Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : March 17, 2022
Accepted : April 10, 2022
Published online : June 30, 2022
doi:10.24659/gsr.9.2_33

Original article

Clinical evaluation of changes in biomarkers by oral intake of NMN
Yuji Morita 1, 2), Hiromi Izawa 1, 3), Atsuyuki Hirano 1), Eriko Mayumi 1),
Seigo Isozaki 2, 4), Yoshikazu Yonei 2)
1) Renais Clinic Nihonbashi, Tokyo, Japan2) Anti-aging Medical Research Center and Glycative Stress Research Center,
Faculty of Life and Medical Sciences. Doshisha University, Kyoto, Japan
3) Jingu-Gaien Woman Life Clinic, Tokyo, Japan4) Antiaging-Bank Co., Ltd, Tokyo, Japan

Abstract

Purpose: The "NAD World" (proposed by Professor Shin-Ichiro Imai of the University of Washington), in which thecontrol of aging, lifespan, and metabolism is systemically integrated through NAD (nicotinamide adenine dinucleotide),
has attracted worldwide attention and is being studied from various angles, however, there are few reports of clinicalstudies in humans. In this study, we investigate the changes in various biomarkers in humans after oral intake ofnicotinamide mononucleotide (NMN), a precursor of NAD, and evaluate its clinical significance.
Methods: Based on the approval to start the study at the ethics review, 17 postmenopausal women (mean age 55.0 years)
without underlying diseases who agreed to participate in the study were subjects and received oral intake of 300 mg/day ofpure NMN for eight weeks. The examination items included body measurements, basal metabolic rate, blood pressure, grip
strength, glycation level (AF value), blood biochemical tests, various hormones, blood SIRT1 mRNA expression and NMN,
NAD and NAM levels, immunological tests (aging, T cell subset including exhausted cells), and skin VAS (visual analog
scale). Results were statistically analyzed and compared at zero and eight weeks (blood biochemistry tests were performed atfour weeks to confirm safety). In addition, we asked the patients to keep a diary during the course of the study to monitor their
NMN intake, their bodily sensation, and side effects.
Results: One of the 17 subjects dropped out at the end of four weeks due to persistent mild headache. The results showedsignificant changes in NAM (nicotinamide/vitamin B3) from 45.2 to 164.7 (p < 0.001), adiponectin from 13.6 to 16.2 (p= 0.004), and skin VAS score (6/7 items, p < 0.001 to = 0.001) before and after intake. Significant differences were also
observed for BMI, AF levels, platelets, HbA1c, HDL-C, amylase, DHEA-s, NAD, and narrowly defined regulatory T cells( p < 0.05 above). In addition, all subjects showed a positive bodily sensation with skin, sleep, and fatigue. There were no
problems with subjective symptoms or laboratory data in the 16 who completed the study.
Conclusion: NMN 300 mg/day orally for eight weeks showed no safety issues and favorable changes in many biomarkers,
suggesting that NMN, a member of the NAD world, may be a promising nutritional material for aging and metabolic control
in humans.

KEY WORDS: nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD), sirtuin, biomarker,

		oral intake, clinical trial

Corresponding to: Yuji Morita, MD
Renais Clinic Nihonbashi
3-3-1 Nihonbashi-Muromachi, Chuo, Tokyo 103- 0022, Japan
TEL : + 81-3-6665-0888 e-mail: y-morita@renais-clinic.com
Co-authors; Izawa H, izawa@woman-life-clinic.com;
Hirano A, a-hirano@renais-clinic.com; Mayumi E, e-mayumi@renais-clinc.com;
Isozaki S, isozaki@antiaging-bank.com; Yonei Y, yyonei@mail.doshisha.ac. jp_ 33 _

Glycative Stress Research 2022; 9 (2) : 33-41(c) Society for Glycative Stress ResearchChanges in Biomarkers by Oral Intake of NMN

Introduction

Medical and physiological research using molecular
genetics and molecular biology has revealed the mechanismsof the life phenomena of ageing and lifespan. Even if
immortality and eternal beauty are not possible, we are
beginning to see pathways to slow down ageing and maintaina youthful and healthy state. The way we age is not the samefor everyone. Physiological ageing, which is the fate of allliving things and unavoidable, is more or less in addition
to ageing beyond the physiological level due to lifestyledisorders. The extra ageing added to such physiologicalageing can be regarded as 'pathological ageing,' which canbe corrected and recovered by lifestyle modification andmedical intervention. In other words, ageing can also be seen
as a lifestyle-related disease. Many diseases sprout from thesoil of such pathological ageing. Therefore, by continuingwith a lifestyle that does not allow them to grow, we can
expect to achieve a youthful, healthy, and long life.
In recent years, as research on ageing has progressed,
studies on sirtuin, which has become widely known as alongevity/anti-ageing gene, and on nicotinamide adenine
dinucleotide (NAD) metabolism have been intensively
pursued worldwide 1- 8). Some of these may be able to control
pathological ageing and bring it closer to physiologicalageing. Concurrently, it will contribute significantly to theprevention and treatment of various systemic diseases.
In this study, we investigated the changes in biomarkers
after oral intake of the NAD precursor NMN (nicotinamidemononucleotide) and examined the possibility that NMN
could be an effective nutritional material for anti-ageing lifeand healthy life extension.

MethodSubjects

The subjects were 17 postmenopausal women aged
between 50 and 80 years (mean age 55.0 years). Exclusion
criteria were as follows: currently undergoing drug treatment/
follow-up for any disease, history of serious illness, historyof gastrointestinal surgery, drug allergy, possible change inlifestyle during the study period, and those deemed unsuitableas subjects for the study by the supervising physician. Subjects
were fully informed in advance and consent was obtained inwriting before the study was conducted. Of the 17 subjects atthe start of the study, one subject dropped out due to persistent
mild headache, and 16 subjects were finally included in the
final analysis.

Test product
Hard capsules containing 150 mg of NMN produced bya yeast-based fermentation process (CNB Medical ResearchInstitute, Tokyo, Japan) were used.

Study protocol.
Two capsules of the test product (containing a total of
300 mg NMN) were taken orally in one batch after breakfast
and tested at three points in the morning before (week zero),four weeks after, and eight weeks after ingestion. During thestudy period, participants were prohibited from changing
their lifestyle, including drinking and smoking habits,
starting new health food intake, and any other items thatmight affect the study. They were also asked to refrain fromexcessive exercise on the day before the test, to refrain fromeating or drinking anything other than water 12 hours before
the test, to avoid staying up late, and not to eat breakfast on
the day of the test as test products only.
At zero-week, height, weight, body mass index (BMI),
body fat percentage, basal metabolic rate, blood pressure,grip strength, and glycaemia were measured and skincondition was assessed using a visual analog scale (VAS).
Blood tests included white blood cell (WBC), WBC fraction,
red blood cell (RBC), hemoglobin (Hb), Ht, MCV, MCH,
MCHC, platelet (PLT), total protein (TP), albumin (Alb),A/G ratio, T-Bil, D-Bil, aspartate aminotransferase (AST),
alanine aminotransferase (ALT), γ -glutamyl transpeptidase(γ -GTP), alkaline phosphatase (ALP), leucine dehydrogenase(LDH), leucine aminopeptidase (LAP), cholinesterase (ChE),
creatine phosphokinase (CPK), total cholesterol ( TC),
triglyceride ( TG), high density lipoprotein-cholesterol(HDL-C), low density lipoprotein-cholesterol (LDL-C),
amylase, blood urea nitrogen (BUN), creatinine (Crea),uric acid (UA), Na, K, Cl, Ca, fasting plasma glucose(FPG), HbA1c, C-reactive protein (CRP), somatomedin C,
dehydroepiandrosterone-sulfate (DHEA-s), cortisol, thyroid
stimulating hormone (TSH), free T3 (FT3), free T4 (FT4),
estradiol ( E 2), free testosterone, insulin, adiponectin, crosslinked N-telopeptide of type 1 collagen (NTX), osteocalcin,
immunological tests ( T- cell sub Sets; T cells, killer T cells,
natural killer cells, regulatory T cells, natural killer (NK)T cells, ageing T cells, exhausted T cells), SIRT1 mRNA
expression, NMN, NAD, NAM (nicotinamide = a type of
niacin (vitamin B3) were measured.
At four weeks, weight, BMI, body fat percentage andblood pressure were measured, as well as blood counts andbiochemical tests and various hormones; at eight weeks, thesame tests as at week zero were performed
The expression level of SIRT1 mRNA in blood wasmeasured from leukemic cells by quantitative RT- PCR
and evaluated using the criteria set by the Analysis Center.
Blood NMN, NAD, and NAM levels were measured bymass spectrometry. The degree of glycation was assessed
for fluorescent advanced glycation end products (AGEs)
deposited in the epidermis to dermis layer of the skin, using
the autofluorescence (AF). The subjects were asked to keepa diary during the course of the study in order to monitor
NMN intake, their bodily sensation, and side-effects.
The above clinical trial was conducted between
September and November 2021.

Statistical analysis
Excel Statistics (Social Information Service, Tokyo,
Japan) was used for statistical analysis and a correspondingt-test was conducted. A two-tailed test with a risk rate ofless than 5 % was considered significantly different.

Ethical standards_ 34 _The study complied with the ethical principles basedGlycative Stress Researchon the Declaration of Helsinki and the Personal Data
Protection Act, and referred to the Ministerial Ordinance on
the Conduct of Clinical Trials of Medicinal Products andthe Ethical Guidelines for Epidemiological Research. The
study was conducted after approval of the ethics and validityof the study by the Ethical Review Committee on Research
Involving Human Subjects of Doshisha University (ApprovalNo.: GSE #2021009) and pre-registration for the clinical
trial was conducted (Registration No.: UMIN #000045347).

markers, HbA1c in glucose metabolism decreased from 5. 34
to 5.23 % and HDL-C in lipid metabolism increased from67.5 to 72.3 mg/dL, both significantly different at p < 0.05
( Fig. 1- a, b). No significant changes were observed in thebone metabolism markers osteocalcin, and type I collagencross-linked-N telopeptide ( NTX).

HormoneNo significant changes in body fat percentage, basal
metabolic rate, blood pressure, or grip strength were observed
over the eight-week course, but BMI increased significantly
from 23.2 to 23.4, p < 0.05.

Adiponectin, which is secreted by fat cells and acts in an
inhibitory manner against lifestyle-related diseases, showed
an increase from 13.6 to 16.2 µg/mL, with a significant
difference at p < 0.01 ( p = 0.004). The adrenal hormone
DHEA-s, considered the mother hormone, also showed asignificant increase from 1,108 to 1, 275 ng/mL, p < 0.05
(Fig. 2-a, b). No significant differences were found for cortisol,
estradiol (E2), free testosterone, thyroid-stimulating hormone(TSH), thyroid hormones (free T3 and T4), somatomedin C,
and insulin.

Bloodchemistry

Immune function

Platelets increased from 25.9 to 27.6 x 10 4 /µL andamylase decreased from 75.6 to 69.8 U/L, showing a significant
difference (both p < 0.05). In terms of metabolism-related

In T cell subsets ( T cells, killer T cells, natural killer cells,
NKT cells, regulatory T cells, killer/ helper- aging T cells,
killer/ helper- exhausted T cells), narrowly defined regulatory

ResultPhysical measurements

a)

b)

( %)
5.45
5.40
5.35
5.30
5.25
5.20
5.15
5.10
5.05
5.00

HDL-C

HbA1c

a)

b)

0W

4W

8W

(mg/dL)
78.0
76.0
74.0
72.0
70.0
68.0
66.0
64.0
62.0
60.0
58.0

0W

4W

8W

Fig. 1. Change in serum HbA1c and HDL-C.
a) HbA1c, b) HDL-C. Results are expressed as mean ± SEM, n = 16, * p < 0.05, paired t test. HbA1c, hemoglobin A1c;
HDL-C, high density lipoprotein-cholesterol; SEM, standard error mean.

a)b)(µg/mL)
20.0
18.0
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0

(ng/mL)

1,600
1,400

DHEA-sAdiponectin

a)

b)

1,200
1,000
800
600
400
200

0W

0

8W

0W

4W

Fig. 2. Change in serum adiponectin and DHEA-s.
a) Adiponectin, b) DHEA-s. Results are expressed as mean ± SEM, n = 16, * p < 0.05, ** p < 0.01, paired t test.
DHEA-s, dehydroepiandrosterone-sulfate; SEM, standard error mean.

_ 35 _

8W

Changes in Biomarkers by Oral Intake of NMN

T cells (CD4 + CD25 int/high CD127 low ) increased from 4.66
to 5.10 % (CD4 + cell area within ratio), showing a significant
increase at p < 0.05 (Fig. 3 ). No other significant differenceswere observed.

Ratio of Treg to CD4+ T cellCD4 + CD25 +int / high CD127 low

NAD synthesis system / sirtuin
Regarding the amount of NAM, NMN, and NAD in the
blood, the main pathway components that synthesize NAD,
NAM, the starting point of the pathway, increased markedly
from 45.2 to 164.7 ng/mL, with a significant difference at p
< 0.01 (p = 0.000). NAD also showed a significant decrease
from 17.4 to 13.7 ng/mL, p < 0.05, while NMN showed nosignificant change from 1.32 to 1.28 ng/mL. SIRT1 mRNA
expression, the blueprint for the sirtuin protein that acts as an
NAD-dependent protein deacetylase (For leukaemia-derived
leukaemic cells, a marked increase was observed in one case,from 6.7 to 33.5. However, the average for all subjects was
5.75Æ 6.19, with no significant change (Fig. 4-a, b, c, d ).

a)

3.0
2.0
1.0
0.0

0W

8W

Results are expressed as mean ± SEM, n = 16, * p < 0.05, pairedt test. Treg: regulatory T cell, CD4 + CD25 int / high CD127 low;
SEM, standard error mean.

b)(ng/mL)

1.80

25.0

1.40

20.0

1.60

1.20

NAD

NMN

4.0

Fig. 3. Ratio of Treg to CD4 + T cell.

(ng/mL)

1.00

0.80

15.0
10.0

0.60
0.40

5.0

0.20

0.00

0W

0.0

8W

0W

8W

d)
(ng/mL)
200
180
160
140
120
100
80
60
40
20
0

9.0

8.0

SIRT1 mRNA expression

7.0

SIRT1

NAM

5.0

b)

a)

c)

6.0

6.0
5.0
4.0

3.0

2.0
1.0

0W

0.0

8W

0W

8W

Fig. 4. Change in serum NMN/NAD/NAM/sirt1mRNA expression.
a) NMN, b) NAD, c) NAM, d) sirt1 mRNA expression. Results are expressed as mean ± SEM, n = 16, * p < 0.05, ** p < 0.01, pairedt test. mRNA expression of sirtuin 1 was evaluated in isolated peripheral mononuclear cell. NMN, nicotinamide mononucleotide;NAD, nicotinamide adenine dinucleotide; NAM, nicotinamide; SEM, standard error mean.

c)d)_ 36 _Glycative Stress Research

Glycative stressSkin

The AF value, which reflects the amount of fluorescentend-glycation products (AGEs) in the skin, decreased from
2.43 to 2.35, with a significant difference at p < 0.05 (Fig. 5 ).

Of the seven VAS items as subjective evaluation (1.
moisture 2. flakiness (dry skin) 3. blemishes 4. tension 5.
make-up application 6. rough skin 7. eruptions), six items
except eruptions decreased, showing a highly significant
change with p < 0.01 ( p = 0.000 to 0.001) ( Table 1 ).

2.60

Subjective changes (bodily sensation)

Skin AF

2.50
2.40
2.30
2.20
2.10
2.00

0W

8W

Fig. 5. Change of skin AF.

Results are expressed as mean ± SEM, n = 16, * p < 0.05,
paired t test. Skin AF value was measured by AGE reader mu.
AF, autofluorescence; AGE, advanced glycation endproduct;
SEM, standard error mean.

The following effects were observed in bodily sensationof the subjects after eight weeks of oral intake: improvementof skin (reduction of spots, fine lines and freckles, skinmoisturizing, tautness, less noticeable lines, whitening),
improvement of sleep (better sleep, less difficulty staying
asleep, better wakefulness in the morning), fatigue andphysical fitness (less fatigue, faster recovery from fatigue,
better mobility, faster running laps, less muscle pain after
exercise).
Other positive results included warmer body (improvementof sensitivity to cold), less eye strain, better night vision, lessallergic eye symptoms, less weight gain, less drunkenness,
improved constipation, shinier hair, faster hair growth,
increased appetite, better mood, less joint pain, and improved
quick-thinking.
On the contrary, headache, feeling tired, diarrhea,dizziness, and drowsiness, all very mild but transient in twopatients ( Table 2).

Table 1. Skin changes evaluated by visual analog scale.
Questions
How is your skin moisturized ?
Is your skin dry ?
Do you care about age spot ?
How is your skin firm & elastic ?
How about a make-up routine ?
Do you care about pimples ?
How about rough skin ?

Before intake(0 week)

After intake(8 weeks)p value

52.1 ±
46.5 ±
74.5 ±
58.1 ±
53.9 ±
6 9.1 ±
46. 5 ±

31. 6 ±
25.1 ±
52.8 ±
31.8 ±
30.1 ±
70.1 ±
23.0 ±< 0.001< 0.001
0.001< 0.001< 0.001
0.865< 0.001

5.1
5.3
4.6
4.3
5.0
5.2
5.5

4.3
4.7
6.0
4.1
4.4
7.1
4.1

Table 2. Subjective change (Bodily sensation)
SKIN
Age spots, wrinkles, and freckles have decreased / Nasolabial fold becomes inconspicuous
Skin becomes moisturized/Skin is getting whiter
SLEEP
Better sleep quality/Not easy to wake up in the middle of the night
Awaking is getting better

PHYSICAL PERFORMANCE
Not easy to get tired/Fatigue recovery has become faster /Body becomes flexible
Run faster/ Less muscle pain after exercise

OTHER
Reduced eye strain/ Better night vision/ Reduced allergies around the eyes
Body becomes warm / Not easy to get fat/Not easy to get a hangoverConstipation improved/Hair grows faster /Hair becomes smooth/Appetite improved
Reduced joint pain/Feel better/ Mind becomes clear
Headache, malaise, diarrhea, dizzy, sleepiness (transient symptoms 1-2 participants)_ 37 _

Changes in Biomarkers by Oral Intake of NMN

Discussion
A paper published in Nature in 2000 by Imai S et al.,
who were part of Professor Guarente's research team at
Massachusetts Institute of Technology, opened the door wide
for the 21st century in aging research 9). Their research using
yeast revealed that sirtuin protein, which is activated by NAD,
acts as a protein deacetylase that regulates the expression ofvarious genes and is deeply involved in aging and metabolism.
Since then, research on the involvement of sirtuin genes
and the NAD synthesis and consumption systems in various
biological functions has progressed dramatically.
While many basic studies and animal studies have beenreported, clinical studies in humans using NR (nicotinamide
riboside), a type of vitamin B3 that is converted to NAD
via NMN, have also been reported 10 -12). However, none ofthese studies has demonstrated any clear anti-aging effect,
and it is possible that the reduced absorption of NR may
have led to its weakened effect, since much of it is broken
down by intestinal bacteria when taken orally. Against thisbackdrop, in April 2021, the Imai group at the Universityof Washington published the world's first clinical research
paper using NMN, an NAD precursor, in Science 13). This wasa randomized, placebo-controlled, double-blind study of
25 obese postmenopausal women with borderline diabetes.
Subjects were randomized to receive placebo or NMN (250mg/day) for ten weeks. No adverse events were observed,
and the NMN group showed increased skeletal muscle
insulin sensitivity, insulin signaling, and muscle remodeling.
However, these changes were seen only in muscle, not inliver reserves or adipose tissue, and the effects were not as
dramatic as those seen in the mice. The results of a second
clinical study involving an increased number of men are
awaited.
In June of the same year, another clinical study of 48
amateur runners was reported from the Guanghou Sports
University in China 14). The study compared NMN at baseline
and after six weeks of intervention between a placebo groupand four groups divided into three doses of 300, 600, and1200 mg/day of NMN. There were no changes in bodycomposition or cardiac function indices such as body fat
mass, body fat percentage, slow fat weight, or BMI regardlessof NMN dose, but there was a dose-dependent increase inskeletal muscle oxygen utilization in the medium- and highdose groups. No adverse events were observed in the highdose group. These results suggest that muscle is one of thetissues most sensitive to NMN and that exercise training
with NMN supplementation may be an exercise strategy to
improve endurance in athletes.
In a clinical study at the University of Tsukuba publishedin January 2022, 108 elderly subjects were divided into fourgroups (NMN morning intake, afternoon intake, placebo
morning intake, and afternoon intake) and examined theeffects of NMN 250 mg/day for 12 weeks on sleep quality,
fatigue, and physical performance 15). In particular, the NMN
afternoon intake group showed the greatest improvement inlower limb motor function and sleepiness. These findings
suggest that afternoon NMN intake may be more effectivein improving lower limb function and reducing sleepiness inthe elderly, and may be beneficial for mental and physicalhealth.
These clinical studies showed differences in organ

sensitivity to NMN in humans, effects on exercise capacity,
sleep, and fatigue, timing of intake, and safety of intake, andlong-term continuous use. This study provided findings that
may be useful in daily medical practice, such as changes
in various biomarkers ( i.e., blood biochemistry, glucose
metabolism, lipid metabolism, hormones, and immunity)
measured as clinical tests as well as safety, and bodily
sensation.
NAD acts as a coenzyme that catalyzes redox reactions
during energy production and is also important for posttranslational modifications such as NAD-dependent protein
deacetylation by sirtuin and induction of DNA damage
repair reactions by ADP-ribosylation catalyzed by PARPs.
Since NAD cannot be taken in directly as a nutrient, it issynthesized in vivo by two pathways: from NAM and fromtryptophan or NA (nicotinic acid, a type of vitamin B3
like NAM and NR). The former, the major pathway, is thesalvage pathway through the recycling of NAM, a reaction
product after NAD acts as a coenzyme or substrate, whichis resynthesized via the intermediate NMN. There is also aknown pathway from NR via NMN in the salvage pathway;
NMN is also found in broccoli, edamame, and cucumbers,
and dietary NMN is also used. The latter is a newly synthesized
de novo pathway ( Fig. 6 ). The salvage pathway consists ofthe conversion of NAM to NMN by NAMPT (nicotinamide
phosphoribosyltransferase) and NMNAT (nicotinamidemononucleotide adenyltransferase) is a two- step enzymatic
reaction from NMN to NAD.
It is known that NAMP T, which acts in the initialstage, is the rate-limiting enzyme for this reaction, whichis regulated in response to environmental and nutritional
conditions, and declines with age. The amount of NAD in the
body declines accordingly, and after middle age, the amountof NAD in the body is less than half of its peak level.
The clinical studies reported so far have not examined
NAD and sirtuin in blood. In this study, we measured NMN,
NAD, NAM, and SIRT1 mRNA expression in blood with one
major objective of exploring the metabolic status of these in
humans.
There were no changes in leukocyte SIRT1 mRNA
expression and blood NMN levels before and after oral
intake of 300 mg/day of NMN over an eight week period.
However, there was a marked increase in the amount of
NAM and a decrease in the amount of NAD ( Fig. 4-a, b,
c, d ). NMN is known to have an extremely short residence
time in the blood and is rapidly absorbed into tissues andconverted to NAD in minutes 16). Therefore, the amount of
NMN in the blood decreases dramatically over time, making
its measurement difficult. Intracellularly increased NAD
activates redox reactions and enzymatic reactions by sirtuin,
PARPs, and is itself metabolized to NAM. The rate-limiting
enzyme NAMPT, which acts in the metabolism of NAM to
NMN, is not increased, so NAM levels remain high. Thissequence of events was formed during the course of NMN
ingestion.
NAD consu mption is known to be as rapid as that of
NMN on a minute-by-minute basis 17). Since SIRT1 mRNAis also rapidly degraded after its expression to replenish therapidly diminishing sirtuin working in concert with NAD,its intracellular localization time is likely to be short. In this_ 38 _Glycative Stress Researchsirtuinde novo
pathwaysalvagepathway

ADP ribose

Fig. 6. NAD synthetic pathway.

Fig. 6

Trp, tryptophan; NA, nicotinic acid; NAD, nicotinamide adenine dinucleotide; NMN, nicotinamidemononucleotide; NAM, nicotinamide; NR, nicotinamide riboside; NAPT, nicotinic acidphosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; NMNPT,
nicotinamide phosphoribosyltransferase; NRK, nicotinamide riboside kinase; ADP, adenosine diphosphate.

study, NMN was ingested orally only once, after breakfast,and since it would be rapidly expressed and degraded
thereafter, the timing of the measurement would causesignificant differences in expression levels. Therefore, theSIRT1 mRNA expression in blood is not suitable for a onepoint comparison before and after long-term intake, as inthis study, because it only looks at the time when the blood
sample was taken. The purpose of this study should be to
examine short-term changes over time.
In experiments with BRASTO mice, in which SIRT1
was genetically engineered to be overexpressed only in the
hypothalamus, the appearance of symptoms and diseases
associated with aging was delayed, and healthy life expectancy
was increased by 16.4 % in females and 9.1 % in males
compared to the target group 18). This is 13 to 14 years forfemales and 7 to 8 years for males in human terms, indicating
the possibility of changing the current situation in which
healthy life expectancy has not kept pace with the increase inlife expectancy. For the purpose to extend healthy life span, it
is desirable not only to reduce oxidative and glycative stress,
which accelerate aging and induce/promote diseases, butalso to keep the various metabolisms such as sugar, lipid, andbone, as well as hormones and immune system, in balance incooperation without any of them becoming weak points.
In this study, there was a decrease in HbA1c, whichindicates the status of blood glucose levels for approximately
the past two months ( Fig. 1-a). Glycation, a reaction between
reducing sugars such as glucose and fructose and proteins
under non-enzymatic conditions, produces terminal glycation
products (AGEs) via HbA1c and other intermediates, resulting
in qualitative and functional degradation of proteins in the
body 19). As an evaluation of the degree of glycation, AF values,
which reflect the amount of fluorescent AGE accumulated in

the skin, also showed a significant decrease (Fig. 5 ) 20). While
LDL-c values were unchanged, HDL- C values increased,
indicating a positive effect on lipid metabolism, which isconsistent with previous reports of improved glucose andlipid metabolism ( Fig. 1-b) 6, 18).
It is known that SIRT1 promotes the expression of
adiponectin, which has a suppressive effect on lifestyle-relateddiseases 21). The increase in adiponectin secretion from adipose
tissue (especially visceral fat), which is highly sensitive to
NMN, and the increase in DHEA-s, the mother hormone
secreted by the adrenal gland, may indicate that NMN actson the hormonal system (Fig. 2-a, b).
The lymphocytes, the mainstay of immune cells, also
become exhausted and undergo functional aging due tolifestyle disorders and aging 22, 23). Regulatory T cells (Treg),
one type of Tregs, control excessive immune responses andhave attracted attention as a countermeasure for autoimmune
diseases such as ulcerative colitis and Crohn's disease,allergic rhinitis, and conjunctivitis 24). In the present study, we
examined T lymphocyte subsets, including Tregs, exhausted,and senescent cells, which were not found in previous
clinical studies. Narrowly defined Tregs were significantly
increased, but neither exhausted nor senescent T cells showedsignificant changes (Fig. 3 ). Since the immune system takes
time to adjust, eight weeks may not be sufficient time. In
recent years, while research has been conducted on antiaging therapies by eliminating senescent cells, a vaccine
targeting senescent cells has been developed 25). It has beenshown to reduce senescent T cells and improve glucose
tolerance in mice. Future validation in humans is awaited.
For the skin, which is easily recognized as an aging
phenomenon, there was a clear improvement in all subjects( Table 1 ) 26, 27). In addition to skin changes, many examinees
also commented on the improvement effects of various_ 39 _

Changes in Biomarkers by Oral Intake of NMN

discomforts and modifications, such as poor sleep quality
and fatigue ( Table 2). It can be said that NMN intake is anutritional material that is likely to cause not only changes
in various biomarkers but also subjective changes of bodily
sensation.
It is also known that in conditions of high glycationstress, such as in diabetics, the TCA cycle in mitochondria is
disrupted and various disorders are induced 28-30), presumably
due to NAD shortage. NMN supplementation appears to bean effective treatment for NAD deficiency.

Conclusion
The positive changes in various biomarkers observedwith oral intake of NMN suggest that NMN may be a
promising nutritional material with the potential to control
ageing and extend healthy life expectancy.

Acknowledgments

There is hope for supplementation of NMN, an
important component of the NAD world [ageing, lifespan,and metabolic control are systemically integrated via NAD 31)]
proposed by Dr Shinichiro Imai, University of Washington,
but it is known that increased NAD activates sirtuin evenwith calorie restriction 32). Resveratrol, which is found in red
wine, was briefly discussed as an activator of the sirtuin
gene, but today the majority of opinions are negative.
However, a clinical study was reported in 2020 33) that showedthat oral intake of red wine extract containing resveratrol(resveratrol 19.2 mg/day) for eight weeks increased SIRT1mRNA expression extracted from blood mononuclear cells
and increased insulin sensitivity. This is an interesting result
and a follow-up study is expected. In addition, lifestylemodification should be started with daily habit, which is
expected to have no small effect, as resistance training andaerobic exercise increase NAMPT, the rate-limiting enzyme
in the NAD synthesis pathway.

Authors of this study would like to thank the participants
for their cooperation. This study was presented at the 22nd
Meeting of Japan Society of Anti-Aging Medicine on Jun,
2022, Osaka, Japan.

Reference9) Imai S, Armstrong CM, Kaeberlein M, et al. Transcriptional

1) Guarente L. Sirtuins, aging, and metabolism. Cold Spring

Harb Symp Quant Biol. 2011; 76: 81-90.

2) Imai S, Yoshino J. The importance of NAMPT/NAD/SIRT1

in the systemic regulation of metabolism and ageing.
Diabetes Obes Metab. 2013; 15 Suppl 3(03): 26-33.
3) Tummala KS, Gomes AL, Yilmaz M, et al. Inhibition of
de novo NAD(+) synthesis by oncogenic URI causes liver
tumorigenesis through DNA damage. Cancer Cell. 2014;
26: 826-839.
4) Pulla VK, Sriran DS, Soni V, et al. Targeting NAMPT
for therapeutic intervention in cancer and inflammation:
Structure-based drug design and biological screening.
Chem Biol Drug Des. 2015; 86: 881-894.
5) Yao Z, Yang W, Gao Z, et al. Nicotinamide mononucleotide
inhibits JNK activation to reverse Alzheimer disease.
Neurosci Lett. 2017; 647: 133-14 0.
6) Yoshino J, Baur JA, Imai S. NAD + intermediates: Thebiology and therapeutic potential of NMN and NR. Cell
Metab. 2018; 27: 513- 528.
7) Covarrubias A J, Perrone R, Grozio A, et al. NAD +metabolism and its roles in cellular processes during
ageing. Nat Rev Mol Cell Biol. 2021; 22: 119-141.
8) Ramanathan C, Lackie T, Williams DH, et al. Oraladministration of nicotinamide mononucleotide increase
nicotinamide adenine dinucleotide level in an animal
brain. Nutrients. 2022; 14 (2): 300.

Conflict of Interest Statement
Authors have no conflict of interest in this study.

silencing and longevity protein sir2 is an NAD-depending
histone deacetylase. Nature. 2000; 403(6771): 795- 800.
10) Dollerup OL, Christensen B, Svart M, et al. A randomized
placebo-controlled clinical trial of nicotinamide ribosidein obese men: Safety, insulin-sensitivity, and lipidmobilizing effects. Am J Clin Nutr. 2018; 108: 343-353.
11) Dollerup Ol, Chubanava S, Agerholm M, et al. Nicotinamide
riboside does not alter mitochondrial respiration, content
or morphology in skeletal muscle from obese and insulinresistant men. J Physiol. 2020; 598: 731-754.
12) Remie CM E, Roumans KHM, Moonen MPB, et al.
Nicotinamide riboside supplementation alters bodycomposition and skeletal muscle acetylcarnitine
concentrations in healthy obese humans. Am J Clin Nutr.
2020; 112: 413-426.
13) Yoshino M, Yoshino J, Kayser BD, et al. Nicotinamidemononucleotide increases insulin sensitivity in prediabetic
women. Science. 2021; 372(6547): 1224 -1229.
14) Liao B, Zhao Y, Wang D, et al. Nicotinamide mononucleotide
supplementation enhances aerobic capacity in amateur
runners: A randomized, double-blind study. J Int Soc Sports
Nurt. 2021; 18: 54.
15) Kim M, Seol J, Sato T, et al. Effect of 12-week intake ofnicotinamide mononucleotide on sleep quality, fatigue, andphysical performance in older Japanese adults: A randomized,
double-blind placebo-controlled study. Nutrients. 2022;
14 (4): 755.

_ 40 _Glycative Stress Research16) Mills KF,

Yoshida S, Stein LR, et al. Long-term
administration of nicotinamide mononucleotide mitigatesage-associated physiological decline in mice. Cell Metab.
2016; 24: 795-806.
17) Rechsteiner M, Hillyard D, Olivera BM. Turnover at
nicotinamide adenine dinucleotide in cultures of humancells. J Cell Physiol. 1976; 88: 207-217.
18) Satoh A, Brace CS, Rensing N, et al. Sirt1 extend life spanand delays aging in mice through the regulation of Nk2
homeobox 1 in the DMH and LH. Cell Metab. 2013; 18:416-430.
19) Morita Y. Glycative stress measures. Official Journal
Japan Society for Dental Anti-Aging. 2019; 12: 138-142.
(in Japanese)20) Morita Y, Yagi M, Ishizaki K, et al. Evaluation of theglycative stress by non-invasive skin AGEs. Glycative StressRes. 2019; 6: 92-102.
21) Majeed Y, Halabi N, Madani AY, et al. Sirt1 promotes
lipid metabolism and mitochondrial biogenesis in
adipocytes and coordinates adipogenesis by targeting key
enzymatic pathways. Sci Rep. 2021; 11(1): 8177.
22) Rodriguez IJ, Ruiz NL, León ML, et al. Immunosenescencestudy of T cells: A systematic review. Front Immunol. 2021;
11: 6 04591.
23) Ando M, Ito M, Srirat T, et al. Memory T cell, exhaustion,and tumor immunity. Immunol Med. 2020; 43: 1-9.
24) Yu N, Li X, Song W, et al. CD4+CD25 + CD127 low/- T
cells: A more specific Treg population in human peritoneal
blood. Inflammation. 2012; 35; 1773-1780.
25) Yoshida S, Nakagami H, Hayashi H, et al. The CD153
vaccine is a senotherapeutic option for preventing theaccumulation of senescent T cells in mice. Nat Commun.
2020; 11(1): 2482.
26) Tan CYR, Tan CL, Chin T, et al. Nicotinamide prevents
UVB- and oxidative stress-induced photoaging in humanprimary keratinocytes. J Invest Dermatol. 2021: S0022202X (21)02475-1.
27) Zhou X, Du HH, Long X, et al. β-nicotinamidemononucleotide (NMN) administrated by intraperitoneal
injection mediates protection against UVB-induced skindamage in mice. J Inflamm Res. 2021; 14: 5165-5182.
28) Nagai R, Mori T, Yamamoto Y, et al. Significance ofadvanced glycation end products in aging-related disease.
Anti-Aging Med. 2010; 7: 112-119.
29) Nagai R, Brock JW, Blatnik M, et al. Succination ofprotein thiols during adipocyte maturation: A biomarker
of mitochondrial stress. J Biol Chem. 2007; 282: 3421934228.
30) Frizzell N, Rajesh M, Jepson MJ, et al. Succination of thiolgroups in adipose tissue proteins in diabetes: Succination
inhibits polymerization and secretion of adiponectin. J Biol
Chem. 2009; 284: 25772-25781.
31) Imai S. The NAD World: A new systemic regulatory
network for metabolism and aging: Sirt1, systemic NAD
biosynthesis, and their importance. Cell Biochem Biophys.
2009; 53: 65-74.
32) Michan S. Calorie restriction and NAD+/sirtuin counteract
the hallmarks of aging. Front Biosci (Landmark Ed). 2014;
19: 1300-1319.
33) Kitada M, Ogura Y, Monno I, et al. Supplementation
with red wine extract increases insulin sensitivity
and peripheral blood mononuclear sirt1 expression in
nondiabetic humans. Nurtrients. 2020; 12: 3108.

_ 41 _