Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : September 30, 2023
Accepted : October 20, 2023
Published online : December 31, 2023
doi:10.24659/gsr.10.4_145

Original article

Glycative stress: Molecular impacts and defense mechanisms
Yoshikazu Yonei 1), Masayuki Yagi 1), Kenji Sato 2), Mari Ogura 1, 3)
1) Anti-Aging Medical Research Center / Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, 1-3 TataraMiyakodani, Kyoto 610- 0394, Japan
2) Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
3) Department of Food and Nutrition, Kyoto Bunkyo Junior College, Kyoto, Japan

Abstract : Glycative stress has drawn attention in recent years as a factor that significantly affects tissues and organs of
the human body and is directly related to health maintenance and aging-related diseases. Glycative stress refers to theexcessive formations of carbohydrate-derived aldehydes or fatty-acid-derived aldehydes in the body. These aldehydes impact
diverse substances such as proteins, lipids, and bases in cells, and subcellular organelles. Proteins in the body are modified
by non-enzymatic procedure and abnormal proteins, i. e., carbonylated proteins, advanced glycation end-products (AGEs),
are formed and stored intra- and extra-cellularly. AGEs induce endoplasmic reticulum stress, causing the deterioration ofcellular functions Although glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glyoxalase, and aldehyde dehydrogenase(ALDH ) are prepared for protection in the body as a defensive measure against aldehydes, a co-enzyme, nicotinamide
adenine dinucleotide (NAD) is consumed to a large extent through metabolic process. As a result, a tricarboxylic acid cycle(TCA cycle) in mitochondria, where NAD is needed, is unable to function smoothly. Fumaric acid is elevated and important
proteins such as GAPDH and adiponectin undergo succinylation, which causes the deterioration of functional proteins.
The present study reviewed the involvement of glycative stress in the insulin secretion of pancreatic β cell and Alzheimer's
dementia (AD). For AD protection, it is important to inhibit peroxidation of lipids which exist abundantly in the brain andinhibit glycative modification of amyloid β (Aβ) and tau proteins. We hope that further extensive research on anti-glycation
will be undertaken, and deeper understanding and increased participation will be encouraged. Studies on glycative stress arenecessary to lead to a social implementation as a practical science.

KEY WORDS: glycative stress; carbohydrate-derived aldehydes; fatty-acid- derived aldehydes; nicotinamide adenine				dinucleotide (NAD); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); insulin resistance;Aβ clearance; melatonin; exercise resistance

1. Introduction

2. Impacts of glycative stress

Glycative stress is a condition in which carbohydratederived aldehydes and fatty-acid-derived aldehydes areformed in excess in the body: the former is derived fromreducing sugar and alcohols and the latter from lipids. Whenthese aldehydes react to proteins, carbonylated proteins andadvanced glycation end-products (AGEs) are formed withinorganisms, and further, bind with RAGE (receptor for AGEs).
Consequently, proinflammatory cytokine is produced, anddegenerative changes and tissue disorders are caused 1- 3).
The present study reviewed impacts of glycative stress at themolecular level and defensive mechanisms against it.

2. 1. Reason and mechanism

Major causative factors for excessive formation ofaldehyde are ⅰ) glucose spikes (associated with diabetes,
impaired glucose tolerance and insulin resistance), ⅱ ) highfat diet (HFD), and ⅲ) a high rate of alcohol consumption.
Reducing sugars such as glucose and fructose have cyclic
structures with 99 % or higher ratio. Some part of reducing
sugar is in a ring opening with a straight-chain structure.
Aldehyde group (- CHO) and keto group (- C = O) are
exposed. As a result, reactivities are elevated. A rapid rise inblood glucose (glucose spike) triggers a chain reaction thatsimultaneously produces a variety of aldehydes 4 - 6). We havetermed this phenomenon "aldehyde spark.

Correspondence: Professor Yoshikazu Yonei MD, PhD.
Anti-Aging Medical Research Center/ Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1- 3, Tatara Miyakodani, Kyotanabe, Kyoto, 610- 0394 Japan;
TEL: +81-774-65-6394 e -mail: yyonei@mail.doshisha.ac.jp
Co-authors: Yagi M, myagi@mail.doshisha.ac.jp;
Sato K, sato.kenji.7x@kyoto-u.ac.jp; Ogura M, m-ogura@po.kbu.ac.jp_ 145 _

Glycative Stress Research 2023; 10 (4): 145-158(c) Society for Glycative Stress ResearchGlycative Stress: Molecular Impacts and Defense Mechanisms

Aldehydes are highly reactive and undergo chemical
reactions readily with proteins, lipids, and bases.
Structural changes and functional changes are induced.
Alcohol-consumption-induced acetaldehyde (A A),
postprandial-hyperglycemia-induced glyceraldehyde (GA),3-deoxyglucosone (3DG), glyoxal (GO), and methylglyoxal(MGO) are carbohydrate-derived aldehydes 7) . Fatty-acidderived aldehydes, which are caused by the oxidation oflipids, are malondialdehyde (MDA), hydroxynonenal (HNE),
acrolein (Acro), and methylglyoxal (MGO). MGO is produced
from both carbohydrate and fatty acid. Findings indicate thatin organs with high lipid composition, fatty-acid-derived
aldehydes are deeply related to pathological mechanisms.
Impacts of glycative stress at the molecular level are shownin Fig. 1.

2. 2. High fat diet (HFD) and fatty-acid-derived
aldehyde

In experiments of mice fed a HFD, a fatty liver was
generated and oxidative stress induced the increase of lipid
peroxidation and the decrease of cysteine 8). Furthermore,another important issue is the decline of the protein levelof glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in
the liver. GAPDH in the glycolysis system provides another'()*+,-./$
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phosphate group to glyceraldehyde - 3 -phosphate (G3P).
Decreased activities of GAPDH promote the increase of
GA an MGO in the blood 7). When lipids are oxidized dueto oxidation stress to form lipid peroxide, the formation of
fatty-acid-derived aldehydes is caused simultaneously 9 -11).
Aldehydes pass through cell membranes easily, transfer into
hepatic cells and further, permeate through mitochondrial
inner membranes. Abnormal proteins, which undergo nonphysiological post-translational modifications of proteinslead to carbonylation, AGE modification, and succinylation(the formation of S-(2-succinyl) cysteine), resulting in an
increase in abnormal proteins 12 -14). Therefore, endoplasmic
reticulum (ER) stress increases. When AGEs bind with
RAGE, which express in Kupffer cells and microphages,
AGE/RAGE signals are activated to form proinflammatory
cytokine and cause inflammatory responses 1). Increased
diverse factors include proinflammatory cytokine ( IL - 1[interleukin 1], IL- 6 and TNF-α [tumor necrosis factor-α]),chemokine ( MCP - 1 [monocyte chemoattractant protein 1]),cell adhesion molecule, and matrix metalloproteinase 15 -16).
That is, when inflammation is added to a non-alcoholic fatty
liver disease, this can be a potential trigger for progression tosteatohepatitis.
It has been verified that not all molecular entities of
AGE ligands bind with RAGE. N ε-(carboxymethyl) lysine

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HFD, high fat diet; HAFD, high animal-fat diet; DM. diabetes mellitus; NAD, nicotinamide adenine dinucleotide; GA, glyceraldehyde;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ALDH, aldehyde dehydrogenase; MDA, malondialdehyde; HNE, hydroxynonenal; Acro,acrolein; MGO, methylglyoxal; 3DG, 3-deoxyglucosone; GO, glyoxal; AA, acetaldehyde; AGEs, advanced glycation endproducts; RAGE,
Receptor for AGEs; WBC, white blood cell; Aβ, amyloid β; 2SC,S-(2-succinyl)cysteine; ER, endoplasmic reticulum; TCA, tricarboxylic acid.

_ 14 6 _Glycative Stress Research(CML), GA, and AGEs that are modified by glycolaldehyde
can bind with RAGE 17).
Among HFD, excessive consumption of animal fat due
to a high animal-fat diet (HAFD) is a considerable concern
nowadays from the viewpoint of addiction. HAFD attenuates
the function of leptin, an adipocyte-derived hormone. Leptin
resistance is induced, and the body constitution is changed
to have a difficulty in losing weight 18-21). The arcuatenucleus located in the appetite center, the hypothalamus, isconsidered a major site for the regulation of homeostasis
of appetite, which hormones and the autonomous nervous
system are responsible for. This is called the metabolic/hunger control system. HAFD causes an inflammationof the hypothalamus as well as ER stress, inducing thedisorder of the metabolic/hunger control system. Therefore,
the judgement of the brain is impaired regarding caloric
requirement 19). The body not only craves animal fat but also
become lazy, detesting exercise.
In HAFD-fed mice, inflammatory activated microglia
infiltrate the hypothalamus within a short period of time,causing progressive brain damage and leukocyte migration,
eventually leading to a chronic inflammatory state 20). We
speculate that lipids abundant in brain tissue, once oxidized,
may enhance fatty-acid- derived aldehyde production,
resulting in increased susceptibility to protein modification,
which in turn may induce ER stress in brain neurons.

C peptide and A-chain. These basic amino acids (Arg andLys) are removed due to the function of carboxypeptidase E.
Consequently, insulin and C peptide are produced. During
elevated glycative stress, short-chain aldehydes in the bloodincrease and transfer into β cells to attack proinsulin andform carbonylated proinsulin. Arg and Lys have two aminobases (-NH 2) are susceptible to carbonylation modification
resulting in a conformational change at the cleavage sitesthat causes resistance against carboxypeptidase E, resulting
in a decrease in insulin formation 22-27). Some of the insulin
stored in β cells undergo modifications to be glycatedinsulin: N-terminus (Phe), Arg and Lys residues and aminobases (-NH 2). Glycated insulin is secreted due to a glucose
stimulation as (non-glycated) insulin is secreted 27).
IRI includes insulin (glycation/non-glycation) andproinsulin (glycation/non-glycation). Healthy persons have
0.05 – 0.4 ng/mL of proinsulin in fasting time, and ratios to
IRI values are 5 – 48% 28). In diabetic patients, it has beenreported that 9% of insulin is glycated insulin 25). Whenglycative stress is accelerated, compositional ratios arealtered. Ratios of glycated insulin increase and that of active
insulin decrease. However, decreased glycative stress via
exercise loading leads to the decrease of glycated proinsulin
and glycated insulin in β cells. As a result, it is suggested thatthe formation of insulin increases, the compositional ratioof glycated insulin decreases, and that of insulin increases.
Fig. 3 shows an assumed schematic diagram where IRI
changes when glycative stress is accelerated and improved 29).
Through physical exercise, proinsulin ratios in IRI decline
and IRI values also decline. Ratios of glycative modificationsin proinsulin and insulin decrease. As a result, it is assumed
that even though IRI values decrease, insulin activities
increase and therefore, insulin resistances improve. To prove
this hypothesis, measurements in IRI are required regarding
insulin and proinsulin, and with or without glycation. This isa further task and challenge.

3. Molecular impacts of glycative stress

3. 1. Molecular impacts of glycative stress on insulin

In pancreatic β cells, insulin is synthesized via preproinsulin and proinsulin (Fig. 2) 3). Proinsulin is cleaved dueto the function of proprotein convertase at the ends of Arg
31– Arg 32, which connect insulin B-chain and C peptide,
as well as at the ends of Lys 64 –Arg 65, which connect;*1"-#$)"-;*%9:*1"-#$)"-?:4/4,-6/.9(2'"-

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The figure quoted from Reference 3. The values of IRI (immunoreactive insulin), which we usually use in clinical sites, are measured
using antigenicity against specific parts of insulin peptide. Immunological cross-reactivity is shown. There are cross-reactions inproinsulin and intermediate products in biosynthesis systems, and the obtained measurement values do not represent only insulin.

_ 147 _Glycative Stress: Molecular Impacts and Defense Mechanisms

IRI

Glycative
stress

Gly

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Gly

ActiveExercise－～±
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InsulinFig. 3. IRI compositional changes by exercise (speculated schematic).

If physical exercise decreases glycative stress, expectedly insulin action would increase despite the decrease in
IRI. IRI, immune reactive insulin; Gly, glycated proinsulin or insulin. The figure quoted from Reference 29.

Fig 3
3. 2. Involvement of glycative stress in Alzheimertype dementia (AD)Neuropathological characteristics of the brain of
AD patients consist of cerebral atrophy, senile plaque andneurofibrillary tangle. Senile plaques mainly consist of Aβ.
Aβ is a peptide with a molecular weight of 4,300 – 4,500,
consisting of approximately forty amino acids (Aβ40, Aβ42).
Aβ is produced via cleave from amyloid-β precursor protein(APP) through the excision with β- and γ-secretase (Fig. 4) 30).
The brain is a rich organ in terms of lipid content, andproteins conjugated with fatty-acid-derived aldehydes suchas Acro and MGO are observed in brains of patients withAD 31 - 41).
When Aβ is incubated under the presence of glucose,
it is glycated and undergoes AGE modification (AGE-Aβ).
Aβ has lysine residues at the numbers 16 and 18 in the aminoacid sequence of glucose (Lys; K) and arginine residue atthe number 5 (Arg; R). These amino acid sequence positions
are related to glycation. Furthermore, Aβ is incubated underthe existence of glucose and is aggregated. It is possible that
AGE-Aβ is a “seed” that promotes the aggregation of Aβ 42).
It is reported that regarding protein in frontal lobe
tissues containing senile plaque protein, AD patients havethree times as much AGEs as healthy aged people 43). Whereas,
when Aβ-glucose reaction system is incubated with theaddition of aminoguanidine, which is an inhibitor against
glycation, the formation of Aβ aggregation is reduced 43). The
existence of AGEs such as pentosidine, pyrraline and CML
N ε -arboxymethyl lysine is observed in senile plaque 44, 45) .
There is a possibility that AGE-Aβ, where Aβ is altered due
to glycation, is related to factors for the formation of protein
cross-linking and the promotion of Aβ aggregation.
Tau protein of paired helical filaments (PHF), which
was collected from brains of AD patients, was examined andtau protein that was modified by AGEs was observed, whilesoluble tau protein collected from people without Alzheimertype dementia or dementia did not show AGE modification.
It is reported that tau protein has thirteen lysine residues inamino acid sequence and six lysine residues are glycatedout of thirteen 45). As glycation positions of tau proteins arelocated in binding sites with microtubules, binding functions
are damaged. Glycated tau protein induces oxidative stresssuch as active oxygen and IL-6, and nerve cell functions aredamaged 46).
In an in vitro experiment system of tau protein, it isdemonstrated that glycated tau protein generates PHF-likefibers but non-glycated tau protein does not generate fibers 47).
AGEs such as pentosidine, pyrraline and CML are observedin PHF. Glycated tau protein induces the formation of
APP and Aβ. Intracellular transport is reduced due to thephosphorylation and glycation of tau protein, and APP
cannot be secreted. As a result, APP is deposited in cells.
The glycation of Aβ and tau protein facilitatively affects
the deposit and aggregation of Aβ, and at the same time,
exacerbates the deposit of tau.
Normally, Aβ is rapidly degraded and discharged asbrain waste (Aβ clearance). However, when abnormal Aβ is
generated due to modification of Aβ structure by oxidativeand glycative stress, insoluble fibers are formed anddeposited 48, 49). Specifically, protein cross-linking is formed,
which induces Aβ-peptide polymerization. This formationof abnormal Aβ polymer causes refractory, aggregation ofneurotoxin and decline of Aβ clearance. Furthermore, whenAβ is not discarded and is accumulated in the brain, Aβsenile plaques (amyloid pathology) are generated. Abnormal
Aβ with intensive neurotoxin clings to healthy nerve cells,
which affects nerve cells. With this as a trigger, tau protein,which is a microtubule connected protein, induces theformation of neurofibrillary tangle with filamentation andcondensation in cytoplasm. As a result, the brain gradually
atrophies and Alzheimer-type dementia progresses. Thisis the concept that unites glycative stress with the “amyloidcascade hypothesis.” 50-52). In this concept, the importance ofAβ clearance is emphasized.
We conducted an experiment to examine impacts ofglycative stress and sleep quality on phagocytic activity of
microglia, using the primary microglia cultured cell (Fig. 5) 30).

_ 14 8 _Glycative Stress Research

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Fig. 4. Aβ cascade: Impacts of glycative stress and protection by sleep quality/melatonin.
The figure based on data from Reference 30. Aβ, amyloid β; APP, amyloid-βprecursor protein.

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Fig. 5. Aβ phagocytosis by microglia: Fluorescence microscopy image.
a ) Aβ phagocytosis, b) Glycated-Aβ phagocytosis, c ) Aβ phagocytosis with melatonin (100 μM ). Images (x 100) of cultured cells 10 days"-D$J

after seeding (8 days after addition of Aβ). The bar indicates 200 μm. Fluorescence-labelled Aβ ( TAMRA-Aβ, Cosmo Bio, Japan) and ratderived microglia primary culture cells (Cosmo Bio, Japan) were used. Red color portions indicate Aβ-derived fluorescence uptaken by
microglia. Note that Aβ phagocytosis was inhibited by Aβ glycation and accelerated by the addition of melatonin. Glycated-Aβ was preparedby MGO treatment (10 mM, one day). Figures quoted from Reference 30. Aβ, amyloid β; MGO, methylglyoxal.

_ 14 9 _Glycative Stress: Molecular Impacts and Defense Mechanisms

Glycated Aβ was prepared via MGO or Acro treatment. Thisis an example case of impacts at the molecular level. Thesefindings confirmed that phagocytic activities of microglia
remarkably deteriorated toward glycated Aβ. Contrarily,
microglial phagocytosis for glycated Aβ are reinforced
when we added melatonin, which is related to the quality ofsleep. However, since the amount of melatonin used in theexperiment was higher than the physiological range in the
middle-aged or elderly, the melatonin action may be limited.
Further verification of this point is needed.
In Aβ cascade, the importance of Aβ clearance has
been an increasing interest in recent years. This finding of
our study has provided a distinct possibility that “glycative
stress decreases Aβ clearance, and melatonin, which isrelated to the improvement of sleep quality, promotes Aβclearance.” Furthermore, this finding has indicated that
one of the mechanisms where glycative stress reduces Aβclearance is the deterioration of microglial phagocytosis.

mechanisms cannot provide effective approaches. It isreported that oxidized protein hydrolase (OPH), which is a
type of serine protease, functions for degradation of AGEs
53-55). Components that promote OPH activities are explored 56).
In the body, major defenses function in the stage ofaldehydes. Typical components are ALDH, glyoxalase, and
GAPDH.
GAPDH exists in abundance, accounting for 10 – 20%
of intracellular proteins. In a glycolysis system, GAPDH,
with NAD as a coenzyme, converts G3P into D -1, 3diphosphonooxypropanoic acid (1, 3-BPG) 7, 57). This reaction
requires energy that is obtained from reduction process fromcoenzyme NAD to NADH.
Livers and kidneys are fortresses for the defense systemagainst glycative stress. Notably, GAPDH are contained
abundantly in hepatocytes and podocytes of the kidney.
When this enzyme is inhibited, intracellular and extracellular
GA is increased, cellular necrosis is induced, and defense
activities against aldehydes are deteriorated, resulting inincrease of GA blood concentration (Fig. 6 ) 7). Exploratory
research had started to identify components for the elevationof GAPDH activity. A report is presented on potentiation
effects of a chlorella extract to promote GAPDH activities 58).
Glyoxalase system, with an assistance of reduced
glutathione (GSH), has a function of metabolization from
MGO to lactic acid. This enzyme is largely contained inlivers and kidneys playing an important role for the defense
against glycative stress 59, 60).
Aldehyde dehydrogenase (ALDH) is known for 19
types of isoenzymes. ALDH2 has the most expression

4. Defensive mechanism against
glycative stress
4. 1. Defensive mechanism

In cell60

Glyceraldehyde (nmol/mL)

Glyceraldehyde(nmol/initial cell number)

The body prepares their own defense mechanisms
against glycative stress. However, once AGEs have formed,
the body has difficulties in degradation of protease andproteosome as well as individual countermeasures for thediversity of AGEs. Therefore, degradation and removal

50
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Fig. 6. Effect of KA on HCT116 cell intracellular and medium GA.

100 μM

HCT116 cells (1 x 10 5 ) were inoculated on a 24 well plate and incubated in a high glucose DMEM medium containing antibiotics and 10%
fetal bovine serum. KA was added to the medium one day after inoculation. Cells were harvested 7 days after inoculation and washed withthe medium. Intracellular GA was extracted with 75% ethanol. The intracellular ( a ) and medium ( b ) GA was quantified by the method ofMartin-Morales et al. (Reference 8). Cells after 6- day incubation with KA ( c : 0 μM, d : 20 μM, and e : 100 μM ) were photographed (lower).
Figures quoted from Reference 7. KA, koningin acid; GA, glyceraldehyde.

_ 15 0 _Glycative Stress Research

quantity, and is contained in livers mostly 61, 62). ALDH2 not
only metabolizes acetaldehyde to acetic acid but also has
detoxification for diverse aldehydes. To name a few, there
are metabolizations from GA to glyceric acid, from glycol
aldehyde to glycolic acid, and from MGO to pyruvic acid.
ALDH2 activities are classified into low, moderate, and highactivity. ALDH2 with high activity has a stronger defense
function against glycative stress.
We are interested in the phenomenon that someindividuals are more prone to aldehyde spikes and others
less when compared to those who exhibit the same level ofblood glucose spikes. If an aldehyde-trapping substance,
which readily reacts with aldehydes, exists in the blood inlarge quantity, aldehydes can be detoxified. For instance,
GO is involved in pathogenesis of hepatopathy induced bymethimazole, a thyroid inhibitor. There is a possibility that
hepatopathy could be prevented by GO trapping. We pay
attention to amino acid in blood, holding and verifying ahypothesis, “Persons whose concentration of total aminoacid are high tend not to have aldehyde spark”.

4. 2. Involvement of glycative stress in NAD

We conducted a clinical trial with an oral administrationof 300 mg/day of nicotinamide mononucleotide (NMN),
which is a precursor of NAD, to seventeen post-menopausal
women (mean age: 55.0) for eight weeks. Research data ofglycative-stress-related indicators showed that HbA1c andintensity of AGE-derived skin autofluorescence (SAF)
significantly decreased, and high molecular weight adiponectin
significantly increased 65). SAF, which is a non-invasive
measurement by an AGE reader for skin fluorescence intensity,
assumed AGE accumulation, is frequently utilized as anindicator for glycative stress 66, 67). The research participants
were in good health, but their metabolism waned incomparison with the younger generation in their thirties.
The finding of this examination indicated a possibility that
due to the supply of NMN, NAD in mitochondria reachedsufficient quantity, which smoothed the TCA cycle, and thesuccinylation of GAPDH and adiponectin were reduced.
It was suggested that the supply of NMN is an effective
measure for a state of insufficient NAD.

4. 3. Significance of improvement of “quality of sleep”

TCA cycle of mitochondria is inhibited in the stateof high blood glucose/high aldehyde, and fumaric acidincreases, which induces the formation of S-(2-succinyl)
cysteine [2SC] of thiol group in cysteine, which comprises
peptide 12 -14) . This is a type of unphysiological post translational modification of protein.
In mitochondria, NAD is involved in reactions of TCA
cycle at three points. In the state of high blood glucose/high aldehyde, among aldehyde metabolizing enzymes,
GAPDH and ALDH metabolize aldehydes utilizing NAD.
Consequently, NAD is depleted in the state of excessive
aldehyde. Therefore, it is considered that the cycle cannot
function without hindrance.
It is reported that examples of succinylated proteinsare 2SC-GAPDH 13, 63), 2SC-adiponectin 11), 2SC-heat shockprotein (2SC-HSP) 10), kelch-like ECH-associated protein 1(KEAP1) 64), and mitochondrial aconitase (ACO2) 64). It isclarified that they largely affect intracellular metabolism.
Disorders of TCA cycle induce the formation of 2SCGAPDH. Metabolism activities of GA are deteriorated, andblood concentrations of GA increasingly elevate 7, 13, 63). This
results in a vicious circle; the high aldehyde state by glycative
stress induces the decline of GAPDH activity, which induces
the increase of aldehydes 3) .
In visceral fat, TCA cycle disorder induces theformation of 2SC-adiponectin (trimer). When Cys-39 thiolgroup (-SH) of adiponectin (trimer) is succinylated, disulfide
linkages are not formed. Thus, hexamer, which has highmembrane permeability, are not formed, and the bloodconcentration of high-molecular-form adiponectin (hexamer)decreases 11). Adiponectin may represent a therapeutic strategy
for insulin resistance. When glycative stress is intense, bloodconcentration of adiponectin decreases, insulin resistance isexacerbated, and subsequently glucose tolerance decreases. A
vicious cycle is induced where glycative stress is increasingly
aggravated.
These findings indicated that intervention at an earlystage is necessary for prevention of onset and progression ofglycative-stress-induced diseases.

Core measures against glycative stress are dietetic
therapy, exercise therapy and “quality of sleep”. The diet isclassified into the three categories according to the steps ofglycative reaction: ⅰ ) the blood glucose spike control/aldehyde
trap, ⅱ) the inhibition of AGE formation and ⅲ) the promotionof AGE degradation. We recommend efficient ingestion offood ingredients that promote these effects. For carbohydrates,
which account for approximately 60 % of total caloric intake,we recommend whole-grain foods. To counter animal fat
dependency, γ-oryzanol, contained in unpolished brown rice,
could provide positive effects 21, 68 -70).
“ Quality of sleep” and glucose tolerance affectinteractivity. Forty percent of diabetes patients with intensive
glycative stress have a sleep disorder. Patients with sleep
apnea syndrome (SAS) and a lowered quality of sleep havecomplications of obesity and diabetes with a high frequency 71).
A glycative stress indicator of SAF is affected in terms oflong or short sleep time. SAF values of persons with shortsleep period shift upward. 72) To reduce glycative stress, it isimportant to simultaneously consider the independentdisorders of carbohydrate metabolism and sleep disorders forprevention and treatment.
Metabolism of melatonin is gaining attention as a key
factor to link “quality of sleep” and glucose tolerance 73).
In vitro experiments have shown that melatonin does notinhibit AGE formation 74), but it does promote the breakdown
of AGEs 75). When melatonin is administered at night,
postprandial blood glucose level is subdued on the following
morning 76). These integrated activities reduce glycative
stress.
Fig. 7 77) compiles mechanisms that melatonin improved
glucose and lipid metabolism, reducing glycative stress.
First, melatonin has antioxidation activity in vitro andin vivo 78 - 88). This is based on two actions: a direct actionof removing reactive oxygen species (ROS), and an actionof increasing antioxidant enzymes such as superoxide
dismutase (SOD) and glutathione peroxidase 89, 90). Furthermore,metabolites, such as N-acetyl-N-formyl-5-methoxykynuramine(AFMK) and N1-acetyl-5-methoxykynuramine (AMK), haveantioxidation activities in vitro.

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Fig. 7. Melatonin and glycative stress.

"-D$N

The figure quoted from Reference 77. ROS, reactive oxygen species; AGEs, advanced glycation endproducts;
TG, triglyceride; HDL-C, high-density lipoprotein cholesterol.

Second, melatonin promotes degradation of AGEs invitro 91). In abnormal glucose metabolism such as diabetes,
ER stress in pancreatic islets β cells is exacerbated andinsulin secretion is lowered 92). AGEs increase ER stress in β
cells and decrease the production and secretion of insulin 93).
When AGEs are decreased via degradation, ER stress decreases.
Consequently, it is expected that the lowered insulin secretion
ability is recovered.
Third, melatonin has activities mediated by hormones.
Melatonin reduces the secretion of glucocorticoid such as
cortisol from the adrenal cortex. Glucocorticoid promotes theacceleration of protein catabolism and gluconeogenesis, andat the same time, accelerates insulin resistance. This causes
high blood glucose. Cortisol holds 95% of glucocorticoid
activities and has a direct action for lipid metabolism.
Cortisol, which is secreted due to acute stress, has an activity
for lipolysis and promotes mitochondria usage of glucose,lipids, and amino acid. However, in a chronic elevated stateof cortisol, the accumulation of lipids is caused with the
inhibition of peroxisome proliferator-activated receptor
γ (PPARγ) in white lipids 94).
Orexin, which is a type of brain hormone, has a sleep
arousal effect. Orexin is in low values during sleep at night
and is secreted from dawn to daytime (orexin amplitude).
Orexin secretion is affected by blood glucose. Low blood
glucose promotes secretion, and high blood glucose inhibits
secretion 95 -97). Orexin amplitude is decreased due to aging.
Orexin and blood glucose alternation affect interactively.

Therefore, it is important to maintain the orexin amplitudewith the purpose of a healthy alternation of blood glucose.
Melatonin lowers orexin secretion which is elevated during
night and regains the amplitude of orexin.
Fourth, melatonin plays a role in the improvement oflipid metabolism in vivo. In an experiment with animals
with hyperlipidemia, melatonin administration decreased
triglyceride (TG) and low-density lipoprotein cholesterol(LDL-C). An experiment with normal rats indicated thathigh-density lipoprotein cholesterol (HDL-C) was increased
98 -100). Melatonin administration improves insulin resistanceand some are mediated by insulin activity. Rats whose pineal
body was removed suggested that melatonin secretion wasdiminished and abnormal glucose metabolism was exhibited101). It is confirmed that melatonin plays an important role
to maintain homeostasis of glucose metabolism. When
melatonin is ingested before bedtime, postprandial blood
glucose level after breakfast is subdued the next morning 76).
Four mechanism paths of melatonin are mentioned
above. Some are mediated by insulin activities and others
directly act to glucose metabolism.
Activities toward pancreatic β cells are complicatedin vivo. In a state of hyperinsulinemia such as prediabetes
and early-stage type Ⅱ diabetes, melatonin decreases insulin
while fasting 102). However, insulin activities are intensifiedin skeletal muscles, and intake of glucose is activated 103, 104).
It can be considered that insulin resistance decreases, where
the homeostasis model assessment of insulin resistance_ 152 _Glycative Stress Research(HOMA-IR) decreases. In a state of intense oxidative stressand glycative stress, the formation of AGEs increases, ER
stress in β cells increases, and the formation and secretion of
insulin decreases. In these conditions, melatonin, which has
anti-oxidation and anti-glycation activities (to promote thedegradation of AGEs), expectedly functions to complement
insulin secretion in pancreatic β cells. The deteriorationof insulin secretion is a risk factor for onset of type Ⅱdiabetes 105). Melatonin is facilitative in the hypertrophy andactivation of brown adipose tissue in vivo 106). It is consideredthat melatonin is involved in not only glucose metabolism but
also energy metabolism.
Impairment of “quality of sleep” and circadian rhythm
disorders increase the risk of onset of AD 107). Melatonin,
which is secreted during the night, has activities of antioxidation 108) and anti-glycation 90) (to promote the degradationof AGEs). Melatonin plays a role protecting the brain during
sleep from oxidation and glycative stress. Furthermore,melatonin has in vitro activities to inhibit the formation ofAβ 109), to inhibit the aggregation of Aβ 110, 111), to decreaseneurotoxicity 112, 113), to improve Aβ clearance 114), and in vivo
activities to maintain memory 115, 116), as is reported. Melatonin,by regulating the secretase expression control network,
inhibits APP processing and Aβ formation 109). From theseperspectives, melatonin attracts attention as a factor that links“quality of sleep” to AD 117). Therefore, educational programs
for lifestyle regarding “quality of sleep” must be included in
the list of preventive measures of AD, improving “quality ofsleep” with increased melatonin secretion.

4. 4. Physical exercise: impacts of glycative stress on
skeletal muscles and exercise-resistancePhysical exercise for type Ⅱ diabetes is recognized toplay a role of improvements in insulin resistance, glucose
tolerance, and lipid metabolism.
When insulin receptors on the surface of myocyte bindwith insulin, glucose transporter 4 (GLUT-4) in cytoplasm
transfer to membranes (translocation), and glucose is taken
into cells via GLUT- 4. When skeletal muscle motions (slidingof myosin and actin) occur, gene expression of GLUT-4
increases and intracellular GLUT- 4 in cells increases 118).
At the time of insulin stimulation, the number of GLUT4 on membranes increases. This raises the rate of glucose
assimilation, which is a mechanism of the improvement of
insulin resistance through exercise. As it is an exceedingly
effective as a glycative stress countermeasure, this isemployed as a part of therapeutic plans for type Ⅱ diabetes.
Approximately 70 % of glucose is uptaken into skeletal
muscles and is utilized. However, muscle mass of persons
who live a normal daily life is deprived by one percent per
year. A pathological condition is designated as sarcopenia.
However, there is a case that is designated as dynapenia,
where the loss of muscle strength is not caused by muscle
mass decrease. Some patients with diabetes maintain theirskeletal muscle mass but lose muscle strength.119 -121). It isconsidered that muscle proteins (myosin and actin) undergo
glycative modification and then, muscle contractile function
is impaired. There is an increasing interest of glycative stressas a causative factor of the deterioration of skeleton muscularfunctions 122).
The first report was that increased blood CML level

was a risk factor of the deterioration of muscle strength andwalking ability 123-125). Ensuing epidemiological studies on
middle-aged and older persons have suggested that there isa correlation between the increase of blood AGEs and SAF
and the decrease of muscle strength and motor function. It
is indicated that younger persons with high SAF level havedecreased muscle strength and physical endurance. Thesefindings suggest that impacts of glycative stress on motor
functions and skeleton muscle functions occur in spite of age
122, 126, 127).
Primary factor of AGE-accumulation-related
deterioration of decreased motor functions and skeletonmuscle functions is glycative modification of muscle contractile
proteins such as myosin, actin, and tropomyosin decreasing
muscle contractile functions 128-131). Further, involvement
is suggested regarding the structural alternations of
extracellular matrix, the decrease of ATPase activity 129, 132),
and the decrease of motor nerve transmission function 133).
In an experiment, where mice were fed a diet containing
a large quantity of AGEs for sixteen weeks, which is fivetimes-volume, the following is recognized: the decreaseof expressions in myogenic factor 5 (Myf5) and myogenic
differentiation 1 ( MyoD), which plays a role to promote theformation of muscular tissue, and the decrease of signaltransduction of insulin-like growth factor 1 (IGF-1), whichplays a role to promote the formation of protein, thus eventuallycausing the decrease of muscle mass 134, 135). An experimenton mice fed with MGO for twenty weeks suggested thatalong with muscle mass decrease, the gene expressions ofinflammatory cytokines, i.e., IL-1β, IL-6, were confirmed.
A possibility was suggested that glycative stress induces
protein catabolism via inflammatory signals 136).
There are differences among individuals regarding
health promotion effects. Patients with diabetes and older
adults are individually different. It is reported that one outof five people do not gain sufficient exercise benefits 137-140).
Glycated muscle proteins, which are modified due toglycative stress, result in the accumulation of AGEs. Thisdecreases skeletal muscle contractile functions and at thesame time, muscular tissue formation. Consequently, “exerciseresistance” is induced. This state means that benefits ofexercise are unlikely to be gained 122). Therefore, it is essential
to start an appropriate exercise habit before exercise
resistance is established.

5. Conclusion
Glycative stress comprehensively affects the body, fromhead to toe, diverse tissues and organs. Maintenance of thehuman body and prevention of aging-related diseases are
significantly related to glycative stress. Lifestyle habits areimportant for practical measures against glycative stress. We
can uncover a clue from lifestyle habits, in dietary education(nutrition, functional food and chrono-nutrition), physicaleducation (loaded muscle exercise and aerobic exercise)and intellectual education (sleep and stress management).
The point is that untreated long-term glycative stress leads
to a vicious cycle of progression of aging-related diseases.
Exercise-resistance is induced due to impacts on skeletonmuscles. Thus, it is difficult in gaining benefits of exercise.

_ 153 _Glycative Stress: Molecular Impacts and Defense Mechanisms

As an example of AD, it is difficult to treat advanced AD andto restore health. Thus, preventive measures are necessary.
To prevent lipid peroxide of the brain due to oxidation stressand similarly, to prevent Aβ glycation from the perspectiveof countermeasures against glycative stress, we should pay
attention to diverse countermeasures. In addition to physicalexercise, aldehyde control, nutrition therapies, improvementof “sleep quality”, and understanding of melatonin are
required. We hope that further extensive research on antiglycation will be undertaken, and deeper understanding
and increased participation will be encouraged. Studies
on glycative stress are required to lead to a social
implementation as a practical science.

Funding

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The present study was supported by Ministry of
Education, Culture, Sports, Science and Technology with
Grants-in-Aid for Scientific Research. (JSPS KAKENHI# 26350917, #17K01880, 20K11593)

Conflicts of Interest
The authors declare that they have no conflict ofinterest.

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