Journal of Advanced Research 37 (2022) 267–278

Nicotinamide mononucleotide (NMN) as an anti-aging health product – Promises and safety concerns
Harshani Nadeeshani a, Jinyao Li b, Tianlei Ying c, Baohong Zhang d, Jun Lu a,e,f,g,h,i,j,⇑
a School of Science, Faculty of Health and Environmental Sciences, Auckland University of Technology, Auckland 1010, New Zealand
b Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, Xinjiang, China
c Key Laboratory of Medical Molecular Virology of MOE/MOH, Shanghai Medical College, Fudan University, 130 Dong An Road, Shanghai 200032, China
d School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
e School of Public Health and Interdisciplinary Studies, Faculty of Health and Environmental Sciences, Auckland University of Technology, Auckland 0627, New Zealand
f Institute of Biomedical Technology, Auckland University of Technology, Auckland 1010, New Zealand
g Maurice Wilkins Centre for Molecular Discovery, Auckland 1010, New Zealand
h College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518071, Guangdong Province, China
i College of Food Engineering and Nutrition Sciences, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, China
j College of Food Science and Technology, Nanchang University, Nanchang 330031, Jiangxi Province, China

h i g h l i g h t s

●Provides an overview of promises and safety concerns of NMN as an anti- aging product.
●Shows that NMN’s beneficial effects supported by in vivo studies.
●Reveals that there is a lack of NMN’s clinical safety and efficacy studies
●Suggests that proper clinical investigations are urgently needed on the effectiveness and safety of NMN supplementation.

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

a b s t r a c t

Article history:
Received 26 June 2021
Revised 2 August 2021
Accepted 4 August 2021
Available online 11 August 2021

Keywords:
Age-induced diseases Anti-aging
Nicotinamide adenine dinucleotide Nicotinamide mononucleotide Supplement

Background: Elderly population has been progressively rising in the world, thus the demand for anti- aging heath products to assure longevity as well as to ameliorate age-related complications is also on the rise. Among various anti-aging health products, nicotinamide mononucleotide (NMN) has been gain- ing attentions of the consumers and the scientific community.
Aim of review: This article intends to provide an overview on the current knowledge on promises and safety concerns of NMN as an anti-aging health product.
Key scientific concepts of review: Nicotinamide adenine dinucleotide (NAD+) levels in the body deplete with aging and it is associated with downregulation of energy production in mitochondria, oxidative stress, DNA damage, cognitive impairment and inflammatory conditions. However, NMN, as the precursor of NAD+, can slow down this process by elevating NAD+ levels in the body. A number of in vivo studies have indicated affirmative results of therapeutic effects for various age-induced complications with NMN sup- plementation. One preclinical and one clinical study have been conducted to investigate the safety con- cerns of NMN administration while a few more human clinical trials are being conducted. As there is a

Peer review under responsibility of Cairo University.
* Corresponding author at: Faculty of Health and Environmental Sciences, Auckland University of Technology, Auckland 0627, New Zealand.
E-mail address: [email protected] (J. Lu).

https://doi.org/10.1016/j.jare.2021.08.003
2090-1232/© 2022 The Authors. Published by Elsevier B.V. on behalf of Cairo University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

large influx of NMN based anti-aging products on the market, proper clinical investigations are urgently needed to find out the effectiveness and safety of NMN supplementation.
© 2022 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The successful control of communicable diseases in the 20th century led to a sharp rise in the mean life expectancy of many countries. In 2019, number of persons, aged 65 or over was 702.9 million in the world and it is projected to be 1548.9 million by 2050 [1]. Percentage of global population aged 65 years or over in 2019 and future projections according to the medium-variant projection is illustrated in Fig. 1. Along with increasing elderly pop- ulation, the prevalence of age-related diseases such as atheroscle- rosis, hypertension, osteoarthritis, neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, diabetes mellitus and cancers has gone up leading to heavy global socioeconomic and medical burden [2].
Therefore, age management medical practices have been mush- rooming in the world for recommending nutritional supplements, various drugs, exercise programs, hormone therapies and other treatments to mitigate the effect of aging. Consequently, the con- sumer demand and the global market value for anti-aging health products are on the rise [3]. Excessive demand of consumers and high profit margin for manufacturers are the major driving force behind the release of anti-aging health products without adequate safety testing [4]. Thus, careful comprehensive and stepwise scien- tific preclinical and clinical investigations are crucial to be conducted.
Among various anti-aging health products, nicotinamide mononucleotide (NMN) has been gaining an increasing attention as a promising anti-aging product. The mitochondrial decay, which is responsible for aging, can be reversed by the increased levels of nicotinamide adenine dinucleotide (NAD+) in the body. NMN is a precursor of NAD+ that acts as an intermediate in NAD+ biosynthe- sis, while dietary supplements of NMN are found to increase the NAD+ levels in the body [5]. NMN is a bioactive nucleotide formed by the reaction between a nucleoside comprising nicotinamide and ribose and a phosphate group [6]. It naturally presents in a variety of plant and animal food sources. Furthermore, several studies have been carried out to investigate the potential of biotechnolog- ical production and purifying NMN from bacterial and yeasts [7].
Other than anti-aging potential of NMN, a wide range of phar- macological activities have been identified in a number of in vivo studies. The link between NMN and the incidence of Alzheimer’s disease, obesity and associated complications, cerebral and cardiac ischemia, and age- and diet-induced type 2 diabetes has been studied extensively [8]. Though, previous attention of scientific community has been paid on NMN only as an intermediate in

NAD+ biosynthesis, recently, a number of pharmacological activi- ties triggered by increasing NAD+ levels in the body, especially anti-aging activity have been taken the centre of attention. As a result, a number of studies including cell culture, animal models and human clinical trials have been conducted to investigate the promises and the safety concerns of using NMN as an anti-aging health product and the potential of using NMN as a supplement to avoid age-related disease conditions. Hence, this review intends to present the most recent advances and current knowledge on promises and safety concerns of the use of NMN as an anti-aging health product, its other pharmacological and therapeutic uses and mechanism of action underlying the anti-aging properties with an interest to stimulate further research and offer an insight to the possibility of translating successful preclinical and clinical anti- aging outcomes of NMN into an effective treatment of aging and age-related diseases.

What is nicotinamide mononucleotide (NMN)?

Nicotinamide mononucleotide (NMN) exists as a and b anome- ric forms while it is identified as nicotinamide ribotide, nicotinamide-1-ium-1-b-D-ribofuranoside 50 -phosphate, b- nicotinamide ribose monophosphate and 3-carbamoyl-1-[5-O- (hydroxyphosphinato)-b-D-ribofuranosyl] pyridinium, among others [9]. The b form is the active anomer and NMN is naturally structured as a result of a reaction, which catalysed by nicoti- namide phosphoribosyltransferase enzyme, between a phosphate group and a nucleoside comprising nicotinamide (an amide form of vitamin B3) and ribose [10]. NMN is a bioactive nucleotide with a pyridine base and its molecular weight is 334.22 g/mol. It is fairly acidic and water-soluble compound. The solubility has been reported to be 1.8 mg/mL [11] (Fig. 2).
NMN is mainly located in the nucleus, mitochondria and cyto- plasm, whereas in the human body, it can be found in placenta tis- sue and body fluids such as blood and urine [9]. It is naturally found in a variety of fruits and vegetables including immature soy- bean pods, cabbage, cucumber, broccoli, tomato, mushroom and

Nicotinamide base

25
20
15
10
5
0
2019

9.1% 11.7% 15.9%

2030

22.6%

Ribose sugar

Year

2050

2100

Phosphate group

Fig. 1. Percentage of global population aged 65 years or over according to the medium-variant projection (UN, 2019).

Fig. 2. Chemical structure of nicotinamide mononucleotide (NMN).

avocado as well as in raw beef and shrimp. The NMN content in these vegetables and fruits are 0.25–1.88 mg/100 g and 0.26–1.6 0 mg/100 g, respectively, whereas raw beef and shrimp contain comparatively lower level of NMN than those plant-based food (0.06–0.42 mg/100 g). Based on the evidence of the presence of NMN in red blood cells, it has been put forward that physiologi- cally pertinent NMN contents, which are required for the biosyn- thesis of NAD+ and many physiological functions, are absorbed from daily food sources [10].
NMN is an intermediate of NAD+ biosynthesis. NAD+ is a very important metabolic redox co-enzyme in eukaryotic organisms and is essential component for large number of enzymatic reac- tions. It plays a vital role in a variety of biological processes of the body including cell death, aging, gene expression, neuroinflam- mation and DNA repair, which indicating a significance role of NAD+ in longevity and health of human life [12]. As revealed by many recent studies, deficiency of NAD+ can be compensated by the NMN supplementation that affects a range of pharmacological activities in different disease conditions [13].
Several methods had been used to prepare and purify NMN: incubation of diphosphopyridine nucleotide in a non-phosphate buffer and fluoride with potato pyrophosphatase, synthesis of NMN from nicotinamide by human hemolysates and erythrocytes, and specific hydrolysis of pyrophosphate bond of NAD+ using NAD+ pyrophosphatase and metal catalysts [7]. However, these methods are not rather efficient and they produce low amounts of NMN, giv- ing rise to high price of NMN. Currently, microbial biotechnologies are used to obtain NMN. Nevertheless, innovative methods and optimisations are essential in order to address the high cost and purity issues of NMN. Many studies have been performed using simple and efficient biotechnological production and purification methods using bacteria and yeast to make NMN production cost effective [14].
Though at first, NMN was only considered as a source of cellular energy and an intermediate in NAD+ biosynthesis, currently, the attention of the scientific community has been paid on anti-aging activity and a variety of health benefits and pharmacological activ- ities of NMN which are related to the restoring of NAD+. Thus, NMN has therapeutic effects towards a range of diseases, including age- induced type 2 diabetes, obesity, cerebral and cardiac ischemia, heart failure and cardiomyopathies, Alzheimer’s disease and other neurodegenerative disorders, corneal injury, macular degeneration and retinal degeneration, acute kidney injury and alcoholic liver disease [15].

Mechanism underlying the anti-aging activity of NMN

Aging, as a natural process is identified by downregulation of energy production in mitochondria of various organs such as brain, adipose tissue, skin, liver, skeletal muscle and pancreas due to the depletion of NAD+ [16]. NAD+ levels in the body decrease as a con- sequence of increasing NAD+ consuming enzymes when aging [17].
+

There are three different biosynthesis pathways to produce NAD+ in mammalian cells including de novo synthesis from trypto- phan, salvage and Preiss-Handler pathways. Among these three pathways, NMN is an intermediate by-product in salvage pathway, and it is involved in NAD+ biosynthesis through salvage and Preiss- Handler pathways as illustrated in Fig. 3 [15]. The salvage pathway is the most efficient and the main route for the NAD+ biosynthesis, in which nicotinamide and 5-phosphoribosyl-1-pyrophosphate are converted to NMN with the enzyme action of NAMPT followed by conjugation to ATP and conversion to NAD by NMNAT [21]. Fur- thermore, NAD+ consuming enzymes are responsible for degrada- tion of NAD+ and consequent formation of nicotinamide as a by- product [13]. In the Preiss-Handler pathway, initially, nicotinic acid is converted to nicotinic acid mononucleotide (NAMN) by nicotinic acid phosphoribosyl-transferase enzyme (NAPRT) activity followed by biosynthesis of nicotinic acid adenine dinucleotide (NAAD+) from NAMN using nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT 1/2/3). Subsequently, NAAD+ is trans- formed to NAD+ by NAD+ synthetase (NADS) using ATP and ammo- nia [22].
Chronic inflammation and oxidative stress, which come along with aging, are the causes for reduction and inhibition of NAMPT-mediated NAD+ biosynthesis [23]. The depletion of NAD+ contents along with aging, which particularly of nuclear origin, is associated with interruption of mitochondrial regulation of PCG-
1a/b-independent pathway of oxidative-phosphorylation as well,
causing pseudohypoxia. This incident can be overturned by raising the NAD+ content [24].
Apart from reducing the functions of mitochondria, biological changes such as cognitive impairments, DNA damage and sirtulin gene inactivation, are brought about by aging which can be evaded by enhancing NAD+ count in the body [24]. Apart from NMN sup- plementation, NAD+ levels in the body can be increased as a response to conditions related to lower energy intake [25], calorie restriction, fasting, lack of glucose content in the body and exer- cise. Nevertheless, NAD+ levels decrease as a consequence of intake of high-fat diets and aging [26].

These NAD consuming enzymes include NADase (CD38/CD157),

poly (ADP-ribose) polymerase (PARP), NAD+ dependent acetylase (sirtuins), BST and tankyrase (TNKS) [18]. Sirtuins consume NAD+ in order to execute a variety of functions such as deacetylation, deglutarylase, lipoamidase, demalonylase and desuccinylase activ- ities. Regulation of longevity, aging and age-associated physiologi- cal changes is one of the substantial aspects of sirtuin biology [19], while CD38 utilises NAD+ to produce cyclic ADP-ribose and nicoti- namide. Apart from that, PARP expends NAD+ to form branched ADP-ribose polymers which assists in DNA repairing [20]. This depleted NAD+ level by NAD+ consuming enzymes can be compen- sated by administration of NMN to the body since NMN is an inter- mediate compound of the NAD+ biosynthesis.

Fig. 3. NAD+ biosynthesis pathways in which NMN involves. Biosynthetic path- ways of NAD+ in mammalian cells in which NMN involves are Preiss–Handler and salvage pathways, and the salvage pathway is the major source of NAD+. NAMPT- nicotinamide phosphoribosyltransferase; ATP-adenosine triphosphate; ADP-ade- nosine diphosphate; NMNAT-nicotinamide mononucleotide adenylyltransferase; NRK-nicotinamide riboside kinases; NAPRT-nicotinate phosphoribosyltransferase; NADS-nicotinamide adenine dinucleotide synthetases.

Currently, several studies have shown that NMN, the NAMPT reaction product, is able to be utilised to trigger the SIRT1 activity [27]. It has been shown that when there is not adequate NAD+ levels, SIRT1 becomes unable to block hypoxia-inducible factor 1 alpha (HIF-1) and elevated levels of which blocks the communica- tion between mitochondria and nucleus at the cellular level and between adipose tissue and hypothalamus at the systemic level [28]. The resulting interruption in mitochondria and nuclear com- munication causes swift reduction in mitochondrial function that leads to the development of age-associated complications and dis- eases. Nevertheless, the particular communication and mitochon- drial function can be restored by the administration of NMN as the NAD+ precursor [24]. Causes for reducing NAD+ levels when aging and mechanism underlying anti-aging activity of NMN are illustrated in Fig. 4. The nutraceutical industry has already started to market NMN aggressively as a highly efficient and viable anti- aging health product to enhance the NAD+ levels [15], thus to pro- vide longevity to the general population. In addition, many studies are carried out to investigate the potential anti-aging activity of NMN and their applicability and usability.

Promises and efficacy as an anti-aging health product

Many studies have been carried out to investigate the promises and efficacy of NMN as an anti-aging health product for managing and regulating aging and age-associated complications and diseases using cell culture, animal models and human clinical

investigations. In vivo studies, which have been carried out to investigate anti-aging therapeutic effects of NMN administration, are summarised in Table 1, including animal models, given NMN dose, duration and observed effects. According to Yoshino et al. [23], during the aging process, NAMPT and NAD+ levels signifi- cantly decrease in various organs and NMN administration could enhance NAD+ levels (from 500 to 1550 pmol/mg-tissue), insulin secretion, insulin sensitivity and lipid profile in age-induced type
2 diabetic mice. Administration of NMN also can restore gene expression linked to circadian rhythm, inflammatory response and oxidative stress, and improve hepatic insulin sensitivity, par- tially by SIRT1 activation.
De Picciotto et al. [29] found that NMN supplementation was capable of restoring NAD+ levels (by threefold), vascular SIRT1 activity, maximum carotid artery endothelium-dependent dilation, and nitric oxide-mediated carotid artery endothelium-dependent dilation in mice. Kawamura et al. [30] have reported that NMN retained in animals for longer period than nicotinamide. NMN resulted in a higher yield of NAD+ (80 nmol/g of liver tissue) in sal- vage biosynthesis pathway activating higher response of SIRT1 than nicotinamide.
Mills et al. [10] found that devoid of any apparent deleterious effect or toxicity, NMN effectively suppressed aging-induced body weight gain and ameliorated eye dysfunction in mice. It main- tained healthy plasma lipid profile, insulin sensitivity, physical activity, energy metabolism and other physiopathologies. Addi- tionally, NMN supplementation averted alterations in age-

NMN
supplementation

Increase NAD+ biosynthesis (Salvage and Preiss- Handler pathways)

Increase NAD+ levels in body

Reverse the aging process

Fig. 4. Causes for reducing NAD+ levels when aging and mechanism underlying anti-aging activity of NMN. DNA damage, chronic inflammation, oxidative stress and increasing NAD+ consuming enzymes (sirtuins, CD38/CD157, PARP, TNKS and BST) accelerate NAD degradation. The reduced levels of NAD+ cause downregulation of energy production in mitochondria, leading to aging and various age-associated diseases. NMN supplementation can reinstate NAD+ levels in the body through biosynthesis pathways, reversing the aging process and preventing age-associated diseases.

Table 1
Anti-aging therapeutic effects of NMN administration in vivo.

Therapeutic effect/ age-related complication Model NMN dose and duration Effect Reference
Age- and diet- Age-induced and IP — 500 mg/kg body weight/day, age Enhanced insulin secretion, insulin sensitivity and lipid profile in [23]
induced diabetes high-fat-induced induced mice – 11 days, high-fat age-induced type 2 diabetic mice
diabetic mice induced mice – 7–10 days Improved hepatic insulin sensitivity and glucose tolerance in high-
fat-induced diabetic mice
Age-associated Aged (26– Drinking water — 300 mg/kg body Restored maximum carotid artery endothelium-dependent [29]
vascular 28 months) C57BI/ weight/day, 8 weeks dilation and nitric oxide-mediated carotid artery endothelium-
dysfunction and 6 mice dependent dilation
oxidative stress Reduced vascular oxidative stress, normalised aortic stiffness and
activated vascular SIRT1 activity
Anti-aging activity
and longer Wistar rats
(7 weeks) IP — 45 mmol/kg body weight, Single Retained in the body for longer period than nicotinamide Resulted
a higher yield of NAD+ activating higher response of SIRT1 than [30]
retention in the nicotinamide and better anti-aging activity and longevity than
body nicotinamide
Alzheimer’s disease APP(swe)/PS1(DE9)
double transgenic Subcutaneously — 100 mg/kg body
weight/every other day, 28 days Enhanced mitochondrial bioenergetic functions
Reduced expression of amyloid precursor protein (APP) [47]
(AD-Tg) mice
Age-associated Wild-type C57BL/ Drinking water — 100 and 300 mg/kg Suppressed aging-induced body weight gain [10]
physiological 6N mice body weight/day, 12 months Ameliorated eye functions, healthy plasma lipid profile, insulin
decline sensitivity, physical activity, energy metabolism and other
physiopathologies
Averted alterations in age-associated gene expression Enhanced
mitonuclear protein imbalance and mitochondrial oxidative
metabolism in skeletal muscles

Therapeutic effect/age- related complication

Model NMN dose and duration Effect Reference

Alzheimer’s disease Intracerebroventricular
infusion of Ab1-42 oligomer in Wistar rats

IP — 500 mg/kg body weight/day, 10 days

Restored learning and cognition
Enhanced energy metabolism and neuron survival Eliminated ROS accumulation

[48]

Age-associated susceptibility to acute kidney injury
Age-associated decline in DNA repair capacity

Aged (20 months) wild-type 129S2/Sv and C57BL/6 mice

Aged (20–26 months) C57BL/ 6J mice

IP — 500 mg/kg body weight/day, 4 days

IP — 500 mg/kg body weight/day, 7 days

Boosted NAD+ and SIRT1 levels in aged kidneys Protected aged kidneys from both ischemia–reperfusion- and cisplatin-induced acute kidney injuries
Reduced DND damage
Protected against changes in haemoglobin and white blood cell count including lymphocytes

[31]

[32]

Alzheimer’s disease APP(swe)/PS1(DE9) double
transgenic (AD-Tg) mice

Subcutaneously — 100 mg/kg body weight/every other day, 28 days

Improved of behavioural measures of cognitive impairments Reduced inflammatory responses, synaptic loss, amyloid plaque burden and b-amyloid production by inhibition of JNK activation

[49]

Age-related vascular aging

Age-related cognitive and behavioural dysfunction

Aged (18 months) C57BL/6J mice

Aged (20 months) C57BL/6 mice

Drinking water — 400 mg/kg body weight/day, 2 months

Per oral — 300 mg/kg body weight/day, 3 weeks

Increased endurance
Improved blood flow in elderly mice by increasing capillary density
Mitigated age-related decline in the sensory processing of hypersensitivity, some aversive stimuli and other related behaviours

[34]

[35]

Aging-induced cognitive impairment

Aged (24 months) Wistar rats IP — 100 mg/kg body weight/
every other day, 28 days

Stimulated neuroprotective effects Reduced aging-induced cognitive decline
Alleviated age-associated memory and learning impairments

[36]

Therapeutic effect/age-related complication

Model NMN dose and
duration

Effect Reference

Promoting micro-RNA profile in the aorta, epigenetic rejuvenation and anti-atherogenic effects

Aged (24 months) C57BL/6 mice IP — 500 mg/kg body weight/day, 14 days

Overturned age-associated transformations in micro-RNA profile in the aged mouse aorta

[37]

Alzheimer’s disease C57BL/6 mice and transgenic mice with mitochondria-targeted yellow fluorescence protein (mito-eYFP) expression in neurons

IP — 62.5 mg/kg, Single

Enhanced mitochondrial bioenergetics
Overturned physiological decline Restrained postischemic NAD+ depletion and cellular death Inhibited mitochondrial excessive fragmentation
Reduced mitochondrial protein acetylation and ROS in the hippocampus

[50]

Skeletal aging Aged (12 months) C57BL/6J mice Drinking water —
300 mg/kg body
weight/day, 3 months

Protected from skeletal aging Reduced osteogenesis
Increased adipogenesis by regulating mesenchymal stromal cells

[38]

(continued on next page)

Table 1 (continued)
Therapeutic effect/age-related complication

Model NMN dose and
duration

Effect Reference

Age-associated cerebromicrovascular dysfunction and cognitive decline

Rescuing female fertility during reproductive aging

Aged (24 months) C57BL/6 mice IP — 500 mg/kg body weight/day, 14 days

Aged (12–14 months) C57BL/6 female mice Drinking water — 2
g/L, 4 weeks

Restored NAD+ levels mitochondrial energetic
Reduce oxidative stress
Reversed cerebrovascular endothelial dysfunction
Improved neurovascular coupling responses and cognitive performance Restored NAD+ levels and fertility Rejuvenated oocyte quality Supported the embryo development reversing the adverse consequences of elevated maternal age

[39]

[43]

Therapeutic effect/age- related complication

Model NMN dose and
duration

Effect Reference

Mitochondrial function and cardioprotection in myocardial ischemia/ reperfusion injury

Promoting neurovascular rejuvenation

Oocyte quality reduction with advanced maternal age

Aged Wistar rats (22–24 months old) IP — 100 mg/kg
body weight/ every other day, 28 days

Aged (24 months) C57BL/6 mice IP — 500 mg/kg
body weight/day, 14 days

Aged (64–68 weeks) ICR female mice IP — 200 mg/kg
body weight/day, 10 days

Enhanced hemodynamic parameters
Decreased dehydrogenase release and infarct size Ameliorated mitochondrial membrane potential Declined mitochondrial ROS and oxidative stress Restored NAD+/NADH ratio
Promoted SIRT1activatio in the neurovascular unit Reversed age-associated alterations in neurovascular transcriptome which predicted to be mediated by the involvement of genes in anti-apoptosis, anti-inflammatory and mitochondrial rejuvenation pathways
Restored NAD+ levels
Increased ovulation, fertilisation capability and meiotic competency
Promoted cytoplasmic and nuclear maturation Suppressed accumulation of DNA damage and ROS Declined apoptosis

[40]

[37]

[42]

Werner syndrome young
(Day 2) and old (Day 10) wrn-1(gk99) and wild type N2 Caenorhabditis elegans and Drosophila melanogaster worms
Ataxia telangiectasia Ataxia-telangiectasia mutated
B6;129S4-Atmtm1Bal/J mice

IP-Intraperitoneal.

1 mM from the L4 stage

Drinking water —
12 mM, 2 weeks

Extended lifespan and health-span
Improved proliferative potency and number of mitotic cells by the NAD+ repletion

Normalized neuromuscular function Delayed memory loss
Extended lifespan
Stimulated neuronal DNA repair
Improve mitochondrial quality via mitophagy

[52]

[54]

associated gene expression in main metabolic organs, while enhancing mito-nuclear protein imbalance and mitochondrial oxidative metabolism in skeletal muscles. Guan et al. [31] elabo- rated that NMN supplementation restored reduced contents of renal protective molecule, SIRT1 and its cofactor, NAD+. The height- ened NAD+ and SIRT1 levels in kidneys of aged mice protected aged kidneys from both ischemia–reperfusion- and cisplatin-induced acute kidney injuries.
As explained by Li et al. [32], nudix homology domains (NHDs) are binding domains of NAD+ and through binding to them, NAD+ is able to regulate protein–protein interactions. The modulation of these interactions may lead to protecting the human body from aging, radiation and cancer. PARP1 is a critical protein that involves in DNA repairing. The inhibition of PARP1 is prevented by binding of NAD+ to the NDH domain of DBC1 (deleted in breast cancer 1) nuclear protein. Nevertheless, when NAD+ concentration is reduced with the age, DBC1 is progressively bound to PARP1, lead- ing to accumulate DNA damage. This process of DNA damage can be reversed by restoring NAD+ levels in the body. Results of this in vivo study showed that, NMN treatment increased hepatic NAD+ contents, disrupted DBC1-PARP1 complex, reduced DNA damage and defended against changes in haemoglobin and white blood cell count including lymphocytes.
Tsubota [33] showed that NMN, as a sirtuins activating agent had protective effects against age-related ocular diseases such as glaucoma, dry eye and macular degeneration. As elaborated by Das et al. [34], reduction of blood flow and capillary density with

aging is a main cause of morbidity and mortality, whereas NMN as a NAD+ precursor can reverse these to a certain extent by trig- gering sirtuin deacylases (SIRT1-7). NMN supplementation could increase NAD levels in liver and gastrocnemius tissues by nearly
5 and 1 folds, respectively. Johnson et al. [35] observed that in vivo occurrence of age-associated cognitive and behavioural dys- functions was induced by declined NAMPT-mediated NAD+ biosyn- thesis causing 40% gradual decrease of NAD+ levels in the hippocampus, predominately in CA1 region. It showed that, even by short term supplementation of NMN, NAD+ levels could be restored, while mitigating the age-related changes in the sensory processing of hypersensitivity, several other aversive stimuli and other associated behaviours, enhancing the quality of later lives. A prospective downstream effector was identified, namely, calcium/calmodulin-dependent serine protein kinase, which got reduced in hippocampus along with age-related NAD+ drop. The promoter activity of this effector was regulated in a SIRT1 reliant manner, while its expression could be enhanced by NMN supplementation.
Hosseini et al. [36] reported that in vivo intraperitoneal injec- tion of NMN and NMN together with melatonin stimulated neuro- protective effects and alleviated age-associated memory and learning impairments. Furthermore, the administration of them separately or in combination enhanced mitochondrial function and decreased apoptosis cell count both in hippocampus and pre- frontal cortex regions of aged rats. Kiss et al. [37] illustrated that age-associated NAD+ decline was linked with mis-regulation of

vascular mico-RNA expression, NMN intraperitoneal treatments resulted in anti-aging transformations mouse aorta mico-RNA expression profile. It was predicted that epigenetic rejuvenation and anti-atherogenic effects were some of regulatory conse- quences of NMN. NMN treatment distinctively expressed mico- RNAs in aged vessels.
The role of NMN in fighting against age-associated disorders, such as skeletal aging associated with reduced osteogenesis and increased adipogenesis by regulating mesenchymal stromal cells (MSCs), was studied by Song et al. [38]. MSCs are non- hematopoietic stem cells that contain regeneration capacity. NMN supplementation led to self-renewal of MSCs along with decreased adipogenesis and strengthened osteogenesis through upregulating SIRT1 activity in mice. In addition, NMN has been identified as a promising and potential therapeutic agent for skele- tal aging which is able to regulate bone-fat imbalance via SIRT1 and rejuvenation and expansion of aged MSCs.
Tarantini et al. [39] found that age-related increase of oxidative stress and cerebromicrovascular dysfunction, which exacerbated neurovascular coupling responses and age-related cognitive decline, were caused by reduced NAD+ availability with aging. NMN supplementation could restore tissue NAD levels by fold change of 1 and overturn these processes which led to improved cognitive performance in aged mice. Hosseini et al. [40] examined the individual and the combined outcome of NMN preconditioning and melatonin postconditioning on mitochondrial function and cardioprotection in myocardial ischemia/reperfusion injury of aged Wistar rats, because ischemic heart diseases are the foremost rea- sons for mortality and disability in elderly. This treatment amelio- rated mitochondrial membrane potential, declined mitochondrial ROS and oxidative stress and restored the balance between the oxi- dised and reduced forms of NAD. The consequences of the com- bined therapy of NMN and melatonin on these beneficial effects were greater than those of individual treatments.
According to Kiss et al. [41] in vivo NMN supplementation enhanced NAD+ levels followed by promoting SIRT1 activation and improving neurovascular functions and cognitive perfor- mances. These protective effects caused by NMN treatments on neurovascular function were predicted to be mediated by the involvement of genes in anti-apoptosis, anti-inflammatory and mitochondrial rejuvenation pathways which are attributable to versatile sirtuin-regulated anti-aging alterations in the neurovas- cular gene expression.
Miao et al. [42] showed that NMN supplementation improved the oocyte quality through restoring NAD+ levels (nearly 50%) in mice. It increased ovulation, fertilisation capability and meiotic competency, while promoting cytoplasmic and nuclear maturation in order to maintain euploidy. Furthermore, NMN reinstated mito- chondrial functions of aged oocytes to mitigate accumulation of DNA damage and reactive oxygen species, leading to low levels of apoptosis. This finding is echoed by Bertoldo et al. [43] who fur- ther indicated that restoration of fertility in aged mice and other benefits of NMN treatment could be recapitulated by the trans- genic overexpression of SIRT2. Apart from rejuvenating oocyte quality of aged animals, NMN supported the embryo development, reversing the adverse consequences of elevated maternal age on developmental milestones by increasing NAD levels in ovarian tis- sue from nearly 200 to 300 pmol/mg. Fu and Zhang [44] conducted an experiment for a patent application for using b-NMN in prepa- ration of anti-aging health-care products or drugs using aged mice. It was found that the NMN administration could extend the life span of mice by ~29%. Aging is the most influential determinant and the greatest known risk factor for neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) [45]. It has been illustrated that elevation of NAD+, sirtuins, CD38, PARP and SARM1 (sterile alpha and TIR motif containing 1) protein levels

through NMN supplementation were capable of inducing neuronal mitophagy to ameliorate cognitive decline in Caenorhabditis ele- gans model of AD [46].
Long et al. [47] suggested that depletion of cellular and mito- chondrial NAD+ contents induced mitochondrial abnormalities, activation of NAD glycohydrolases and bioenergetic failure as well as cell death. NMN supplementation decreased mitochondrial frag- mentation and mutant amyloid precursor protein levels (38%) in mice without observing any detrimental side effects, while it enhanced mitochondrial bioenergetic functions.
Wang et al. [48] indicated that intraperitoneal administration of NMN restored learning and cognition in AD model Wistar rats, while enhancing energy metabolism and neuron survival and pre- venting ROS accumulation and the central biomarker (amyloid beta) induced neuronal death. Yao et al. [49] has also reported improvements of behavioural measures of cognitive impairments and reduction of multiple AD-linked pathological characteristics such as inflammatory responses, synaptic loss, amyloid plaque burden and b-amyloid production by inhibition of JNK activation in mice after subcutaneous administration of NMN.
According to Klimova et al. [50], in vivo NMN administration enhanced mitochondrial bioenergetics, overturned physiological decline and restrained postischemic NAD+ depletion and cellular death. Furthermore, increasing NAD+ levels in mitochondria (from 3.12 to 5.51 nmol/mg) by NMN supplementation caused many metabolic changes such as SIRT3-mediated decline in mitochon- drial protein acetylation, which defended mitochondria by detri- mental effects of ROS and excessive and impetuous fragmentation. In addition, according to Lu et al. [51], NMN could cause considerable beneficial effects, attenuate apoptosis and enhance mitochondrial inhibitor induced declining of energy metabolism in PD-like neuropathological and behavioural changes as resulted in cellular model of PD, utilising rotenone-treated PC12 cells. They investigated increased ATP1 and NAD+ levels (30%) in PC12 cells, necrotic and apoptotic cell death and cell survival with NMN supplementation.
Fang et al. [52] investigated that approximately two folds reple- tion of NAD+ through NMN unusually delayed accelerated aging and extended lifespan of Caenorhabditis elegans and Drosophila mel- anogaster models of Werner syndrome. A short-term treatment of NAD+ precursor, nicotinamide riboside (NR) improved cochlear health, restored outer hair cell loss and prevented hearing loss in CSBm/m mice [53]. It showed that, as a precursor of NAD+, NMN may have the same potential to prevent the age-related and Cock- ayne syndrome-related hearing loss. Fang et al. [54] demonstrated that boosting NAD+ through NMN supplementation evidently extended in vivo lifespan, normalized neuromuscular functions, stimulated neuronal DNA repair and ameliorated neuropathologi- cal defects.

Safety concerns and challenges

The ultimate goal of geroscience is to discover approaches to boost natural defence mechanisms and prolong healthy lifespan through better management of the risks posed to cells and tissues of humans. Due to the continuous enhancement of the living stan- dards of people, the desire of healthy longevity has become pro- gressively stronger. Conversely, there is a possibility to achieve the maximum life span specified by the nature through decelerat- ing the rate of aging. Using tiny molecules to slow down the aging process and enhancing aging-associated outcomes is a flourishing research area at the present [55]. Furthermore, at the present, the number of anti-aging medicines and health products are com- mercially available and popular among elderly consumers [56]. Along with the current concerns on aging as a natural biological

process, many researches on longevity are being conducted to understand and manage the aging process through anti-aging interventions, while complying with gerontological and biogeron- tological aspects.
Fu and Zhang [44] have applied for a patent application for using b-NMN in the preparation of healthcare products or anti- aging drugs as a single dose of 1–500 mg/kg body weight/day of b-NMN. The healthcare products or anti-aging drugs was in the forms of injections, enteric-coated preparations, aqueous solutions, granules, capsules or tablets. A number of NMN anti-aging health products have been produced and launched by various pharmaceu- tical, nutraceutical, biotechnology and health food companies. The quantity of NMN in available commercial products vary from 50 to 150 mg/capsule, whereas some consumers take two 150 mg cap- sules per day [57]. Nowadays, there are NMN products, marketed as supplements for anti-aging and longevity in the form of cap- sules, which are very high in dose such as ≥500 mg. The safety of these doses cannot be assessed since required clinical and toxi- cological studies have not been completed yet to establish the rec- ommended safe levels for long term administration. Nevertheless, their safety and efficacy are uncertain and unreliable since most of them have not been backed up by rigorous scientific preclinical and clinical testing. This issue has been arisen as manufacturers are hesitant to pay for research and clinical trials due to potential lower profit margin, and there is no authorising agency to regulate NMN products because it is often sold as functional food product rather than heavily regulated therapeutic drug. Therefore, more strict approval process has been demanded by consumer advocacy groups requesting regulatory agencies to set standard and restric- tions for marketing anti-aging health products, considering safety, health and wellbeing of consumers [58]. NMN should not be con- sidered as a panacea for the elderly, because boosting NAD levels when not required may yield some detrimental effects. Therefore, the dose and frequency of NMN supplementation should be care- fully prescribed depending on the type of age-related deficiency and all other confronting health conditions of the people [59]. Other NAD precursors have been studied to discover the efficacy for diverse age-related deficiencies and they are used for particular deficiencies, only after they are proven for effectiveness and safe to use. Therefore, the same principle should be applied to NMN as well [60,61].
Grozio et al. [62] reported that NMN was speedily absorbed in the small intestine by a specific transporter, which was encoded by the Slc12a8 gene as demonstrated in in vitro and in vivo studies. Even though a large number of preclinical studies have provided a highly promising possibility for developing NMN as an evidence- based anti-aging health product, its safety and effectiveness on human physiology remains unclear. The toxicological and clinical evidence to back up its utility is currently scarce. Despite this, NMN supplements are already available in the market and consid- erably used by consumers as anti-aging health products. Therefore, in addition to animal models, human trails to investigate the safety and efficacy of NMN should be conducted focusing on toxicological parameters and safe metabolite levels in the human body [8].
Increased attention on potential pro-longevity effects of NAD+ precursors over the recent years has led to increased consumption of nicotinamide either as a clinical treatment or a dietary supple- ment, raising concerns on the safety of their long-term usage. Nicotinamide has been found to cause adverse effects on several organs in the human body such as kidney, liver, pancreatic b- cells and cells in plasma and induce nausea, stomach discomfort and headaches [63,64]. In addition, high dose of nicotinamide sup- plementation is associated with decreased insulin sensitivity and increased oxidative stress [64]. However, NMN supplementation has been found to have significant recovering effects on hepatocyte functions and liver pathologies in early-stage of ethanol toxicity,

instead of causing adverse effects to the liver [65]. It has also been found to improve insulin sensitivity and oxidative stress in animal models [23,29].
Although, nicotinamide supplementation induced DNA damage and oxidative DNA damages in various tissues of the body such as renal and hepatic tissues [66], NMN has been proven to reduce DNA damage and accumulation of ROS [32,42]. Supplementation of nicotinamide caused neurodegeneration of dopaminergic neu- rons and structural brain changes and behavioural deficits in rats [67]. Supplementation of NMN has been demonstrated to stimu- late neuroprotective effects and ameliorate cognitive decline and behavioural dysfunctions [35,36]. Rolfe [68] reported that adminis- tration of nicotinamide and niacin may cause development of PD, whereas supplementation of NMN has been found to be a promis- ing therapeutic remedy for PD [51].
NR is also a precursor of NAD+ and administration of NR has the capability to elevate body NAD+ levels. According to the results of clinical trials that have been performed to assess the safety of NR administration, NR supplementation increased LDL cholesterol levels in the body [69] and enhanced fatty liver conditions [70] as adverse effects. Nevertheless, NMN administration was observed to improve plasma lipid profile, ameliorating free fatty acids, triglycerides and cholesterol levels and lower intrahepatic triglyceride levels in mice [10]. Shi et al. [71] have shown that excessive dose of NR generated white adipose tissue dysfunction and heightened insulin resistance in mice. However, contradictory results have been reported with NMN supplementation as it reduced adiposity and enhanced insulin sensitivity [24,72].
NMN as another precursor of NAD+, has the potential to encoun- ter similar safety concerns and challenges as other NAD+ precur- sors. Thus, further clinical studies on the safety and toxicology of NMN are urgently needed. Mehmel et al. [60] argued that NR is a more suitable NAD+ precursor as an anti-aging treatment. This highlights the needs to study adverse effects of nicotinamide and NMN, even though NMN appears to have considerable beneficial pharmacological actions in preclinical studies. Yoshino et al. [57] conducted a clinical trial, supplementing 250 mg/day of NMN for 10 weeks to 25 prediabetes women who were overweight or obese in order to identify effect on body composition, gene expression profile, insulin signalling and insulin sensitivity and observed potential metabolic benefits without any adverse effects.
A toxicological study for NR chloride has been done in a clinical setting, and the no observed adverse effect level (NOAEL) was 300 mg/kg/day and the lowest observed adverse effect level was 1000 mg/kg/day [73]. No such data is available for human admin- istration of NMN yet. However, Cros et al. [74] investigated sub- chronic oral toxicity, acute oral toxicity, genotoxicity and
mutagenicity of high purity synthetic form of NMN (NMN-C®)
using female Sprague-Dawley rats (7 weeks old). According to the results, NOAEL of NMN-C® was ≥1500 mg/kg/d. At an oral administration dose of 2666 mg/kg of NMN-C®, no treatment- associated adverse effects or mortality were nor resulted. NMN-C® did not show toxic effects and appeared to be safe over a 90-day sub-chronic period of repeated oral administration at
doses of 375, 750 and 1500 mg/kg/d.
Conze, Crespo-Barreto, and Kruger [75] determined safety of a synthetic form of NR namely, NiagenTM using in vitro and rat toxi- cological study, and found that it was not genotoxic. The lowest observed adverse effect level for NiagenTM was 1000 mg/kg body weight/day and NOAEL was 300 mg/kg body weight/day. Further- more, NR has been examined in six clinical trials, where it has been established that it is safe for short-term (8 days) and long-term (6 weeks) supplementation in compliance with confirmed oral availability [60]. These values have not been established regarding NMN supplementation. Furthermore, recently, NR has been granted Generally Recognised as Safe (GRAS) status by the US Food

and Drug Administration (FDA), which is yet to be achieved for NMN.
Mills et al. [10] reported that in vivo long-term administration of NMN mitigated age-associated physiological decline, effectively without generating noticeable toxicity, deleterious side effects or raised mortality. Tsubota [76] reported that the first phase I human clinical study (UMIN000021309) has been initiated aiming at eval- uating the bioavailability, mechanism of action and the safety of NMN in the human body. This has been a collaborative study between Keio University School of Medicine in Tokyo and Wash- ington University School of Medicine in St. Louis. Using ten healthy volunteers, the time course of blood NMN concentration and the safety of NMN administration were assessed, which was intended for developing anti-aging nutraceutical. However, the research team has been unsuccessful in detecting NMN in plasma samples. Irie et al. [77] conducted a non-blinded clinical trial using 10 healthy men to investigate the safety of oral NMN administration and the pharmacokinetics of nicotinamide metabolites at the Keio University School Medicine, Japan. They found that single oral administration of 100, 250 and 500 mg of NMN doses was well- tolerated and safe since it did not cause any observable clinical symptoms or changes in body temperature, oxygen saturation, blood pressure and heart rate. In addition, significant changes in ophthalmic, ocular fundus and neurological system parameters were not observed after NMN administration. There were no changes in the results of laboratory analysis of urine and blood as well as sleep quality and score before and after the NMN admin- istration. Oral administration of NMN increased serum bilirubin contents and decreased blood glucose, chloride and serum crea- tinine levels, but within the normal range. NMN administration did not increase nicotinamide in blood to the level which causes adverse effects associated with high dose of nicotinamide. Since the safety of single oral administration of NMN has only been con- sidered in this study, further clinical investigations are essential to be performed to assess the safety and efficacy of long-term admin- istration of NMN. The organs and the tissue NMN levels have not been analysed in this study, which should be considered in future
clinical studies.
As stated by Hong et al. [15], three more human clinical studies are being carried out to evaluate the safety concerns of NMN administration. One study is a phase II study (UMIN000030609), which has been initiated by the Keio University School of Medicine, Japan to evaluate the safety of long-term administration of NMN, pharmacokinetics and metabolites of NMN, and its effect on glu- cose metabolism in healthy adults. Another study is being per- formed to assess the effect of long-term intake of NMN on different hormones in healthy individuals (UMIN000025739) by the Hiroshima University, Institute of Biomedical and Health Sciences. The third clinical study (UMIN000036321) has been designed to assess the consequences of oral administration of NMN on the body composition in elderly people at the University of Tokyo Hospital. These clinical trials are still ongoing and there is no result published yet. Yoshino et al. [23] have also highlighted the importance of more comprehensively assessing potential adverse effects of NMN by conducting preclinical and clinical stud- ies, considering different dietary conditions as well.
According to Di Stefano et al. [78], inhibitors of NMN synthesis- ing enzyme (NAMPT), provided robust functional and morpholog- ical protection of injured synapses and axons, regardless of reducing NAD. But, exogenous NMN eliminated this protection with the accumulation of NMN after nerve injury and NMNAT2 degradation advanced axon degeneration. They suggested that the relationship between the increase of NMN and axon degenera- tion could be a long-hypothesised toxic and deleterious factor. Poljsak and Milisav [79] reported that both NR and NMN, as vita- min B3 forms and NAD+ precursors were not detected to be

cancerogenic. Radenkovic and Verdin [80] also have stated that since no study has reported rigorous side effects and they have been in use for many years, therapeutic interventions including NMN, NR, vitamin B3 and niacin supplementation, which increase NAD+ levels, are likely to be fairly safe for the human use.
Radenkovic and Verdin [80] further explained that side effects of long-term NAD upregulation are complicated to identify and quantify, but they still can exist. In addition, it was illustrated that prolonged high dose of vitamin B3 compounds, including NMN may possibly have long-term side effects. According to Rolfe [68], NAD upregulation has the possibility to make worse the senescence-associated secretory phenotype (SASP) generated by senescent cells in aged tissues. A suggested mechanism to cause this side effect was the suppression of 50 adenosine monophosphate-activated protein kinase and tumor protein p53, directing to stimulate nuclear factor kappa B protein transcription factor through p38 mitogen-activated protein kinases and raised expression of inflammatory cytokines. The cellular senescence bur- den enlarged with aging, while the produced ASAP was a responsi- ble factor for different age-related pathologies [81].
However, evidences to support these potential side effects of long-term NMN usage and NAD upregulation are lacking. In fact, the usage of anti-aging interventions is expected to be initiated at comparatively younger and healthier age than very old age. Therefore, identification and quantification of adverse effects and challenges of long-term utilisation of anti-aging health products is extremely essential and critical since these anti-aging products are apparently used for a long time. According to Yu et al. [82], NMN treatment (400 mg/kg) obstructed the exercise-induced ben- efits of a mouse model of diet-induced obesity such as reduced hepatic triglyceride accumulation, glucose stimulated insulin secretion from islets and glucose tolerance. This finding should be further investigated thoroughly since the exercise is one of the key components for maintaining health and wellbeing of the elderly.

Conclusions

NMN is a precursor of NAD+ and an intermediate of NAD+ biosynthesis, which is achieved through three pathways. NMN is an intermediate by-product in two of them. NAD+ levels in the body are depleted with aging as a result of activities of NAD+ con- suming enzymes. Depletion of NAD+ level is associated with down- regulation of energy production in mitochondria, increasing oxidative stress, DNA damage, cognitive impairments and inflam- matory diseases. NMN, as the precursor of NAD+, has been seen to likely reverse these age-related complications and slow down the rate of aging by enhancing NAD+ levels in the body.
Many studies have been done to explore NMN’s anti-aging effects in various cells and tissues. Most of the works have been done in vitro or in animal models. However, published reports about NMN’s long-term safety and clinical efficacy of anti-aging effects in humans are scarce. From the above review, it can be seen that only very few pre-clinical and clinical studies have been con- ducted to investigate the safety of long-term administration of NMN. A few more human clinical trials are being conducted to evaluate the safety concerns of NMN supplementation and the out- comes are yet to be available.
However, many NMN anti-aging health products are already available in the market and manufacturers are aggressively mar- keting the products using in vitro and in vivo results from the liter- ature. Therefore, the first priority should be to establish toxicology, pharmacology and safety profiles of NMN in humans, including healthy and diseased. For NMN’s anti-aging efficacy, the most fea- sible route to obtain data will probably through long-term follow-

ups of people who consume NMN regularly. Such research should be supported by NMN manufacturers as they have the moral responsibility to provide efficacy results of their products.

Funding

Funding support from Education New Zealand, New Zealand- China Tripartite Research Collaboration Fund – AUT 13772 to Pro- fessors Jun Lu, Jinyao Li, Tianlei Ying and Baohong Zhang; Professor Jun Lu also has received funding support from EZZ Life Science Holdings Pty Ltd.

Compliance with Ethics Requirements

This article does not contain any studies with human or animal subjects.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper.

Acknowledgement

The authors would like to thank Professor Terrence Madhujith, Professor of Food Science and Technology & Department Chair, Department of Food Science and Technology, Faculty of Agricul- ture, University of Peradeniya, Peradeniya, Sri Lanka, for proofread- ing this manuscript.

References

[1]United Nations (UN), Department of Economic and Social Affairs, Population Division. World population ageing 2019: highlights (ST/ESA/SER.A/430). New York, USA: United Nations; 2019.
[2]Rea IM, Gibson DS, McGilligan V, McNerlan SE, Alexander HD, Ross OA. Age and age-related diseases: role of inflammation triggers and cytokines. Front Immunol 2018;9:586.
[3]Diamanti-Kandarakis E, Dattilo M, Macut D, Duntas L, Gonos ES, Goulis DG, et al. Aging and anti-aging: a combo-endocrinology overview. Eur J Endocrinol 2017;176:283–308.
[4]Reisman NR. Anti-aging medicine: the legal issues: legal issues associated with the current and future practice of anti-aging medicine. J Gerontol A Biol Sci Med Sci 2004;59(7):B674–81.
[5]Yamamoto T, Byun J, Zhai P, Ikeda Y, Oka S, Sadoshima J. Nicotinamide mononucleotide, an intermediate of NAD+ synthesis, protects the heart from ischemia and reperfusion. PLoS ONE 2014;9(6):e98972.
[6]Bieganowski P, Brenner C. Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans. Cell 2004;117(4):495–502.
[7]Marinescu GC, Popescu R, Dinischiotu A. Size exclusion chromatography method for purification of nicotinamide mononucleotide (NMN) from bacterial cells. Sci Rep 2018;8(1):1–11.
[8]Poddar SE, Sifat EK, Haque S, Nahid NA, Chowdhury S, Mehedi I. Nicotinamide mononucleotide: exploration of diverse therapeutic applications of a potential molecule. Biomolecules 2019;9(1):34–49.
[9]National Centre for Biotechnology Information (NCBI). PubChem Compound Summary for CID 14180, Nicotinamide mononucleotide. [cited 2020 Sep 22]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Nicotinamide- mononucleotide.
[10]Mills KF, Yoshida S, Stein LR, Grozio A, Kubota S, Sasaki Y, et al. Long-term administration of nicotinamide mononucleotide mitigates age-associated physiological decline in mice. Cell Metab 2016;24(6):795–806.
[11]Virtual Computational Chemistry Laboratory [homepage on the Internet]. ALOGPS 2.1. 2001–2016. [updated 2016; cited 2021 Aug 01]. Aqueous solubility calculation. Available from: http://www.vcclab.org.
[12]Hou Y, Lautrup S, Cordonnier S, Wang Y, Croteau DL, Zavala E, et al. NAD+ supplementation normalizes key Alzheimer’s features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency. Proc Natl Acad Sci U S A 2018;115(8):E1876–85.
[13]Okabe K, Yaku K, Tobe K, Nakagawa T. Implications of altered NAD metabolism in metabolic disorders. J Biomed Sci 2019;26(1):34–47.
[14]Marinescu GC, Popescu RG, Stoian G, Dinischiotu A. b-nicotinamide mononucleotide (NMN) production in Escherichia coli. Sci Rep 2018;8(1):1–11.
[15]
Hong W, Mo F, Zhang Z, Huang M, Wei X. Nicotinamide mononucleotide: a promising molecule for therapy of diverse diseases by Targeting NAD+ metabolism. Front Cell Dev Biol 2020;8:246.
[16]Mouchiroud L, Houtkooper RH, Moullan N, Katsyuba E, Ryu D, Cantó C, et al. The NAD+/sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling. Cell 2013;154(2):430–41.
[17]Fang EF, Lautrup S, Hou Y, Demarest TG, Croteau DL, Mattson MP, et al. NAD+ in aging: molecular mechanisms and translational implications. Trends Mol Med 2017;23(10):899–916.
[18]Camacho-Pereira J, Tarragó MG, Chini CC, Nin V, Escande C, Warner GM, et al. CD38 dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism. Cell Metab 2016;23(6):1127–39.
[19]Hall JA, Dominy JE, Lee Y, Puigserver P. The sirtuin family’s role in aging and age-associated pathologies. J Clin Invest 2013;123(3):973–9.
[20]Cantó C, Menzies KJ, Auwerx J. NAD+ metabolism and the control of energy homeostasis: a balancing act between mitochondria and the nucleus. Cell Metab 2015;22(1):31–53.
[21]Imai SI. Dissecting systemic control of metabolism and aging in the NAD World: the importance of SIRT1 and NAMPT-mediated NAD biosynthesis. FEBS Lett 2011;585(11):1657–62.
[22]Yang Y, Sauve AA. NAD+ metabolism: bioenergetics, signaling and manipulation for therapy. Biochim Biophys Acta Proteins Proteom 2016;1864(12):1787–800.
[23]Yoshino J, Mills KF, Yoon MJ, Imai SI. Nicotinamide mononucleotide, a key NAD+ intermediate, treats the pathophysiology of diet-and age-induced diabetes in mice. Cell Metab 2011;14(4):528–36.
[24]Gomes AP, Price NL, Ling AJ, Moslehi JJ, Montgomery MK, Rajman L, et al. Declining NAD+ induces a pseudohypoxic state disrupting nuclear- mitochondrial communication during aging. Cell 2013;155(7):1624–38.
[25]Cantó C, Jiang LQ, Deshmukh AS, Mataki C, Coste A, Lagouge M, et al. Interdependence of AMPK and SIRT1 for metabolic adaptation to fasting and exercise in skeletal muscle. Cell Metab 2010;11(3):213–9.
[26]Verdin E. NAD+ in aging, metabolism, and neurodegeneration. Science 2015;350(6265):1208–13.
[27]Imai SI. A possibility of nutriceuticals as an anti-aging intervention: activation of sirtuins by promoting mammalian NAD biosynthesis. Pharmacol Res 2010;62(1):42–7.
[28]Lim JH, Lee YM, Chun YS, Chen J, Kim JE, Park JW. Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypoxia-inducible factor 1a. Mol Cell 2010;38(6):864–78.
[29]De Picciotto NE, Gano LB, Johnson LC, Martens CR, Sindler AL, Mills KF, et al. Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice. Aging Cell 2016;15(3):522–30.
[30]Kawamura T, Mori N, Shibata K. b-Nicotinamide mononucleotide, an anti- aging candidate compound, is retained in the body for longer than nicotinamide in rats. J Nutr Sci Vitaminol 2016;62(4):272–6.
[31]Guan Y, Wang SR, Huang XZ, Xie QH, Xu YY, Shang D, et al. Nicotinamide mononucleotide, an NAD+ precursor, rescues age-associated susceptibility to AKI in a sirtuin 1–dependent manner. J Am Soc Nephrol 2017;28(8):2337–52.
[32]Li J, Bonkowski MS, Moniot S, Zhang D, Hubbard BP, Ling AJ, et al. A conserved NAD+ binding pocket that regulates protein-protein interactions during aging. Science 2017;355(6331):1312–7.
[33]Tsubota K. Anti-aging approach for ocular disorders: from dry eye to retinitis pigmentosa and myopia. Nippon Ganka Gakkai Zasshi 2017;121(3):232–48.
[34]Das A, Huang GX, Bonkowski MS, Longchamp A, Li C, Schultz MB, et al. Impairment of an endothelial NAD+-H2S signaling network is a reversible cause of vascular aging. Cell 2018;173(1):74–89.
[35]Johnson S, Wozniak DF, Imai S. CA1 NAMPT knockdown recapitulates hippocampal cognitive phenotypes in old mice which nicotinamide mononucleotide improves. npj Aging Mech Dis 2018;4(1):1–12.
[36]Hosseini L, Farokhi-Sisakht F, Badalzadeh R, Khabbaz A, Mahmoudi J, Sadigh- Eteghad S. Nicotinamide mononucleotide and melatonin alleviate aging- induced cognitive impairment via modulation of mitochondrial function and apoptosis in the prefrontal cortex and hippocampus. Neuroscience 2019;423:29–37.
[37]Kiss T, Giles CB, Tarantini S, Yabluchanskiy A, Balasubramanian P, Gautam T, et al. Nicotinamide mononucleotide (NMN) supplementation promotes anti- aging miRNA expression profile in the aorta of aged mice, predicting epigenetic rejuvenation and anti-atherogenic effects. Geroscience 2019;41 (4):419–39.
[38]Song J, Li J, Yang F, Ning G, Zhen L, Wu L, et al. Nicotinamide mononucleotide promotes osteogenesis and reduces adipogenesis by regulating mesenchymal stromal cells via the SIRT1 pathway in aged bone marrow. Cell Death Dis 2019;10(5):1–12.
[39]Tarantini S, Valcarcel-Ares MN, Toth P, Yabluchanskiy A, Tucsek Z, Kiss T, et al. Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice. Redox Boil 2019;24:101192.
[40]Hosseini L, Vafaee MS, Badalzadeh R. Melatonin and nicotinamide mononucleotide attenuate myocardial ischemia/reperfusion injury via modulation of mitochondrial function and hemodynamic parameters in aged rats. J Cardiovasc Pharmacol Ther 2020;25(3):240–50.
[41]Kiss T, Nyúl-Tóth Á, Balasubramanian P, Tarantini S, Ahire C, Yabluchanskiy A, et al. Nicotinamide mononucleotide (NMN) supplementation promotes neurovascular rejuvenation in aged mice: transcriptional footprint of SIRT1

activation, mitochondrial protection, anti-inflammatory, and anti-apoptotic effects. GeroScience 2020;42:527–46.
[42]Miao Y, Cui Z, Gao Q, Rui R, Xiong B. Nicotinamide mononucleotide supplementation reverses the declining quality of maternally aged oocytes. Cell Rep 2020;32(5):107987.
[43]Bertoldo MJ, Listijono DR, Ho WHJ, Riepsamen AH, Goss DM, Richani D, et al. NAD+ repletion rescues female fertility during reproductive aging. Cell Rep 2020;30(6):1670–81.
[44]Fu R, Zhang Q. Inventors; Hoboomlife Bio-Technology Co Ltd, assignee. Use of b-nicotinamide mononucleotide in preparation of anti-aging drugs or health- care products. United States patent application US 15/310,371; 2017 Sep 21. [cited 2020 Oct 25]. Available from: https://patents.google.com/patent/ US20170266213A1/en.
[45]Wahl D, Anderson RM, Le Couteur DG. Antiaging therapies, cognitive impairment, and dementia. J Gerontol A 2020;75(9):1643–52.
[46]Fang EF. Mitophagy and NAD+ inhibit Alzheimer disease. Autophagy 2019;15 (6):1112–4.
[47]Long AN, Owens K, Schlappal AE, Kristian T, Fishman PS, Schuh RA. Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer’s disease-relevant murine model. BMC Neurol 2015;15(1):1–14.
[48]Wang X, Hu X, Yang Y, Takata T, Sakurai Y. Nicotinamide mononucleotide protects against b-amyloid oligomer-induced cognitive impairment and neuronal death. Brain Res 2016;1643:1–9.
[49]Yao Z, Yang W, Gao Z, Jia P. Nicotinamide mononucleotide inhibits JNK activation to reverse Alzheimer disease. Neurosci Lett 2017;647:133–40.
[50]Klimova N, Long A, Kristian T. Nicotinamide mononucleotide alters mitochondrial dynamics by SIRT3-dependent mechanism in male mice. J Neurosci Res 2019;97(8):975–90.
[51]Lu L, Tang LE, Wei W, Hong Y, Chen H, Ying W, et al. Nicotinamide mononucleotide improves energy activity and survival rate in an in vitro model of Parkinson’s disease. Exp Ther Med 2014;8(3):943–50.
[52]Fang EF, Hou Y, Lautrup S, Jensen MB, Yang B, SenGupta T, et al. NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome. Nat Commun 2019;10(1):1–18.
[53]Okur MN, Mao B, Kimura R, Haraczy S, Fitzgerald T, Edwards-Hollingsworth K, et al. Short-term NAD+ supplementation prevents hearing loss in mouse models of Cockayne syndrome. npj Aging Mech Dis 2020;6(1):1–10.
[54]Fang EF, Kassahun H, Croteau DL, Scheibye-Knudsen M, Marosi K, Lu H, et al. NAD+ replenishment improves lifespan and healthspan in ataxia telangiectasia models via mitophagy and DNA repair. Cell Metab 2016;24(4):566–81.
[55]Tosato M, Zamboni V, Ferrini A, Cesari M. The aging process and potential interventions to extend life expectancy. Clin Interv Aging 2007;2(3):401–12.
[56]Cardona B. Dangers and dilemmas surrounding the consumption of anti- ageing medicine. In: Rodopi BV, editor. Justice for older people. Netherlands: Brill Publishers; 2012. p. 35–45.
[57]Yoshino MJ, Yoshino BD, Kayser G, Patti MP, Franczyk KF, Mills M, Sindelar T, Pietka BW, Patterson SI, Imai S Klein. Nicotinamide mononucleotide increases muscle insulin sensitivity in prediabetic women. Science 2021:eabe9985.
[58]Brower V. A nutraceutical a day may keep the doctor away: Consumers are turning increasingly to food supplements to improve wellbeing when pharmaceuticals fail. EMBO Rep 2005;6(8):708–11.
[59]Rajman L, Chwalek K, Sinclair DA. Therapeutic potential of NAD-boosting molecules: the in vivo evidence. Cell Metab 2018;27(3):529–47.
[60]Mehmel M, Jovanovic´ N, Spitz U. Nicotinamide riboside-the current State of research and therapeutic uses. Nutrients 2020;12(6):1616.
[61]Braidy N, Berg J, Clement J, Khorshidi F, Poljak A, Jayasena T, et al. Role of nicotinamide adenine dinucleotide and related precursors as therapeutic targets for age-related degenerative diseases: rationale, biochemistry, pharmacokinetics, and outcomes. Antioxid Redox Signal 2019;30(2):251–94.
[62]Grozio A, Mills KF, Yoshino J, Bruzzone S, Sociali G, Tokizane K, et al. Slc12a8 is a nicotinamide mononucleotide transporter. Nature Metab 2019;1(1):47–57.
[63]Knip M, Douek IE, Moore WPT, Gillmor HA, McLean AEM, Bingley PJ, et al. Safety of high-dose nicotinamide: a review. Diabetologia 2000;43 (11):1337–45.
[64]Hwang ES, Song SB. Possible adverse effects of high-dose nicotinamide: mechanisms and safety assessment. Biomolecules 2020;10(5):687–708.
[65]Assiri MA, Ali HR, Marentette JO, Yun Y, Liu J, Hirschey MD, et al. Investigating RNA expression profiles altered by nicotinamide mononucleotide therapy in a chronic model of alcoholic liver disease. Hum Genomics 2019;13(1):1–13.
[66]Li D, Tian YJ, Guo J, Sun WP, Lun YZ, Guo M, et al. Nicotinamide supplementation induces detrimental metabolic and epigenetic changes in developing rats. Br J Nutr 2013;110(12):2156–64.
[67]Harrison IF, Powell NM, Dexter DT. The histone deacetylase inhibitor nicotinamide exacerbates neurodegeneration in the lactacystin rat model of Parkinson’s disease. J Neurochem 2019;148(1):136–56.
[68]Rolfe HM. A review of nicotinamide: treatment of skin diseases and potential side effects. J Cosmet Dermatol 2014;13(4):324–8.
[69]
Dellinger RW, Santos SR, Morris M, Evans M, Alminana D, Guarente L, et al. Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD+ levels in humans safely and sustainably: a randomized, double-blind, placebo- controlled study. npj Aging Mech Dis 2017;3(1):1–9.
[70]Dollerup OL, Christensen B, Svart M, Schmidt MS, Sulek K, Ringgaard S, et al. A randomized placebo-controlled clinical trial of nicotinamide riboside in obese men: safety, insulin-sensitivity, and lipid-mobilizing effects. Am J Clin Nutr 2018;108(2):343–53.
[71]Shi W, Hegeman MA, Doncheva A, Bekkenkamp-Grovenstein M, de Boer VC, Keijer J. High dose of dietary nicotinamide riboside induces glucose intolerance and white adipose tissue dysfunction in mice fed a mildly obesogenic diet. Nutrients 2019;11(10):2439.
[72]Uddin GM, Youngson NA, Sinclair DA, Morris MJ. Head-to-head comparison of short-term treatment with the NAD+ precursor nicotinamide mononucleotide (NMN) and 6 weeks of exercise in obese female mice. Front Pharmacol 2016;7:258.
[73]Conze D, Brenner C, Kruger CL. Safety and metabolism of long-term administration of NIAGEN (nicotinamide riboside chloride) in a randomized, double-blind, placebo-controlled clinical trial of healthy overweight adults. Sci Rep 2019;9(1):1–13.
[74]Cros C, Cannelle H, Laganier L, Grozio A, Canault M. Safety evaluation after acute and sub-chronic oral administration of high purity nicotinamide mononucleotide (NMN-C®) in Sprague-Dawley rats. Food Chem Toxicol 2021;150:112060.
[75]Conze DB, Crespo-Barreto J, Kruger CL. Safety assessment of nicotinamide riboside, a form of vitamin B3. Hum Exp Toxicol 2016;35(11):1149–60.
[76]Tsubota K. K, The first human clinical study for NMN has started in Japan. npj Aging Mech Dis 2016;2(1):16021.
[77]Irie J, Inagaki E, Fujita M, Nakaya H, Mitsuishi M, Yamaguchi S, et al. Effect of oral administration of nicotinamide mononucleotide on clinical parameters and nicotinamide metabolite levels in healthy Japanese men. Endocr J 2020;67 (2):153–60.
[78]Di Stefano M, Nascimento-Ferreira I, Orsomando G, Mori V, Gilley J, Brown R, et al. A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration. Cell Death Differ 2015;22(5):731–42.
[79]Poljsak B, Milisav I. Vitamin B3 forms as precursors to NAD+: are they safe? Trends Food Sci Technol 2018;79:198–203.
[80]Radenkovic D, Verdin E. Clinical evidence for targeting NAD therapeutically. Pharmaceuticals 2020;13(9):247–63.
[81]Mendelsohn AR, Larrick JW. Interacting NAD+ and cell senescence pathways complicate antiaging therapies. Rejuvenation Res 2019;22(3):261–6.
[82]Yu J, Laybutt DR, Kim LJ, Quek LE, Wu LE, Morris MJ, et al. Exercise-induced benefits on glucose handling in a model of diet-induced obesity are reduced by concurrent nicotinamide mononucleotide. Am J Physiol Endocrinol Metab 2021;321(1):E176–89.