity of NAM or NAD+, which was measured in tissues isolated at intervals of 5–60 min after intraperitoneal administration of radioactive NAM (0.9 μM). In all the organs the levels of NAM rapidly increased during the initial 10 min and then decreased at varying rates for the following 50-min chase. During this chase period radioactive NAD+ linearly increased in all the organs but varied widely in rates. As a consequence the degree of NAD+ conversion at the final 60-min point appeared quite different from tissue to tissue. This difference in conversion rates appears to be independent of the amount of NAM that entered the cells (note that radioactivity on the y axis is different in each of the four graphs). In the report the conversion that occurred in other tissues fell between the two extremes represented by the cases of the kidneys and skeletal muscles. Reproduced from Hwang ES, Song SB. Nicotinamide is an inhibitor of SIRT1 in vitro, but can be a stimulator in cells. Cell Mol Life Sci. 2017; 74(18):3347-3362. © Springer International Publishing 2017.

FIG. 29.5

spleen tissue it was sustained for over 1 h105 (Fig. 29.5). This, together with change in the NAD+ level, indicates that SIRT1 inhibition can persist only transiently at an early period after NAM administration and soon disappears with some variation in different tissues. In most studies the inhibitory effect of NAM was checked within 12 h of treatment as summarized in Ref. 88. On the other hand, because the pharmacologic effects of NAM are expected to last for days, rather than hours, one may reasonably expect the effects of SIRT1 mobilization to be manifested for a day upon daily administration or intake of NAM at pharmacological doses. At this juncture it is worth mentioning a hypothesis that proposes a hypothetical microdomain that may be formed in the nucleus and facilitate the rapid conversion of NAM and SIRT1 activation (Fig. 29.1). NAMPT and nicotinamide mononucleotide adenytransferase-1 (NAMAT1), two enzymes of the NAD+ savage pathway, have been found to modulate the expression of certain genes in a SIRT1-dependent manner. In addition, a decrease in the expression of these two enzymes induced a substantial decrease in SIRT1 activity while causing only a small decrease in total cellular NAD+ levels.51 Furthermore, NMNAT-1 has been found to interact with SIRT1 located in the promoter regions of target genes. These findings raised speculation that a substrate-channeling system106 may exist in the nucleus where NAMPT and NMNAT-1 localize to a microdomain in close proximity to SIRT1 and facilitate the link between NAM conversion and NAD+ synthesis and its direct supply to SIRT1. This channeling hypothesis could explain the seemingly prompt activation of SIRT1 upon NAM administration.

29.2.3 Factors That May Affect the Cellular Levels of NAD+ and NAM and SIRT1 Activity The fact that different tissues exhibit different extents and rates of change in NAD+ levels after NAM administration indicates that there may be cellular factors driving the shift in the levels of NAM and NAD+. These could cause differences in the direction and extent of the pharmacologic effects of NAM in different tissues and even individuals or disease conditions. This NAM-NAD+ cycle and SIRT1 activity could be important targets for research aiming to achieve better pharmacologic outcomes of NAM treatment. Some such factors are discussed in the following bullet list:

788

29. PHARMACOLOGICAL NICOTINAMIDE: MECHANISMS CENTERED AROUND SIRT1 ACTIVITY

• Activity of NAMPT. NAMPT, the first enzyme in the NAD+ salvage pathway, produces NMN by combining NAM with 5-phosphoribosyl-1-pyrophosphate (PRPP). It is important to note that NAMPT protein levels vary in different tissues107 and are subject to change upon various stresses.58 NAMPT activity is known to be sensitive to cellular ATP level. In human cells at normal physiology a majority of NAMPTs occur in their phosphorylated forms, which have high affinity to NAM,108 linking NAM-NAD+ levels to cellular energy status. Meanwhile, there are different isoforms of NAMPTs. One of them, eNAMTP, an extracellular form (a.k.a. visfatin), is known to have higher NMN synthetic activity than that of its intracellular isoform, and exogenous NMN has been proposed to help in the cellular maintenance of NAD+ levels.109 eNAMPT activity can make NAM treatment easily stimulatory to SIRT1 because the NAD+ level can be increased in cells without any influx of NAM itself (Fig. 29.1). eNAMPT expression is reported to be higher in certain types of cells or tissues.110 It was recently proposed that eNAMPT plays a role against pathological conditions. Monocyte-derived eNAMPT has been shown to play a role in increasing NAD+ levels and SIRT1 activity, and this has been hypothesized to contribute to the survival of cardiomyocytes in pressure-overloaded mice.111 • Activity of PARP. PARPs are the dominant consumers of NAD+ in cells. Cellular NAD+ levels precipitate within a matter of minutes (and thereby SIRT1 activity as well) as a result of PARP activation upon severe DNA damage.112, 113 Although PARP-1 in its inactive state has a KM and a Kcat for NAD+ that are not very different from those of SIRT1,92, 114, 115 NAD+ consumption by activated PARP-1 is dominant over that by SIRT1, as evidenced by the finding that PARP-1 inhibitors partially relieved NAD+ depletion driven by NAMPT inactivation in human cells while SIRT1 inhibitors did not.116 Meanwhile, NAM is a product of PARP activity, and therefore it exerts a similar feedback-inhibitory effect to SIRT1, but the IC50 of PARP-1 is lower than that of SIRT1 (31 μM for PARP-1117 vs 50–180 μM for SIRT1101, 118–120) indicating that NAM more readily inhibits PARP-1 than SIRT1. • Metabolites of NAM. NAM and NAD+ are metabolized into diverse compounds (Fig. 29.1). Some have been shown to affect cell physiology. The levels of N-methylnicotinamide (MeNAM) and its oxidized forms, N-methyl-2-pyridone-5-carboxamide (2-PY) and N-methyl-4-pyridone-5-carboxamide (4-PY), show a steep increase upon NAM administration.121 However, their basal levels are not sufficiently high to affect the levels of NAM or NAD+. Meanwhile, the high serum level of MeNAM has been found to be strongly associated with obesity and diabetes in humans.122 Additionally, inhibiting the generation of MeNAM from NAM has been shown to prevent diet-induced obesity in mice.123 These findings suggest that MeNAM may be an etiologic factor for obesity and diabetes, raising a concern about MeNAM itself and the activity of nicotinamide N-methyltransferase (NNMT). However, no studies have reported obesity as a result of prolonged NAM therapy. NADH is another important metabolite. It inhibits sirtuins with IC50 values of 3–68 mM,120 which are extremely high to influence SIRT1 in cells. However, a slight change in the NADH level can cause a large alteration in the NAD+/NADH ratio and affect SIRT1 activity.124 In addition, the mitochondrial NAD+ redox level affects SIRT3, 4, and 5, which are mitochondrial sirtuins. The NAD+ redox levels in the cytosol and mitochondria are also subject to change along with glycolytic flux, the TCA cycle, and the respiratory chain (ETC), which are in turn regulated by cellular ATP demand and uncoupling in the electron transport chain.125 Therefore, the effect of NAM may be affected by the cellular levels of ATP and oxygen supply. • Cellular modulation of SIRT1 level and activity. SIRT1 protein expression is another factor that sensitizes SIRT1 activity to NAM administration. The treatment of 293T cells with 5 mM NAM inhibited the deacetylation of PGC-1α mediated by endogenous SIRT1 proteins. However, when the SIRT1 protein level was increased via transduction of the gene, PGC-1α in the cells were hypoacetylated upon treatment with NAM even at a dose of 50 mM.126 SIRT1 expression as well as its activity are regulated by endogenous proteins involved in signal transduction and transcription. These proteins include SIRT1 itself, and in many reported cases SIRT1 expression increases with increase in its activity. For example, resveratrol treatment enhanced the SIRT1 mRNA level 1.5-fold.127 Additionally, treatment with 5 mM NAM, while increasing NAD+ levels, upregulated SIRT1 expression by more than 2.5 fold in hepatocytes in a dose-dependent manner.99 SIRT1 activity is also subject to regulation by posttranslational modifications. Phosphorylation by cyclin B-dependent kinase (Cdk1)21 and sumoylation128 have been shown to increase deacetylase activity in vitro. Inversely, S-nitrosylation, a modification brought about by nitric oxide, induces a decrease in activity.129 Through these modifications, cellular SIRT1 activity may become more or less vulnerable to different levels of NAD+ and NAM. • Tissue variation. NAMPT activity appears to vary widely in different tissues, being highest in the liver. Its activity is substantially lower in the kidney, spleen, heart, muscle, brain, and lung and relatively minimal in the intestine and pancreas.107 Thereby, the kinetics of NAM to NAD+ conversion can differ in the cells of these organs, and therefore the activation of SIRT1 and other enzymes of nonredox reactions can differ.

29.3 THERAPEUTIC EFFECTS OF NAM AND UNDERLYING MECHANISMS

789

29.3 THERAPEUTIC EFFECTS OF NAM AND UNDERLYING MECHANISMS The cytoprotective or pharmacological effectiveness of NAM shown or suggested in various disorders and disease conditions in human subjects and animal models is summarized in Table 29.2, along with a brief discussion on the key underlying mechanisms. Most of the mechanisms suggested are plausibly supported by some of the biological TABLE 29.2

Examples of the Pharmacologic Effectiveness of Nicotinamide

Pharmacological application and potentiala

Description of effects

Key mechanisms suggested

Antiinflammation Alleviation of arthritis and inflammatory conditions

Attenuates the expression and secretion of proinflammatory cytokines; attenuates neutrophil chemotaxis and mononuclear cell infiltration; reduces antigen presentation

(1) SIRT1-mediated downregulation of proinflammatory transcription factors, NF-κB2, 3 HIF-1α,4 AP-15; PARP-1-mediated necrotic cell death and transcriptional activation by NF-κB6, 7; adipogenesis-induced secretion of inflammatory cytokines8 (2) SIRT4-, 5-, and 6-mediated modulation of NF-κB transcriptional activity (3) Maintenance of low-level ROS production via SIRT1- and 3-mediated enhancement of mitochondria integrity9

Immune modulation Lessening of autoimmune disorders and toxin-induced shock

Attenuates the proliferation of B and T cells and antibody secretion

(1) Attenuation of STAT signaling and immune response through inactivation of PARP14, an ADP-ribosyl transferase functioning in STAT6 promoter activation10 (2) Attenuation of MHC-II expression and T cell proliferation by lowering STAT signaling and SIRT1-mediated PARP inactivation11

Antidiabetes Prevention of the onset and progression of diabetes

Preserves β-cells and their function

(1) β-cell protection by SIRT1 through attenuation of PARP-1-induced apoptosis and necrosis; direct inhibition of PARP-1; suppression of gluconeogenesis through SIRT-1-mediated degradation of TORC212; increase in ATPdependent insulin secretion via UCP2 downregulation13 (2) SIRT1- and 3-mediated mitochondria quality enhancement9

Antiskin aging and tumorigenesis Protection from inflammatory diseases; acceleration of wound healing; suppression of skin cancer

Enhances the biogenesis of epidermal barrier; reduces melisma; attenuates inflammation in irradiated lesion; reduces skin cancer

(1) NAM-mediated increase in synthesis of sphingolipid in epidermal permeability barrier through the expression of serine palmitoyl transferase14; improvement of matrix and moisture retention by increasing the synthesis of collagen and other components in the dermis matrix15; suppression of the migration of melanosomes to keratinocytes in the damaged dermis16 (2) SIRT1-mediated acceleration of keratinocyte differentiation17

Neuroprotection Treatment of dementia and neurological diseases; protection against cerebral ischemia, multiple sclerosis, and Alzheimer disease

Protects neurons from oxidative damage via NAD+ and ATP preservation as well as through attenuation of inflammation; enhances postischemic neovascularization

(1) NAM-mediated inhibition of PARP-1 and SIRT1 leading to maintenance of cellular NAD+ and ATP levels18–20; direct supply of NAD+ (2) SIRT1-mediated attenuation of inflammation and apoptosis through inactivation of p5321, 22; increase in the activity of α-secretase, which produces a neuroprotective segment of APP23, 24; upregulation of the expression of prosurvival factors and downregulation of proapoptotic factors25; enhancement of proliferation of endothelial progenitor cells26 (3) SIRT3-mediated closure of mitochondrial mPTP9, 27 Continued

790 TABLE 29.2

29. PHARMACOLOGICAL NICOTINAMIDE: MECHANISMS CENTERED AROUND SIRT1 ACTIVITY

Examples of the Pharmacologic Effectiveness of Nicotinamide—cont’d

Pharmacological application and potential

Description of effects

Key mechanisms suggested

Antidepression Enhanced recovery from behavioral deficits, mental disorders, and discomforts ranging from schizophrenia to ADHD

Increases plasma serotonin level; improved memory and cognitive decline in AD model mice

NAM may directly increase:

Anti-HIV and AIDS Lessening HIV production; alleviation of AIDS

Inhibits HIV production in lymphocytes; alleviates chronic immune inflammation in infected individuals; increases CD4+ cell counts in patients with AIDS

(1) NAM-mediated PARP inhibition attenuated PARP-1-mediated integration of proviral DNA in chromosomes10, 33; activation of proviral promoter, LTR34; actin filament rearrangements for transport of proviral DNA and progeny virions35 (2) SIRT-1 activation alleviates the NF-κB proinflammatory pathway (3) NAM treatment saves patients from experiencing severe diminution of tryptophan in plasma by alleviating tryptophan usage for NAD+36

Antimetastasis and adjuvant to radiation therapy

Suppresses metastasis; enhances efficiency of radiotherapy when administered at the time of radiotherapy (ARCON)

(1) SIRT1-mediated inactivation of HIF-1α and NF-κB, key transcription factors for prometastatic environments37 (2) NAM-mediated increase of NAD+/NADH ratio inhibited tumor growth and metastasis38 (3) NAM-mediated acceleration of blood flow and oxygen supply to tumors proposed to enhance the efficiency of radiotherapy39

Renal protection Retardation of diabetic nephropathy development; amelioration of hyperphosphatemia in hemodialysis patients

Reduces renal cell apoptosis, inflammation, and fibrosis; protects hemodialysis patients from hyperphosphatemia

(1) SIRT1 activation and PARP inactivation prevented NF-κB-induced renal cell apoptosis, inflammation, and fibrosis40 (2) NAM-mediated inhibition of Na+-dependent phosphate cotransporters in renal tubule and intestine led to increase in renal and fecal Pi excretion41 and decrease in phosphate uptake42, 43 (however, it is unknown how NAM inhibits the transporters in renal tubules and the intestine)

(1) Serotonin production by sparing tryptophan and thereby the supply of NAD+ from the salvage pathway28, 29; antioxidative capacity, improving clinical symptoms in psychiatric disorders30; improving NAD+/NADH ratio in patients with schizophrenia31 (2) Reduction in the levels of phosphorylated tau implicated in AD32

a

NAM has had applications in many disease conditions in humans, some of which originated from the results of tests in mouse models. The results in this table are from both human and animal models.

activities of NAD+ or NAM. Moreover, the mechanisms involving the activation of sirtuins, particularly SIRT1, seem to underlie many of the therapeutic effects of NAM, as shown in the following examples.

29.3.1 Antiinflammatory Effect Taking NAM or nicotinic acid substantially reduces pain and swelling and improves joint flexibility in patients with arthritis.93 For over 50 years NAM has been known as a treatment for arthritis and for its promising effects against inflammatory and autoimmune disorders.94, 130 NAM appears to suppress the formation of proinflammatory tissue environments. The treatment downregulates the expression of a host of proinflammatory cytokines in cultured cells and mice131, 132 as well as the generation of superoxide by NADPH oxidase in activated neutrophils and monocytes.133 It suppresses neutrophil chemotaxis and mononuclear cell infiltration and inhibits antigen presentation.134, 135

29.3 THERAPEUTIC EFFECTS OF NAM AND UNDERLYING MECHANISMS

791

Moreover, NAM prevents IFN-γ-stimulated iNOS induction in macrophages, where nitric oxide acts as a potent mediator of inflammation.132, 136 There are several different routes for NAM-mediated suppression of inflammation. Of them, SIRT1 activationmediated downregulation of the transcription factors necessary for the expression of proinflammatory genes appears to be the most prominent. NF-κB is a transcription factor that plays a central role in inflammation and immune response by activating a host of genes, including those encoding for cytokines, chemokines, cell adhesion molecules, immunoglobulins, and major histocompatibility complex (MHC) molecules.137 The RELA/p65 subunit of NF-κB is deacetylated by SIRT1 and its transcriptional activity gets downregulated.2, 3 SIRT1 may also downregulate NF-κB via inactivation of PARP-1, which is required for the transcriptional activity of NF-κB.6, 7 The involvement of PARP-1 in NF-κB activation suggests NAM can inhibit inflammation directly. However, data do not support this direct role of NAM. It has been shown that the stimulation of NF-κB activity in cytokine expression requires PARP-1 expression but not PARP-1 activation nor its binding to DNA.138 Furthermore, 6-(5H)-phenanthridinone, a PARP inhibitor, suppressed PARP activation but failed to inhibit cytokine expression.139 Moreover, PARP-1 acetylation has been shown to be necessary for interaction with NF-κB and its transcriptional activation, which is inhibited by histone deacetylases including SIRT1.140 Therefore, NAM appears to suppress NF-κB transcriptional activity mostly through SIRT1 activation. SIRT1 deacetylates and inactivates other proinflammatory transcription factors such as HIF-1α4 and AP-1 subunits.5 HIF-1α plays a role in inflammatory cell recruitment and activation of the inflammatory response through crosstalk with NF-κB,141 whereas AP-1 transcribes diverse genes of inflammatory cytokines upon microbial infection.142 SIRT1 also plays a role in reducing systemic inflammation, but does so in an indirect manner. It inhibits the expression of proliferator-activated receptor γ (PPARγ),143 the master regulator of adipogenesis, and thereby suppresses fat accumulation.144 This antiadipogenesis effect can also be antiinflammatory because adiposity includes systemic increase in the secretion of proinflammatory cytokines from macrophages.8 Finally, SIRT1 activation may also alleviate inflammation by enhancing mitochondrial quality and thereby reducing the generation of ROS, major stimulants of inflammation.145 On the other hand, SIRT1 activity is downregulated in an inflammatory environment. NF-κB induces the expression of microRNA 34a (miR-34a),146 which suppresses the translation of SIRT1 mRNA.147 In addition, the production of ROS and nitric oxide regulates SIRT1 through modifications such as cysteine oxidation, carbonylation, S-glutathionylation, and nitrosylation.148, 149 Furthermore, IFN-γ increases the expression of class II transactivator (CIITA), which is recruited to and silences the SIRT1 promoter.150 These mechanisms together may explain the increased inflammatory responses in many chronic metabolic and age-related diseases. The activation of other sirtuins may also contribute to the antiinflammatory effect of NAM. SIRT4 abolishes NF-κBmediated induction of proinflammatory cytokine genes by inhibiting its nuclear localization,151 although it is not clear how a mitochondrial sirtuin modulates NF-κB. SIRT5 is shown to suppress the proinflammatory response in macrophages through succinyl modification of pyruvate kinase isozyme M2 (PKM2), which otherwise forms a complex with HIF-1α to activate IL-1β expression.152 SIRT6 attenuates the activation of NF-κB target genes by deacetylating histone H3 bound to promoters and thereby limits NF-κB accessibility.153–155 Finally, NAM-mediated NAD+ restoration attenuates the activation of NADPH oxidase (NOX) and ROS generation that occurs in cells where the NAD+ pool is depleted.156, 157 This suggests that in situations where the NAD+ redox level is chronically reduced NAM administration helps alleviate inflammation and ROS-induced tissue damage.

29.3.2 Antidiabetic Effect NAM prevented the onset and progression of diabetes in animal models.158, 159 In addition, in clinical trials of varying scales NAM demonstrated beneficial effects on metabolic outcomes.160 It has also been shown to preserve β-cell functions when administered to subjects at high risk for insulin-dependent diabetes mellitus (IDDM) at diagnosis.161–163 Furthermore, it showed much promise in reducing the incidence of diabetes in children or in first-degree relatives of patients with IDDM.164, 165 Encouraged by these findings, a large-scale, randomized clinical trial—the European Nicotinamide Diabetes Intervention Trial (ENDIT)—investigating NAM administered at doses up to a maximum of 3 g/day to 552 individuals at high risk (islet cell antibody-positive relatives of patients with type 1 diabetes) was conducted for 5 years.166 However, it failed to determine any benefit of NAM in the prevention of diabetes. Low dose, delayed administration, and/or a likelihood of increased insulin resistance at puberty were speculated to be reasons for the failure to confirm the effects reported. Nonetheless, no adverse effect on the health of children was confirmed even at high doses, thereby strongly supporting the safety of high-dose NAM administration. Clinical and animal studies have subsequently been conducted, and both null and positive outcomes in the prevention or treatment of type I and II diabetes were reported.167

792

29. PHARMACOLOGICAL NICOTINAMIDE: MECHANISMS CENTERED AROUND SIRT1 ACTIVITY

Deranged NAD+ homeostasis is certainly associated with diabetes, and changes in the activities of PARPs, sirtuins, and mitochondrial integrity play critical roles. The apoptotic death of β-cells is a characteristic etiological event for type I diabetes. Moreover, PARP activation is involved, as evidenced by the fact that PARP-1 knockout mice are resistant to diabetes induced by chemicals.168, 169 This led to speculation that PARP-1 inhibition is a mechanism underlying NAM-mediated prevention of β-cell damage.170 SIRT1-dependent mechanisms have also been suggested. SIRT1 overexpression in β-cells has been shown to enhance insulin secretion and glucose tolerance in transgenic mice, whereas its knockout blunted insulin secretion.171, 172 NF-κB inactivation by SIRT1 has been attributed to islet cell protection from cytokine-mediated cytotoxicity and enhancement of their viability in rats.173 Furthermore, insulin secretion by β-cells is dependent on ATP levels. It is critically regulated by uncoupling protein (UCP) 2, which facilitates proton leak across the mitochondrial inner membrane, thereby lowering ATP production.13 SIRT1 binds to and downregulates the promoter of the UCP2 gene.13 Likewise, SIRT1- and SIRT3-mediated enhancement of mitochondrial biogenesis174 and mitochondrial integrity9 would be beneficial to the viability and function of β-cells. Metformin, a potent drug against type 2 diabetes, appears to work predominantly through SIRT1 activation.175 It alleviates hyperglycemia by inhibiting gluconeogenesis, and this effect can be largely attributed to SIRT1-mediated deacetylation and degradation of TORC2, which activates the expression of gluconeogenic genes.12 On the other hand, there is concern about the SIRT1inhibitory effect of NAM. Treatment with 10 mM NAM blunted glucose-induced increase of insulin secretion in tests on rat and mouse β-cell lines.95 This negative effect of NAM is suspected to be a reason for the failure to determine the benefit of NAM in ENDIT. However, if this is indeed the case in humans, many adverse effects in other diverse applications of NAM would be observed.

29.3.3 Neuroprotection Effect Dementia is the most severe manifestation of pellagra, but has been effectively treated with NAM.1 NAM has also been shown to be protective against various neurological diseases including cerebral ischemia,176, 177 multiple sclerosis,178 and Alzheimer disease.179 Brain cell death induced by various oxidative stresses is prevented or attenuated by NAM administration.22, 180–182 For example, intraperitoneal administration of 200 mg/kg NAM after the onset of ischemia increased brain NAD+ levels and reduced ischemic infarct size.18 The cytoprotective effect of NAM is well known, but most prominent in brain cells. NAM administration also improved behavioral deficits in experimental models of multiple sclerosis.178 In this case demyelinated axon degeneration was prevented. Neurons and brain cells appear to have a special property in NAD+ biology, and the mechanisms underlying neuroprotection resulting from NAM administration may have additional features as a result of this. Neurons have a high demand for ATP. Therefore NAD+, a key element in bioenergetics, is important for their survival and function. Furthermore, brain cells appear to heavily depend on the conversion of NAM to NAD+ for the supply of NAD+ as evidenced by the fact that NAMPT inhibition results in brain tissues being severely depleted in NAD+.183 Nonetheless, neurons are inefficient in NAD+ biosynthesis, and NAD+ is known to be largely supplied from surrounding glial cells.184 These would make neurons particularly vulnerable to enzyme-mediated NAD+ consumption. Indeed, increasing the NAD+ level by NMNAT-1 overexpression (not by NAM administration) has been shown to attenuate neurotoxicity in mice, as also observed by PARP-1 knockout.185 Likewise, reduced PARP activation was noted in every study in which the effect of NAM was observed. Furthermore, PARP-1 inhibition in the brain endothelium protected the blood-brain barrier (BBB) in physiologic and neuroinflammatory conditions.186 Therefore, the provision of NAD+ and the inhibition of NAD+ consumption by PARP could be the most important mechanism underlying the protective effect of NAM against neuronal damage.19 Studies have also indicated that NAD+ degradation, even via SIRT1 activation, may play adverse roles in neurons. Zn2+ neurotoxicity, in which the high amounts of Zn2+ synaptically released underlie ischemia, seizure, and trauma-induced neuron death, was reduced by the administration of benzamide or 3-aminobenzamide (inhibitors of NAD+ catabolism) as well as NAM.187 Toxicity was attenuated by sirtinol and 2-hydroxynaphthaldehyde (SIRT1 inhibitors) and potentiated by resveratrol and fisetin (SIRT1 activators).188 This may well indicate that enzyme-mediated NAD+ consumption even by SIRT1 causes an acute and immediate precipitation in the NAD+ level in neurons, making them more vulnerable to ischemic damage. Additionally, the transient presence of NAM as an inhibitor may cause a beneficial outcome. However, many cell-beneficial effects of SIRT1 activation are also expected to be driven via increased NAD+ levels at later stages. For example, decreased levels or activity of p53 and its downstream apoptotic pathway have been observed in NAM-treated cells.145, 189 Moreover, SIRT1mediated inhibition of inflammation would certainly improve the viability of neurons. Studies investigating Alzheimer disease have also proposed mechanisms involving sirtuin activation. SIRT1 activation by NAD+ or resveratrol in cell culture and transgenic mouse models of Alzheimer disease increased the production and activity of α-secretase,

REFERENCES

793

which produces a nontoxic and possibly neuroprotective segment of APP.23, 24 Furthermore, many pathological phenotypes of diverse neurodegenerative diseases are known to be associated with the formation of mPTP,190 which disrupts ΔΨm and thereby impairs mitochondrial integrity.191–193 In Alzheimer disease Aβ has been shown to facilitate mPTP formation by promoting CypD localization to the inner membrane.194 NAM has been proposed to induce mPTP closure and thereby ΔΨm upregulation.9, 27 The activation of SIRT3, which induces the deacetylation of mitochondrial proteins including CypD, may be involved.9

29.3.4 Factors That May Cause the Side Effects of NAM Daily long-term intake of gram doses of NAM has been shown to be safe, as was the case in ENDIT. However, this only involved healthy young individuals. A wider use of NAM demands further safety assurance, particularly for the elderly. To date, there are two aspects that need to be addressed to ascertain the potential side effects of NAM: one relates to a metabolite of NAM and the other relates to concern about the impact of high NAD+/NADH ratio on tissue energy maintenance. As a metabolite of NAM, MeNAM has been suggested to generate superoxide anions and destroy complex I subunits leading to the dysfunction of energy metabolism,195 although its underlying mechanism is not known. Nicotinamide N-methyltransferase, which is responsible for MeNAM generation, is abundant in the brain, and therefore high levels of NAM may augment MeNAM-induced neurotoxicity.196–198 Epidemiological surveys suggest that high consumption of niacin (and possibly NAM) may be linked to the onset of Parkinson disease (PD).198 However, a direct link between NAM and PD has so far not been clearly determined. Moreover, an increase in the levels of NAM leads to low NADH/NAD+ ratios in mitochondria resulting in a decrease in electrons feeding oxidative phosphorylation. This results in decreased ROS generation and a decrease in ATP production as has been shown in the culture of human fibroblasts.9 The proliferation of fibroblasts in vitro was not affected by NAM, but the physiological activities of cells in certain tissues demanding high levels of ATP can be affected. Therefore, the intake of NAM should be carefully supervised, particularly when it comes to elderly individuals whose cells tend to suffer from functional degeneration of mitochondria in general.

29.3.5 Perspectives There may be another very important application of NAM. The cellular levels of NAD+ decrease during aging. A similar decline in SIRT1 activity may contribute to the development of aging-associated diseases and accelerated degeneration; hence much greater awareness and better countermeasures are called for. Indeed, NAM administration has been shown to promote healthy aging (but not extension of life span itself ) in mice.49 There is a reasonable expectation that this may well be applicable to humans too. Furthermore, many SIRT1-modulatory activities are suggested to underlie the positive effects of calorie restriction on metabolic syndrome. Therefore, NAM administration can also confer beneficial effects on metabolic syndrome. It has been demonstrated that the supplementation of NMN, nicotinic acid, or nicotinamide riboside to aged mice restores the metabolic phenotype to what it was in young mice.69, 85, 199–201 Likewise, NAM and these NAD+ precursor molecules hold great potential to be used as pharmaceuticals.

Acknowledgments This study was supported by a grant from the Korean National Research Foundation (NRF-2013M3A9B6076431).

References 1. Elvehjem CA, Madden RJ, Strong FM, et al. The isolation and identification of the anti-black tongue factor. J Biol Chem. 1938;123:137–149. 2. Yeung F, Hoberg JE, Ramsey CS, et al. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J. 2004;23(12):2369–2380. 3. Chen J, Zhou Y, Mueller-Steiner S, et al. SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling. J Biol Chem. 2005;280(48):40364–40374. 4. Cramer T, Yamanishi Y, Clausen BE, et al. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell. 2003;112(5):645–657. 5. Zhang R, Chen HZ, Liu JJ, et al. SIRT1 suppresses activator protein-1 transcriptional activity and cyclooxygenase-2 expression in macrophages. J Biol Chem. 2010;285(10):7097–7110. 6. Liaudet L, Pacher P, Mabley JG, et al. Activation of poly(ADP-Ribose) polymerase-1 is a central mechanism of lipopolysaccharide-induced acute lung inflammation. Am J Respir Crit Care Med. 2002;165(3):372–377. 7. Hassa PO, Hottiger MO. A role of poly (ADP-ribose) polymerase in NF-kappaB transcriptional activation. Biol Chem. 1999;380(7-8):953–959. 8. Odegaard JI, Chawla A. Mechanisms of macrophage activation in obesity-induced insulin resistance. Nat Clin Pract Endocrinol Metab. 2008;4 (11):619–626.

794

29. PHARMACOLOGICAL NICOTINAMIDE: MECHANISMS CENTERED AROUND SIRT1 ACTIVITY

9. Song SB, Jang SY, Kang HT, et al. Modulation of mitochondrial membrane potential and ROS generation by nicotinamide in a manner independent of SIRT1 and mitophagy. Mol Cell. 2017;40(7):503–514. 10. Mehrotra P, Riley JP, Patel R, et al. PARP-14 functions as a transcriptional switch for Stat6-dependent gene activation. J Biol Chem. 2011;286 (3):1767–1776. 11. LeClaire RD, Kell W, Bavari S, et al. Protective effects of niacinamide in staphylococcal enterotoxin-B-induced toxicity. Toxicology. 1996;107 (1):69–81. 12. Liu Y, Dentin R, Chen D, et al. A fasting inducible switch modulates gluconeogenesis via activator/coactivator exchange. Nature. 2008;456 (7219):269–273. 13. Zhang CY, Baffy G, Perret P, et al. Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes. Cell. 2001;105(6):745–755. 14. Tanno O, Ota Y, Kitamura N, et al. Nicotinamide increases biosynthesis of ceramides as well as other stratum corneum lipids to improve the epidermal permeability barrier. Br J Dermatol. 2000;143(3):524–531. 15. Bissett DL, Oblong JE, Berge CA. Niacinamide: A B vitamin that improves aging facial skin appearance. Dermatol Surg. 2005;31(7 Pt 2):860–865 discussion. 5. 16. Hakozaki T, Minwalla L, Zhuang J, et al. The effect of niacinamide on reducing cutaneous pigmentation and suppression of melanosome transfer. Br J Dermatol. 2002;147(1):20–31. 17. Blander G, Bhimavarapu A, Mammone T, et al. SIRT1 promotes differentiation of normal human keratinocytes. J Invest Dermatol. 2009;129(1):41–49. 18. Liu D, Gharavi R, Pitta M, et al. Nicotinamide prevents NAD+ depletion and protects neurons against excitotoxicity and cerebral ischemia: NAD+ consumption by SIRT1 may endanger energetically compromised neurons. NeuroMolecular Med. 2009;11(1):28–42. 19. Virag L, Szabo C. The therapeutic potential of poly(ADP-ribose) polymerase inhibitors. Pharmacol Rev. 2002;54(3):375–429. 20. Liu D, Pitta M, Mattson MP. Preventing NAD(+) depletion protects neurons against excitotoxicity: bioenergetic effects of mild mitochondrial uncoupling and caloric restriction. Ann N Y Acad Sci. 2008;1147:275–282. 21. Sasaki T, Maier B, Koclega KD, et al. Phosphorylation regulates SIRT1 function. PLoS One. 2008;3(12)e4020. 22. Klaidman LK, Mukherjee SK, Hutchin TP, et al. Nicotinamide as a precursor for NAD+ prevents apoptosis in the mouse brain induced by tertiary-butylhydroperoxide. Neurosci Lett. 1996;206(1):5–8. 23. Kojro E, Fahrenholz F. The non-amyloidogenic pathway: structure and function of alpha-secretases. Subcell Biochem. 2005;38:105–127. 24. Albani D, Polito L, Batelli S, et al. The SIRT1 activator resveratrol protects SK-N-BE cells from oxidative stress and against toxicity caused by alpha-synuclein or amyloid-beta (1-42) peptide. J Neurochem. 2009;110(5):1445–1456. 25. Pallas M, Casadesus G, Smith MA, et al. Resveratrol and neurodegenerative diseases: activation of SIRT1 as the potential pathway towards neuroprotection. Curr Neurovasc Res. 2009;6(1):70–81. 26. Wang ZL, Luo XF, Li MT, et al. Resveratrol possesses protective effects in a pristane-induced lupus mouse model. PLoS One. 2014;9(12)e114792. 27. Chong ZZ, Lin SH, Maiese K. The NAD+ precursor nicotinamide governs neuronal survival during oxidative stress through protein kinase B coupled to FOXO3a and mitochondrial membrane potential. J Cereb Blood Flow Metab. 2004;24(7):728–743. 28. Slominski A, Semak I, Pisarchik A, et al. Conversion of L-tryptophan to serotonin and melatonin in human melanoma cells. FEBS Lett. 2002;511 (1-3):102–106. 29. Tian YJ, Li D, Ma Q, et al. Excess nicotinamide increases plasma serotonin and histamine levels. Sheng Li Xue Bao. 2013;65(1):33–38. 30. Pandya CD, Howell KR, Pillai A. Antioxidants as potential therapeutics for neuropsychiatric disorders. Prog Neuro-Psychopharmacol Biol Psychiatry. 2013;46:214–223. 31. Kim SY, Cohen BM, Chen X, et al. Redox dysregulation in schizophrenia revealed by in vivo NAD+/NADH measurement. Schizophr Bull. 2017;43(1):197–204. 32. Green KN, Steffan JS, Martinez-Coria H, et al. Nicotinamide restores cognition in Alzheimer’s disease transgenic mice via a mechanism involving sirtuin inhibition and selective reduction of Thr231-phosphotau. J Neurosci. 2008;28(45):11500–11510. 33. Murray MF, Nghiem M, Srinivasan A. HIV infection decreases intracellular nicotinamide adenine dinucleotide [NAD]. Biochem Biophys Res Commun. 1995;212(1):126–131. 34. Gimble JM, Duh E, Ostrove JM, et al. Activation of the human immunodeficiency virus long terminal repeat by herpes simplex virus type 1 is associated with induction of a nuclear factor that binds to the NF-kappa B/core enhancer sequence. J Virol. 1988;62(11):4104–4112. 35. Stolp B, Fackler OT. How HIV takes advantage of the cytoskeleton in entry and replication. Viruses. 2011;3(4):293–311. 36. Murray MF. Nicotinamide: an oral antimicrobial agent with activity against both Mycobacterium tuberculosis and human immunodeficiency virus. Clin Infect Dis. 2003;36(4):453–460. 37. Ben-Neriah Y, Karin M. Inflammation meets cancer, with NF-kappaB as the matchmaker. Nat Immunol. 2011;12(8):715–723. 38. Santidrian AF, Matsuno-Yagi A, Ritland M, et al. Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression. J Clin Invest. 2013;123(3):1068–1081. 39. Moding EJ, Kastan MB, Kirsch DG. Strategies for optimizing the response of cancer and normal tissues to radiation. Nat Rev Drug Discov. 2013;12 (7):526–542. 40. Li WJ, Shin MK, Oh SJ. Poly(ADP-ribose) polymerase is involved in the development of diabetic cystopathy via regulation of nuclear factor kappa B. Urology. 2011;77(5):1265 e1–8. 41. Ginsberg C, Ix JH. Nicotinamide and phosphate homeostasis in chronic kidney disease. Curr Opin Nephrol Hypertens. 2016;25(4):285–291. 42. Kempson SA, Colon-Otero G, Ou SY, et al. Possible role of nicotinamide adenine dinucleotide as an intracellular regulator of renal transport of phosphate in the rat. J Clin Invest. 1981;67(5):1347–1360. 43. Katai K, Tanaka H, Tatsumi S, et al. Nicotinamide inhibits sodium-dependent phosphate cotransport activity in rat small intestine. Nephrol Dial Transplant. 1999;14(5):1195–1201. 44. Braidy N, Guillemin GJ, Mansour H, et al. Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats. PLoS One. 2011;6(4)e19194. 45. Massudi H, Grant R, Braidy N, et al. Age-associated changes in oxidative stress and NAD+ metabolism in human tissue. PLoS One. 2012;7(7) e42357. 46. Yang H, Yang T, Baur JA, et al. Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival. Cell. 2007;130(6):1095–1107.

REFERENCES

795

47. Peek CB, Affinati AH, Ramsey KM, et al. Circadian clock NAD+ cycle drives mitochondrial oxidative metabolism in mice. Science. 2013;342 (6158):1243417. 48. Zhang H, Ryu D, Wu Y, et al. NAD(+) repletion improves mitochondrial and stem cell function and enhances life span in mice. Science. 2016;352 (6292):1436–1443. 49. Mitchell SJ, Bernier M, Aon MA, et al. Nicotinamide improves aspects of healthspan, but not lifespan, in mice. Cell Metab. 2018;27(3):667–676 e4. 50. Hasmann M, Schemainda I. FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis. Cancer Res. 2003;63(21):7436–7442. 51. Zhang T, Berrocal JG, Frizzell KM, et al. Enzymes in the NAD+ salvage pathway regulate SIRT1 activity at target gene promoters. J Biol Chem. 2009;284(30):20408–20417. 52. Harris LJ. Nicotinamide and pellagra; a review of the development of knowledge and of the present position. Practitioner. 1952;168(1009):57–59. 53. Yamada K, Hara N, Shibata T, et al. The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry. Anal Biochem. 2006;352(2):282–285. 54. Ghosh D, LeVault KR, Barnett AJ, et al. A reversible early oxidized redox state that precedes macromolecular ROS damage in aging nontransgenic and 3xTg-AD mouse neurons. J Neurosci. 2012;32(17):5821–5832. 55. Kang HT, Lee HI, Hwang ES. Nicotinamide extends replicative lifespan of human cells. Aging Cell. 2006;5(5):423–436. 56. Jang SY, Kang HT, Hwang ES. Nicotinamide-induced mitophagy: event mediated by high NAD+/NADH ratio and SIRT1 protein activation. J Biol Chem. 2012;287(23):19304–19314. 57. Katsyuba E, Auwerx J. Modulating NAD(+) metabolism, from bench to bedside. EMBO J. 2017;36(18):2670–2683. 58. Imai S, Guarente L. NAD+ and sirtuins in aging and disease. Trends Cell Biol. 2014;24(8):464–471. 59. Jeggo PA. DNA repair: PARP—another guardian angel? Curr Biol. 1998;8(2):R49–R51. 60. Berger NA, Petzold SJ. Identification of minimal size requirements of DNA for activation of poly(ADP-ribose) polymerase. Biochemistry. 1985;24 (16):4352–4355. 61. Grube K, Burkle A. Poly(ADP-ribose) polymerase activity in mononuclear leukocytes of 13 mammalian species correlates with species-specific life span. Proc Natl Acad Sci U S A. 1992;89(24):11759–11763. 62. Berger NA. Poly(ADP-ribose) in the cellular response to DNA damage. Radiat Res. 1985;101(1):4–15. 63. Halestrap AP, Richardson AP. The mitochondrial permeability transition: a current perspective on its identity and role in ischaemia/reperfusion injury. J Mol Cell Cardiol. 2015;78:129–141. 64. Yu SW, Wang H, Poitras MF, et al. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science. 2002;297(5579):259–263. 65. Culmsee C, Zhu C, Landshamer S, et al. Apoptosis-inducing factor triggered by poly(ADP-ribose) polymerase and bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia. J Neurosci. 2005;25(44):10262–10272. 66. Oliver FJ, Menissier-de Murcia J, Nacci C, et al. Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADPribose) polymerase-1 deficient mice. EMBO J. 1999;18(16):4446–4454. 67. Zerfaoui M, Naura AS, Errami Y, et al. Effects of PARP-1 deficiency on airway inflammatory cell recruitment in response to LPS or TNF: differential effects on CXCR2 ligands and duffy antigen receptor for chemokines. J Leukoc Biol. 2009;86(6):1385–1392. 68. North BJ, Verdin E. Sirtuins: Sir2-related NAD-dependent protein deacetylases. Genome Biol. 2004;5(5):224. 69. Mouchiroud L, Houtkooper RH, Auwerx J. NAD(+) metabolism: a therapeutic target for age-related metabolic disease. Crit Rev Biochem Mol Biol. 2013;48(4):397–408. 70. Mouchiroud L, Houtkooper RH, Moullan N, et al. The NAD(+)/sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling. Cell. 2013;154(2):430–441. 71. Webster BR, Lu Z, Sack MN, et al. The role of sirtuins in modulating redox stressors. Free Radic Biol Med. 2012;52(2):281–290. 72. Rajendrasozhan S, Yang SR, Edirisinghe I, et al. Deacetylases and NF-kappaB in redox regulation of cigarette smoke-induced lung inflammation: epigenetics in pathogenesis of COPD. Antioxid Redox Signal. 2008;10(4):799–811. 73. Kauppinen A, Suuronen T, Ojala J, et al. Antagonistic crosstalk between NF-kappaB and SIRT1 in the regulation of inflammation and metabolic disorders. Cell Signal. 2013;25(10):1939–1948. 74. Brunet A, Sweeney LB, Sturgill JF, et al. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science. 2004;303 (5666):2011–2015. 75. van der Horst A, Tertoolen LG, de Vries-Smits LM, et al. FOXO4 is acetylated upon peroxide stress and deacetylated by the longevity protein hSir2(SIRT1). J Biol Chem. 2004;279(28):28873–28879. 76. Lee HY, You HJ, Won JY, et al. Forkhead factor, FOXO3a, induces apoptosis of endothelial cells through activation of matrix metalloproteinases. Arterioscler Thromb Vasc Biol. 2008;28(2):302–308. 77. Brenmoehl J, Hoeflich A. Dual control of mitochondrial biogenesis by sirtuin 1 and sirtuin 3. Mitochondrion. 2013;13(6):755–761. 78. Scarpulla RC. Nucleus-encoded regulators of mitochondrial function: integration of respiratory chain expression, nutrient sensing and metabolic stress. Biochim Biophys Acta. 2012;1819(9-10):1088–1097. 79. Kim I, Rodriguez-Enriquez S, Lemasters JJ. Selective degradation of mitochondria by mitophagy. Arch Biochem Biophys. 2007;462(2):245–253. 80. Lee IH, Cao L, Mostoslavsky R, et al. A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. Proc Natl Acad Sci U S A. 2008;105(9):3374–3379. 81. Huang R, Xu Y, Wan W, et al. Deacetylation of nuclear LC3 drives autophagy initiation under starvation. Mol Cell. 2015;57(3):456–466. 82. Ahn BH, Kim HS, Song S, et al. A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci U S A. 2008;105(38):14447–14452. 83. Sol EM, Wagner SA, Weinert BT, et al. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3. PLoS One. 2012;7(12)e50545. 84. Hafner AV, Dai J, Gomes AP, et al. Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy. Aging (Albany NY). 2010;2(12):914–923. 85. Canto C, Houtkooper RH, Pirinen E, et al. The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cell Metab. 2012;15(6):838–847.

796

29. PHARMACOLOGICAL NICOTINAMIDE: MECHANISMS CENTERED AROUND SIRT1 ACTIVITY

86. Bitterman KJ, Anderson RM, Cohen HY, et al. Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J Biol Chem. 2002;277(47):45099–45107. 87. Avalos JL, Bever KM, Wolberger C. Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol Cell. 2005;17(6):855–868. 88. Hwang ES, Song SB. Nicotinamide is an inhibitor of SIRT1 in vitro, but can be a stimulator in cells. Cell Mol Life Sci. 2017;74(18):3347–3362. 89. Sauve AA, Schramm VL. Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry. Biochemistry. 2003;42(31):9249–9256. 90. Anderson RM, Bitterman KJ, Wood JG, et al. Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature. 2003;423(6936):181–185. 91. Sandmeier JJ, Celic I, Boeke JD, et al. Telomeric and rDNA silencing in saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway. Genetics. 2002;160(3):877–889. 92. Feldman JL, Dittenhafer-Reed KE, Kudo N, et al. Kinetic and structural basis for acyl-group selectivity and NAD(+) dependence in sirtuincatalyzed deacylation. Biochemistry. 2015;54(19):3037–3050. 93. Hoffer A. Treatment of arthritis by nicotinic acid and nicotinamide. Can Med Assoc J. 1959;81:235–238. 94. Namazi MR. Nicotinamide: a potential addition to the anti-psoriatic weaponry. FASEB J. 2003;17(11):1377–1379. 95. Lin QQ, Yan CF, Lin R, et al. SIRT1 regulates TNF-alpha-induced expression of CD40 in 3T3-L1 adipocytes via NF-kappaB pathway. Cytokine. 2012;60(2):447–455. 96. Busch F, Mobasheri A, Shayan P, et al. Sirt-1 is required for the inhibition of apoptosis and inflammatory responses in human tenocytes. J Biol Chem. 2012;287(31):25770–25781. 97. Kaplan NO, Goldin A, Humphreys SR, et al. Pyridine nucleotide synthesis in the mouse. J Biol Chem. 1956;219(1):287–298. 98. Lee HI, Jang SY, Kang HT, et al. p53-, SIRT1-, and PARP-1-independent downregulation of p21WAF1 expression in nicotinamide-treated cells. Biochem Biophys Res Commun. 2008;368(2):298–304. 99. Li J, Dou X, Li S, et al. Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation. Biochim Biophys Acta. 2015;1853(11 Pt A):2929–2936. 100. Clark JB, Pinder S. Control of the steady-state concentrations of the nicotinamide nucleotides in rat liver. Biochem J. 1969;114(2):321–330. 101. Rye PT, Frick LE, Ozbal CC, et al. Advances in label-free screening approaches for studying histone acetyltransferases. J Biomol Screen. 2011;16 (10):1186–1195. 102. Marcotte PA, Richardson PL, Guo J, et al. Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction. Anal Biochem. 2004;332(1):90–99. 103. Smith BC, Denu JM. Chemical mechanisms of histone lysine and arginine modifications. Biochim Biophys Acta. 2009;1789(1):45–57. 104. Hara N, Yamada K, Shibata T, et al. Elevation of cellular NAD levels by nicotinic acid and involvement of nicotinic acid phosphoribosyltransferase in human cells. J Biol Chem. 2007;282(34):24574–24582. 105. Collins PB, Chaykin S. The management of nicotinamide and nicotinic acid in the mouse. J Biol Chem. 1972;247(3):778–783. 106. Srere PA. Complexes of sequential metabolic enzymes. Annu Rev Biochem. 1987;56:89–124. 107. Shibata K, Murata K, Hayakawa T, et al. Effect of dietary orotic acid on the levels of liver and blood NAD in rats. J Nutr Sci Vitaminol (Tokyo). 1985;31(3):265–278. 108. Burgos ES, Schramm VL. Weak coupling of ATP hydrolysis to the chemical equilibrium of human nicotinamide phosphoribosyltransferase. Biochemistry. 2008;47(42):11086–11096. 109. Revollo JR, Korner A, Mills KF, et al. Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme. Cell Metab. 2007;6(5):363–375. 110. Kim D, Lee G, Huh YH, et al. NAMPT is an essential regulator of RA-mediated periodontal inflammation. J Dent Res. 2017;96(6):703–711. 111. Yano M, Akazawa H, Oka T, et al. Monocyte-derived extracellular Nampt-dependent biosynthesis of NAD(+) protects the heart against pressure overload. Sci Rep. 2015;515857. 112. Goodwin PM, Lewis PJ, Davies MI, et al. The effect of gamma radiation and neocarzinostatin on NAD and ATP levels in mouse leukaemia cells. Biochim Biophys Acta. 1978;543(4):576–582. 113. Skidmore CJ, Davies MI, Goodwin PM, et al. The involvement of poly(ADP-ribose) polymerase in the degradation of NAD caused by gammaradiation and N-methyl-N-nitrosourea. Eur J Biochem. 1979;101(1):135–142. 114. Mendoza-Alvarez H, Alvarez-Gonzalez R. Poly(ADP-ribose) polymerase is a catalytic dimer and the automodification reaction is intermolecular. J Biol Chem. 1993;268(30):22575–22580. 115. Banasik M, Stedeford T, Strosznajder RP, et al. The effects of organic solvents on poly(ADP-ribose) polymerase-1 activity: implications for neurotoxicity. Acta Neurobiol Exp (Wars). 2004;64(4):467–473. 116. Pittelli M, Formentini L, Faraco G, et al. Inhibition of nicotinamide phosphoribosyltransferase: cellular bioenergetics reveals a mitochondrial insensitive NAD pool. J Biol Chem. 2010;285(44):34106–34114. 117. Rankin PW, Jacobson EL, Benjamin RC, et al. Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo. J Biol Chem. 1989;264 (8):4312–4317. 118. Yang H, Lavu S, Sinclair DA. Nampt/PBEF/Visfatin: a regulator of mammalian health and longevity? Exp Gerontol. 2006;41(8):718–726. 119. Guan X, Lin P, Knoll E, et al. Mechanism of inhibition of the human sirtuin enzyme SIRT3 by nicotinamide: computational and experimental studies. PLoS One. 2014;9(9):e107729. 120. Madsen AS, Andersen C, Daoud M, et al. Investigating the sensitivity of NAD+-dependent sirtuin deacylation activities to NADH. J Biol Chem. 2016;291(13):7128–7141. 121. Cantoni GL. Methylation of nicotinamide with soluble enzyme system from rat liver. J Biol Chem. 1951;189(1):203–216. 122. Liu M, Li L, Chu J, et al. Serum N(1)-methylnicotinamide is associated with obesity and diabetes in Chinese. J Clin Endocrinol Metab. 2015;100 (8):3112–3117. 123. Kraus D, Yang Q, Kong D, et al. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity. Nature. 2014;508 (7495):258–262. 124. Marcu R, Wiczer BM, Neeley CK, et al. Mitochondrial matrix Ca(2)(+) accumulation regulates cytosolic NAD(+)/NADH metabolism, protein acetylation, and sirtuin expression. Mol Cell Biol. 2014;34(15):2890–2902.

REFERENCES

797

125. Houtkooper RH, Canto C, Wanders RJ, et al. The secret life of NAD+: an old metabolite controlling new metabolic signaling pathways. Endocr Rev. 2010;31(2):194–223. 126. Rodgers JT, Lerin C, Gerhart-Hines Z, et al. Metabolic adaptations through the PGC-1 alpha and SIRT1 pathways. FEBS Lett. 2008;582(1):46–53. 127. Shan T, Ren Y, Wang Y. Sirtuin 1 affects the transcriptional expression of adipose triglyceride lipase in porcine adipocytes. J Anim Sci. 2013;91 (3):1247–1254. 128. Yang Y, Fu W, Chen J, et al. SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress. Nat Cell Biol. 2007;9 (11):1253–1262. 129. Shinozaki S, Chang K, Sakai M, et al. Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65. Sci Signal. 2014;7(351):ra106. 130. Omidian M, Khazanee A, Yaghoobi R, et al. Therapeutic effect of oral nicotinamide on refractory uremic pruritus: a randomized, double-blind study. Saudi J Kidney Dis Transpl. 2013;24(5):995–999. 131. Hiromatsu Y, Sato M, Tanaka K, et al. Inhibitory effects of nicotinamide on intercellular adhesion molecule-1 expression on cultured human thyroid cells. Immunology. 1993;80(2):330–332. 132. Pellat-Deceunynck C, Wietzerbin J, Drapier JC. Nicotinamide inhibits nitric oxide synthase mRNA induction in activated macrophages. Biochem J. 1994;297:53–58 Pt 1. 133. Kroger H, Miesel R, Dietrich A, et al. Synergistic effects of thalidomide and poly (ADP-ribose) polymerase inhibition on type II collagen-induced arthritis in mice. Inflammation. 1996;20(2):203–215. 134. Ferreira RG, Matsui TC, Godin AM, et al. Neutrophil recruitment is inhibited by nicotinamide in experimental pleurisy in mice. Eur J Pharmacol. 2012;685(1–3):198–204. 135. Hiromatsu Y, Sato M, Yamada K, et al. Inhibitory effects of nicotinamide on recombinant human interferon-gamma-induced intercellular adhesion molecule-1 (ICAM-1) and HLA-DR antigen expression on cultured human endothelial cells. Immunol Lett. 1992;31(1):35–39. 136. Fujimura M, Tominaga T, Yoshimoto T. Nicotinamide inhibits inducible nitric oxide synthase mRNA in primary rat glial cells. Neurosci Lett. 1997;228(2):107–110. 137. Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene. 1999;18(49):6853–6866. 138. Hassa PO, Covic M, Hasan S, et al. The enzymatic and DNA binding activity of PARP-1 are not required for NF-kappa B coactivator function. J Biol Chem. 2001;276(49):45588–45597. 139. Ungerstedt JS, Heimersson K, Soderstrom T, et al. Nicotinamide inhibits endotoxin-induced monocyte tissue factor expression. J Thromb Haemost. 2003;1(12):2554–2560. 140. Rajamohan SB, Pillai VB, Gupta M, et al. SIRT1 promotes cell survival under stress by deacetylation-dependent deactivation of poly(ADPribose) polymerase 1. Mol Cell Biol. 2009;29(15):4116–4129. 141. D’Ignazio L, Bandarra D, Rocha S. NF-kappaB and HIF crosstalk in immune responses. FEBS J. 2016;283(3):413–424. 142. Wang A, Al-Kuhlani M, Johnston SC, et al. Transcription factor complex AP-1 mediates inflammation initiated by Chlamydia pneumoniae infection. Cell Microbiol. 2013;15(5):779–794. 143. Backesjo CM, Li Y, Lindgren U, et al. Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells. J Bone Miner Res. 2006;21(7):993–1002. 144. Picard F, Kurtev M, Chung N, et al. Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature. 2004;429 (6993):771–776. 145. Chong ZZ, Lin SH, Li F, et al. The sirtuin inhibitor nicotinamide enhances neuronal cell survival during acute anoxic injury through AKT, BAD, PARP, and mitochondrial associated "anti-apoptotic" pathways. Curr Neurovasc Res. 2005;2(4):271–285. 146. Li L, Yuan L, Luo J, et al. MiR-34a inhibits proliferation and migration of breast cancer through down-regulation of Bcl-2 and SIRT1. Clin Exp Med. 2013;13(2):109–117. 147. Yamakuchi M, Ferlito M, Lowenstein CJ. miR-34a repression of SIRT1 regulates apoptosis. Proc Natl Acad Sci U S A. 2008;105 (36):13421–13426. 148. Caito S, Rajendrasozhan S, Cook S, et al. SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress. FASEB J. 2010;24(9):3145–3159. 149. Zee RS, Yoo CB, Pimentel DR, et al. Redox regulation of sirtuin-1 by S-glutathiolation. Antioxid Redox Signal. 2010;13(7):1023–1032. 150. Li P, Zhao Y, Wu X, et al. Interferon gamma (IFN-gamma) disrupts energy expenditure and metabolic homeostasis by suppressing SIRT1 transcription. Nucleic Acids Res. 2012;40(4):1609–1620. 151. Tao Y, Huang C, Huang Y, et al. SIRT4 suppresses inflammatory responses in human umbilical vein endothelial cells. Cardiovasc Toxicol. 2015;15 (3):217–223. 152. Ye Z, Ye S, Zhou D, et al. A rare variation of celiac trunk and hepatic artery complicating pancreaticoduodenectomy: a case report and literature review. Medicine (Baltimore). 2017;96(48)e8969. 153. Kawahara TL, Michishita E, Adler AS, et al. SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span. Cell. 2009;136(1):62–74. 154. Lappas M. Anti-inflammatory properties of sirtuin 6 in human umbilical vein endothelial cells. Mediat Inflamm. 2012;2012:597514. 155. Wu Y, Chen L, Wang Y, et al. Overexpression of sirtuin 6 suppresses cellular senescence and NF-kappaB mediated inflammatory responses in osteoarthritis development. Sci Rep. 2015;517602. 156. Benavente CA, Jacobson EL. Niacin restriction upregulates NADPH oxidase and reactive oxygen species (ROS) in human keratinocytes. Free Radic Biol Med. 2008;44(4):527–537. 157. Benavente CA, Jacobson MK, Jacobson EL. NAD in skin: therapeutic approaches for niacin. Curr Pharm Des. 2009;15(1):29–38. 158. Nakajima H, Yamada K, Hanafusa T, et al. Elevated antibody-dependent cell-mediated cytotoxicity and its inhibition by nicotinamide in the diabetic NOD mouse. Immunol Lett. 1986;12(2-3):91–94. 159. O’Brien BA, Harmon BV, Cameron DP, et al. Nicotinamide prevents the development of diabetes in the cyclophosphamide-induced NOD mouse model by reducing beta-cell apoptosis. J Pathol. 2000;191(1):86–92. 160. Crino A, Schiaffini R, Ciampalini P, et al. A two year observational study of nicotinamide and intensive insulin therapy in patients with recent onset type 1 diabetes mellitus. J Pediatr Endocrinol Metab. 2005;18(8):749–754. 161. Greenbaum CJ, Kahn SE, Palmer JP. Nicotinamide’s effects on glucose metabolism in subjects at risk for IDDM. Diabetes. 1996;45(11):1631–1634.

798

29. PHARMACOLOGICAL NICOTINAMIDE: MECHANISMS CENTERED AROUND SIRT1 ACTIVITY

162. Pozzilli P, Browne PD, Kolb H. Meta-analysis of nicotinamide treatment in patients with recent-onset IDDM. The nicotinamide trialists. Diabetes Care. 1996;19(12):1357–1363. 163. Manna R, Migliore A, Martin LS, et al. Nicotinamide treatment in subjects at high risk of developing IDDM improves insulin secretion. Br J Clin Pract. 1992;46(3):177–179. 164. Elliott RB, Pilcher CC, Fergusson DM, et al. A population based strategy to prevent insulin-dependent diabetes using nicotinamide. J Pediatr Endocrinol Metab. 1996;9(5):501–509. 165. Olmos PR, Hodgson MI, Maiz A, et al. Nicotinamide protected first-phase insulin response (FPIR) and prevented clinical disease in first-degree relatives of type-1 diabetics. Diabetes Res Clin Pract. 2006;71(3):320–333. 166. Gale EA, Bingley PJ, Emmett CL, et al. European Nicotinamide Diabetes Intervention Trial G. European Nicotinamide Diabetes Intervention Trial (ENDIT): a randomised controlled trial of intervention before the onset of type 1 diabetes. Lancet. 2004;363(9413):925–931. 167. Cabrera-Rode E, Molina G, Arranz C, et al. Effect of standard nicotinamide in the prevention of type 1 diabetes in first degree relatives of persons with type 1 diabetes. Autoimmunity. 2006;39(4):333–340. 168. Masutani M, Suzuki H, Kamada N, et al. Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes. Proc Natl Acad Sci U S A. 1999;96(5):2301–2304. 169. Pieper AA, Brat DJ, Krug DK, et al. Poly(ADP-ribose) polymerase-deficient mice are protected from streptozotocin-induced diabetes. Proc Natl Acad Sci U S A. 1999;96(6):3059–3064. 170. Kolb H, Burkart V. Nicotinamide in type 1 diabetes. Mechanism of action revisited. Diabetes Care. 1999;22(Suppl 2):B16–B20. 171. Moynihan KA, Grimm AA, Plueger MM, et al. Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-stimulated insulin secretion in mice. Cell Metab. 2005;2(2):105–117. 172. Bordone L, Motta MC, Picard F, et al. Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells. PLoS Biol. 2006;4(2):e31. 173. Kim HI, Yu JE, Lee SY, et al. The effect of composite pig islet-human endothelial cell grafts on the instant blood-mediated inflammatory reaction. Cell Transplant. 2009;18(1):31–37. 174. Liu Q, Wang C, Zhou H. Iridium-catalyzed highly enantioselective transfer hydrogenation of aryl N-heteroaryl ketones with N-oxide as a removable ortho-substituent. Org Lett. 2018;20(4):971–974. 175. Caton PW, Nayuni NK, Kieswich J, et al. Metformin suppresses hepatic gluconeogenesis through induction of SIRT1 and GCN5. J Endocrinol. 2010;205(1):97–106. 176. Lee EJ, Wu TS, Chang GL, et al. Delayed treatment with nicotinamide inhibits brain energy depletion, improves cerebral microperfusion, reduces brain infarct volume, but does not alter neurobehavioral outcome following permanent focal cerebral ischemia in Sprague Dawley rats. Curr Neurovasc Res. 2006;3(3):203–213. 177. Sakakibara Y, Mitha AP, Ogilvy CS, et al. Post-treatment with nicotinamide (vitamin B(3)) reduces the infarct volume following permanent focal cerebral ischemia in female Sprague-Dawley and Wistar rats. Neurosci Lett. 2000;281(2-3):111–114. 178. Kaneko S, Wang J, Kaneko M, et al. Protecting axonal degeneration by increasing nicotinamide adenine dinucleotide levels in experimental autoimmune encephalomyelitis models. J Neurosci. 2006;26(38):9794–9804. 179. Bayrakdar ET, Armagan G, Uyanikgil Y, et al. Ex vivo protective effects of nicotinamide and 3-aminobenzamide on rat synaptosomes treated with Abeta(1-42). Cell Biochem Funct. 2014;32(7):557–564. 180. Klawitter V, Morales P, Bustamante D, et al. Plasticity of the central nervous system (CNS) following perinatal asphyxia: does nicotinamide provide neuroprotection? Amino Acids. 2006;31(4):377–384. 181. Ji D, Li GY, Osborne NN. Nicotinamide attenuates retinal ischemia and light insults to neurones. Neurochem Int. 2008;52(4-5):786–798. 182. Neira-Pena T, Rojas-Mancilla E, Munoz-Vio V, et al. Perinatal asphyxia leads to PARP-1 overactivity, p65 translocation, IL-1beta and TNF-alpha overexpression, and apoptotic-like cell death in mesencephalon of neonatal rats: prevention by systemic neonatal nicotinamide administration. Neurotox Res. 2015;27(4):453–465. 183. Wang P, Miao CY. NAMPT as a therapeutic target against stroke. Trends Pharmacol Sci. 2015;36(12):891–905. 184. Penberthy WT, Tsunoda I. The importance of NAD in multiple sclerosis. Curr Pharm Des. 2009;15(1):64–99. 185. Sheline CT, Cai AL, Zhu J, et al. Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+ and loss of NAD+. Eur J Neurosci. 2010;32(6):894–904. 186. Rom S, Zuluaga-Ramirez V, Dykstra H, et al. Poly(ADP-ribose) polymerase-1 inhibition in brain endothelium protects the blood-brain barrier under physiologic and neuroinflammatory conditions. J Cereb Blood Flow Metab. 2015;35(1):28–36. 187. Sheline CT, Behrens MM, Choi DW. Zinc-induced cortical neuronal death: contribution of energy failure attributable to loss of NAD(+) and inhibition of glycolysis. J Neurosci. 2000;20(9):3139–3146. 188. Cai AL, Zipfel GJ, Sheline CT. Zinc neurotoxicity is dependent on intracellular NAD levels and the sirtuin pathway. Eur J Neurosci. 2006;24 (8):2169–2176. 189. Sonee M, Martens JR, Mukherjee SK. Nicotinamide protects HCN2 cells from the free radical generating toxin, tertiary butylhydroperoxide (t-BuOOH). Neurotox Res. 2002;4(7-8):595–599. 190. Rao VK, Carlson EA, Yan SS. Mitochondrial permeability transition pore is a potential drug target for neurodegeneration. Biochim Biophys Acta. 2014;1842(8):1267–1272. 191. Reddy PH, Beal MF. Amyloid beta, mitochondrial dysfunction and synaptic damage: implications for cognitive decline in aging and Alzheimer’s disease. Trends Mol Med. 2008;14(2):45–53. 192. Rui Y, Tiwari P, Xie Z, et al. Acute impairment of mitochondrial trafficking by beta-amyloid peptides in hippocampal neurons. J Neurosci. 2006;26(41):10480–10487. 193. Guo L, Du H, Yan S, et al. Cyclophilin D deficiency rescues axonal mitochondrial transport in Alzheimer’s neurons. PLoS One. 2013;8(1)e54914. 194. Du H, Guo L, Fang F, et al. Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer’s disease. Nat Med. 2008;14(10):1097–1105. 195. Fukushima T. Niacin metabolism and Parkinson’s disease. Environ Health Prev Med. 2005;10(1):3–8. 196. Willets JM, Lunec J, Williams AC, et al. Neurotoxicity of nicotinamide derivatives: their role in the aetiology of Parkinson’s disease. Biochem Soc Trans. 1993;21(Pt 3 (3)):299S. 197. Seifert R, Hoshino J, Kroger H. Nicotinamide methylation. Tissue distribution, developmental and neoplastic changes. Biochim Biophys Acta. 1984;801(2):259–264.

REFERENCES

799

198. Matsubara K, Aoyama K, Suno M, et al. N-methylation underlying Parkinson’s disease. Neurotoxicol Teratol. 2002;24(5):593–598. 199. Satoh A, Stein L, Imai S. The role of mammalian sirtuins in the regulation of metabolism, aging, and longevity. Handb Exp Pharmacol. 2011;206:125–162. 200. Yoshino J, Mills KF, Yoon MJ, et al. Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and ageinduced diabetes in mice. Cell Metab. 2011;14(4):528–536. 201. Ramsey KM, Mills KF, Satoh A, et al. Age-associated loss of Sirt1-mediated enhancement of glucose-stimulated insulin secretion in beta cellspecific Sirt1-overexpressing (BESTO) mice. Aging Cell. 2008;7(1):78–88.