Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : April 18, 2024
Accepted : June 14, 2024
Published online : June 30, 2024
doi:10.24659/gsr.11. 2_79

Original article

From fatty liver to steatohepatitis: Involvement of aldehydes
Yoshikazu Yonei 1), Yoshimasa Saito 2), Masayuki Yagi 1), Maru Ogura 3), Kenji Sato 4)
1) Anti-Aging Medical Research Center/ Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan
2) Division of Pharmacotherapeutics, Keio University Faculty of Pharmacy, Tokyo, Japan3) Kyoto Bunkyo Junior College, Kyoto, Japan
4) Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Abstract

Over the past 80 years, we have been exposed to increased caloric intake associated with higher fat intake, concurrently withless physical activity. These lifestyle changes result in a marked increase in diseases associated with glycative stress, thecondition characterized by excess aldehyde generation, such as obesity, type 2 diabetes, dyslipidemia, metabolic syndrome,and fatty liver/steatohepatitis. We describe our hypothesis as to how aldehydes are involved in the process of progression
from fatty liver to steatohepatitis. Glycative stress is defined as an excess of aldehydes, and oxidative stress as an excess
of free radicals and ROS (reactive oxygen species). In the body, carbohydrate- and fatty acid-derived aldehydes, which areproduced in excess due to hyperglycemia and a high-fat diet, respectively, and modify proteins, lipids, and DNA by glycation.
In our protection system, these aldehydes are, consuming NAD + (nicotinamide adenine dinucleotide) and GSH (glutathione),
catalyzed by GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ALDH (aldehyde dehydrogenase), and GLO (glyoxalase).
Concurrently, oxidative stress is intensified by a decrease in GSH, and fatty acid-derived aldehydes are mostly producedby the oxidation of fatty acids. Therefore, as fatty liver progresses, the production of fatty acid-derived aldehydes increases
significantly. An increase in the NADH / NAD + ratio in hepatocytes leads to a decrease in lipid β-oxidation and theprogression of fat accumulation. DNA modification modifies the promoter region of the low-density lipoprotein receptor
related protein 1 (LRP1) gene, and decreased expression of LRP1 leads to abnormalities in lipid metabolism in hepatocyte.
Once AGEs, products of protein glycation, stimulate RAGE in macrophages and Kupffer cells to increase the secretion of
inflammatory cytokines, inducing inflammation. Glycation of procollagen stimulates Ito cells (hepatic stellate cells), anddecreased GSH stimulates fibroblasts to increase collagen production, thus inducing fibrosis. When NAD + is insufficient, theTCA cycle malfunctions and fumaric acid increases, which results in GAPDH being modified by succinylation and converted
to S-(2-succinyl) cysteine-GAPDH, reducing activity and increasing unprocessed aldehydes, creating a vicious cycle. From
the above, we deduce that the turning point from fatty liver to steatohepatitis is NAD + deficiency caused by excess aldehydes,
which in turn causes damage to the TCA cycle. We believe that measures against glycative stress (excess aldehydes) are
important for preventing steatohepatitis. The involvement of LRP1 remains to be determined.

KEY WORDS: glyceraldehyde, methylglyoxal, fumaric acid, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH),		

aldehyde dehydrogenase (ALDH), glyoxalase, nicotinamide adenine dinucleotide (NAD +), glutathione

Introduction
We, humans, have faced struggles with oxidative stressin the process of evolution for several hundred thousand
years, establishing defense systems against oxidative stress.
This system is particularly elaborate in comparison withother animals. In contrast, it was only several decades

ago when we started to fight glycative stress. Our society
has been changing and we have problems with decreased
physical activity due to developments in traffic systems andhousehold electronic appliances, as well as excessive eating,especially of high fat foods. This led to remarkably increased

Contact Address: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center, Graduate School of Life andMedical Sciences, Doshisha University
1 - 3, Tatara Miyakodani, Kyotanabe, Kyoto, 610- 0394 Japan
TEL&Fax: +81-774- 65- 6394 e-mail: yyonei@mail.doshisha.ac. jp
Co-authors: Saito Y, ysaito@keio. jp; Yagi M, yagi@yonei-labo.com;
Ogura M, m-ogura@po. kbu.ac.jp; Sato K, sato.kenji.7x@kyoto-u.ac.jp

Glycative Stress Research 2024; 11 (2): 79-93(c) Society for Glycative Stress Research_ 79 _

From Fatty Liver to Steatohepatitis: Involvement of Aldehydes

glycative-stress, which is deeply related to diseases, suchas obesity, metabolic syndrome, type 2 diabetes andhyperlipemia. This problem is a worldwide trend. Fatty liver,the topic of the present study, increases not only in morbidityand but also in severity. Cases of steatohepatitis are observed
as the result of the combination in inflammation and fibrosis.
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholicsteatohepatitis (NASH) develop into cirrhosis with the rateof 10 % to 29 % within 10 years and 4 % to 27% of patients
have hepatic cancer 1, 2). Risk factors of fatty liver (obesity,
hyperlipemia, hyperglycemia and smoking) have a possibilityof excessively generated aldehyde involvement. We examine
what occurs in the development process from fatty liverdiseases to steatohepatitis and infer its mechanisms fromthe perspectives of glycative stress. Medical treatments forsteatohepatitis are challenging and complicated comparedwith simple fatty liver. The present attempts would be asignificant step toward the establishment of treatments forsteatohepatitis

1. Differences between oxidative stress andglycative stress

To begin, we reexamine oxidative stress and glycative
stress from the viewpoints of field and classification.
When oxidation-reduction reaction is simply defined aselectron exchange, the boundary between the two types ofstress is confusing. Oxidative stress and glycative stresscan be accurately distinguished with simple definitions
to be understood. Glycative stress refers to the state ofexcessive aldehyde formations in the body. Aldehydes arehighly reactive and react with serum proteins in blood andmembranes of vascular endothelial cells. Aldehydes areconstantly produced and eliminated, and are detected in the
blood in equilibrium. Furthermore, aldehydes easily reactwith protein and lipids in cells through cell membranes.
Moreover, they react with cytosine (DNA constituent base)through nuclear membranes. Products of aldehyde-protein
reactions are designated as advanced glycation endproducts(AGEs) including protein carbonyl 3, 4). Amadori-glycatedphosphatidylethanolamine (Amadori-PE) is a glycation
product of lipids 5).
Oxidative stress refers to the state of excessive free
radical and reactive oxygen species (ROS). Antioxidants
such as Vitamin C and E reduce oxidation reactions. These
antioxidants, however, do not suppress glycation reaction
where aldehydes and biomolecules bond.
The leading role is played by aldehydes in glycative
stress reactions. Aldehydes are mainly classified into
carbohydrate-derived and lipid-derived aldehydes basedon formation paths ( Fig. 1 ) 6, 7). The former is a short chain
aldehyde due to “simultaneous formations of diversified
aldehyde in a chain reaction manner, ” which follow blood
glucose spikes as postprandial high blood glucose 8). We
designate this phenomenon as an “aldehyde spark” 4). Partsof reducing sugars ( i.e., glucose, fructose) become a ringopening aldehyde, where aldehyde groups are exposed.
Consequently, they react with substances around them andinduce injury to vascular endothelial cells. Ring-opening
glucose (the number of carbons: 6 ) are stimulated by otheraldehydes, oxidative stress, heat and ultraviolet radiation,

which induce further formations of short- chain aldehydes(the number of carbons: 2 and 3). Among them, AGEs, which
are produced from glyceric aldehyde (GA), are the most
toxic. This is designated as toxic AGE (TAGE) 9, 10). The
latter, lipid-derived aldehydes, are produced via oxidationof fatty acid. Among them, methylglyoxal (MGO), which isan aldehyde produced from carbohydrates and fatty acids, isrelated to the onset of multiple diseases in the body.
During the demethylation process of epigenetic changes,
formaldehyde is produced from the released methyl groups
11-14). Therefore, formaldehyde levels of 0.05 ~ 0.1 mM arephysiologically detected in the blood of healthy individuals.
It is said that organic mercury (methylated mercury), thecause of Minamata disease, is demethylated in the liver toproduce formaldehyde 15). Naturally, both DNA and histone
proteins are modified by aldehydes.
As measuring fatty-acid-derived short-chain aldehydes
in blood had been difficult, it has been difficult to attract
attention to this aldehyde 6). However, fatty-acid-derived
aldehydes are deeply related to the onset and aggravation of
diseases in brain tissues and bones, particularly bone marrow
where lipids are abundant. Fatty-acid-derived aldehydes areproduced in the body with oxidative stress condition andexert diverse biological effects. It has been regarded that
4-hydroxynonenal (4HNE) and malondialdehyde (MDA)
are causative factors of alcoholic hepatopathy 16, 17). Normal
hepatic parenchymal cells have a small distribution as fat.
Fatty liver, however, has a risk of increasing fatty-acid-derived
aldehydes. Experiments with NASH model mice, where
eicosapentaenoic acid was administered (EPA) to the control
group, an omega-3 unsaturated fatty acid suggested thedepletion of glutathione in liver tissues and the exacerbationof fibrosis in the liver 18). This phenomenon is caused by
the excessive formation of fatty-acid-derived aldehydes. It
was suggested that a large amount of GSH, a cofactor, wasconsumed due to the metabolization of glyoxalase (GLO) 19).
The research of glycative stress started with theexaminations of Maillard reaction in food. The initial
reaction in glycation is a carbonylation reaction, where an
exposed aldehyde group in a ring-opening glucose (-CHO)
and an amino group of protein/peptide (-NH 2) are reacted,
and consequently, Schiff base is formed (a chemical formula
is supposed but this compound does not exist as is transiently
formed). The present study designates reactions of aldehydegroups and amino groups as carbonylation and all thesequential reaction as glycative reaction.
Short-chain fatty acids (acetic acid, butyric acid andpropionic acid), which are produced by mutualistic bacteria
of intestinal flora, contribute to the prevention andprogression of fatty liver 20-23). Short-chain fatty acids havean inhibitory activity for the AGEs formation in vitro (theinhibition of protein carbonylation) 23) and a promotive
activity for base metabolism via specific receptors in vitro 24).
However, oral administrations of short-chain fatty acids
have no effects on receptors because they are subsequently
digested and absorbed 25).
Another type of aldehyde is derived from food anddrinking items for pleasure without the purpose of nutrition.
One is alcohol-derived aldehyde. The other is cigarettesmoking-derived aldehyde ( Table. 1 ). General perception
shows that smoking is related to oxidative stress. However,_ 80 _Glycative Stress Research

the smoke of cigarettes contains multiple aldehydes. The skinof smokers has a high level of skin autofluorescence values( Fig. 2 ) 26). Mortality rate is twice as high in smokers thannon-smokers in the USA. Similarly, smoking increases themortality rate of fatty liver twice as much 27). Moreover, liver

filament formations are remarkably aggravated by smoking
and Fibro Scan ® shows progression of liver sclerosis 28), which
elevates incidence rates of malignant tumors 29). Therefore,
people with drinking and/or smoking habits have more
intense glycative stress.

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Fig. 1. The main characters are aldehyde. Where do they come from?

Aldehydes produced in the body are mainly derived from carbohydrates and fatty acids. Trace amounts of formaldehyde are producedwith the demethylation of DNA and histone proteins. Exogenous aldehydes come from drinking alcohol and smoking. HFD, high fat diet;TG, triglyceride; LDL, low-density lipoprotein; 3DG, 3-deoxyglucosone; AGEs, advanced glycation endproducts,

a)

b)
3.5

Smoking Group
Non-Smoking Group

*

2.5

†

Smoking

SAF**
Nonsmoking

1.5
0.0

0.5

1.0

2.0

3.0

SA F
Teens

20s

30 s

40 s

(Age: 20 ~ 59 years old)

50 s

Age Group

Fig. 2. Aging trends in SAF and the impacts of smoking.

The results are expressed as mean ± standard deviation, † p < 0.1, *p < 0.05, **p < 0.01m Mann-Whitney’s U-test. a) n = 244,b) Subject’s ages between 10 to 59. Smoking group, n = 33; Non-smoking group, n = 177. SAF, skin autofluorescence measured
by AGE Reader (DiagnOptics, The Netherlands). The figures are created based on reference 26).

_ 81 _

From Fatty Liver to Steatohepatitis: Involvement of Aldehydes

Table 1. Toxic substances contained in cigarette smoke.
acetaldehydechromiumlead

N-nitrosomethylethylamine

acetamide

chrysene

MeA-a-C(2-amino-3-methyl-9H- pyridoindole)

N-nitrosomorpholine (NMOR)

acetonecobaltMercury

N-nitrosonornicotine ( NNN)

acroleincoumarinmethyl ethyl ketone

N-nitrosopiperidine ( NPIP)

acrylamide

cresol5- methyl chrysene

N-nitrosopyrrolidine (NPYR)

acrylonitrile

croton aldehyde

4 - (methylnitrosamino) -1(3-pyridyl) -1-butanone (NNK)N- nitrososarcosine ( NSAR)

aflatoxin B

cyclopentapyrene

naphthalene

nornicotine

3-aminobiphenyl

dibenzanthracene

nickelphenolammonia

dibenzopyrene

nicotine

PhIP (2-amino-1-methyl-6phenylimidazo pyridine)anabasine

2,6 - dimethylaniline

nitric oxide ( NOx)polonium 210

o‐anisidine

ethyl carbamate (urethane)

nitrobenzene

propionaldehydearsenicum

ethylbenzene

nitrogen monoxid

propylene oxide

A- α - C

ethylene oxide

nitromethane

pyridinebenzaldehyde

formaldehyde

2-nitropropa vinyl acetate

quinoline

benzanthracenefuran

N-nitrosoanatabine(NAB)

resorcinolbenzeneGlu-P-1(2-amino-6-methyl pyrrolidone)

N- nitrosoanatabine( NAT)

selenium

benzofuran

Glu-P-2(2-aminodipyrrolid )N- nitrosodiethanolamine( NDELA)styrenebenzopyrene

glycidaldehyde

N-nitrosodiethylamine 2

o-toluidine

beryllium

glycidolN-nitrosodimethylamine( NDMA)

toluene1, 3- butadiene

hydrazine

N-nitrosomethylethylamine

Trp-P-1 (3-amino-1,4dimethyl -5H- pyridoindole)

butylaldehydehydrogen cyanide

N- nitrosomorpholine( NMOR)

Trp-P-2 (1-methyl-3amino-5H- pyridoindole)

cadmiumhydroquinone 3

N-nitrosonornicotine( NNN)

uranium 235

carbon monoxide

indenopyrene

N- nitrosopiperidine( NPIP)uranium 238

catechol

IQ(2-amino-3-methylimidazole)N-nitrosopyrrolidine( NPYR)

vinyl acetate

chlorinated dioxin /furan

isoprene

N-nitrososarcosine( NSAR)

vinyl chloride

Red letters indicate aldehydes. Not only carcinogens and oxidation-inducing substances, but also diverse aldehydes are contained.

_ 82 _Glycative Stress Research

The human body has anti-glycation systems. (Fig. 3 )Among them, the liver plays the vital role. In hepatocyte,
defense systems for aldehydes exist abundantly, such as
glyceraldehyde - 3 -phosphate dehydrogenase (GAPDH),
glyoxalase, (GLO), and aldehyde dehydrogenase (ALDH)(Fig. 4 ).

・ GAPDHGAPDH accounts for approximately 20 % for protein
in hepatocyte and is the most highly distributed enzyme
in the body. GADPH plays the key role in glycative stressdefense 30, 31). This exists as a tetramer in cells and serves as aglycolytic enzyme. GAPDH is related to the GA metabolism.
Glyceraldehyde (GA), which is moved into or formed inhepatocyte, is intensively phosphorylated by triokinase 32, 33),

EnzymesMaterials

Organs/Cells /Organelles【Aldehyde metabolism】
GAPDH
ALDH
Glyoxalase I &II【Aldehyde trapping】
amino acids【AGE breaking】
OPH【AGE generation control】
short chain fatty acids

2. Liver as a defense system against glycative stress【Soluble RAGE】【AGE breaking】
melatonin

Fig. 3. Anti-glycation system in human.

【Phagocytosis】
fibroblasts・glial cells
reticuloendothelial cells
macrophage・monocyte【Organelles】
RAGE
scavenger receptors for AGEs
lysosome・proteasome【Excretion organs】
liver ( hepatocyte)
kidney (podocyte)

Glycation products are processed through various cells/intracellular organelles, metabolized and subsequently eliminated by the liverand kidneys. Hepatocytes are rich in aldehyde metabolizing enzymes, i.e., GAPDH, ALDH, and GLO, while the AGEs metabolizing
enzyme OPH is expressed in skin and various tissues in addition to the liver. Various materials are involved in the anti-glycation system.
Representative endogenous substances are shown in parentheses. Amino acids trap aldehyde, short-chain fatty acids derived from intestinalbacteria inhibit AGEs formation, and melatonin promotes AGE cleaving as well as its antioxidant effect. GAPDH, glyceraldehyde-3phosphate dehydrogenase; ALDH, Aldehyde dehydrogenase; GLO, glyoxalase; OPH, oxidized protein hydrolase; AGEs, advanced glycation
endproducts; RAGE, receptor for AGEs.

Fig. 4. Glucose metabolic pathway and involvement of aldehydes in hepatocytes.

In hepatocytes, aldehyde excess activates GAPDH, ALDH2, and glyoalase, while glucose excess activates the polyol pathway. Whenaldehydes enter the cell, they are promptly phosphorylated and retained inside the cell to be metabolized by enzymes. Much NAD + and
GSH are consumed as compensation for enzymatic activity. The influence extends to mitochondrial function. GLUT2, glucose transporter
2; AL, aldehydes; GA, glyceraldehyde; MGO, methylglyoxal; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ALDH, aldehyde
dehydrogenase, SDH, sorbitol dehydrogenase; LDH, lactate dehydrogenase; G6P, glucose-6-phosphate; F1P, fructose 1-phosphate; F6P,
fructose 6-phosphate; G3P, glyceraldehyde-3-phosphate; 3PG, 3-phosphoglycerate.

_ 83 _

From Fatty Liver to Steatohepatitis: Involvement of Aldehydes

and converted into GAP, which is membrane-impermeable.
GAP is metabolized by GAPDH with the assistance of NAD+and is converted into 3-phosphoglycerate (3PG) ( Fig. 5 ). It
seems like there is a mission in the hepatocyte to metabolize
all GA and protect our body from GA. Although GAPDH is
located in the glycolytic pathway, it is not a rate determining
enzyme because of its large amount. It is suggested that theprotection system is ready for emergent risks of the mass
production of GA.
GAPDH is a protein with moonlighting functions.
GAPDH, which has a high affinity to DNA, has a functionof DNA repair enzyme, as a monomer of uracil-DNA
glycosylase 34, 35). When intensive glycative stress induces theexcessive formation of intracellular aldehydes, abnormal
proteins are increasingly produced from intracellular protein
due to aldehyde modification and consequently, increased
endoplasmic reticulum ( ER) stress induces insufficient
processing activities of proteasome. This would be “ the statein the direction to cell death.” In this condition, GAPDH
changes its behavior from the role of guardian to thepromoter of apoptosis 36). GAPDH moves into mitochondria,
and induces the acceleration of membrane permeability, theloss of transmembrane potential and the release of apoptosis
inducer 37, 38). This is regarded as a trigger of apoptosis 30).
When GAPDH forms monomers and dimers via nuclear
translocation, it serves to function as a protein promoting
apoptosis 39- 41). GAPDH in cells binds with integral
membrane proteins, endoplasmic reticulum Ca 2 + pump.
Consequently, Ca 2 + intake of cytoplasm increases 42, 43).
Finally, cells result in apoptosis.
・Aldehyde dehydrogenase (ALDH)
The ALDH super family, which has 19 types of
isozymes, plays a significant role for sustaining biological

functions 44, 45). Among them, ALDH2 is a detoxifying
enzyme with a high affinity for acetaldehyde produced byalcohol drinking 46). This enzyme expresses in the liver andthe kidney as well as mitochondrial matrix in cells. ALDH
degrades multiple types of aldehydes including acetaldehyde(Fig. 5 ) 47-50). Glyoxal, methylglyoxal and others of aldehydes
are also substrates of ALDH2 51). It has been recognized that
ALDH2 metabolizes fatty-acid-derived aldehydes such as
4HNE and MDA 52- 54). NAD +, a cofactor, is required for
ALDH to exert enzyme reactions. Therefore, in the conditionof intense glycative stress, that is, the state of excessive
formation of aldehydes, a multitude of NAD + are consumed.
Excessive aldehyde formation induces the shortage ordepletion of NAD + (nicotinamide adenine dinucleotide)in the process of metabolization by GAPDH and ALDH,
as NAD + is necessary for metabolization. Individuals who
drink alcohol (excessive ethanol consumption) should be
careful because NAD + is also consumed when ethanol isconverted to acetaldehyde by alcohol dehydrogenase.
When superfluous glucose increases in cells, the polyol
pathway is activated. This is an alternative pathway for extra
glucose which the glycolytic pathway does not manage toprocess, where extra glucose is converted into fructose notusing a glycolytic pathway. Glucose is converted into sorbitol
via aldose reductase and subsequently, sorbitol is converted
into fructose via sorbitol dehydrogenase (SDH). However,SDH catalyzes reactions by consuming NAD +. This assists
leading to shortage or depletion of NAD +. Some partsof fructose are converted into GA and the amount of GA
increases. This leads to an increase in aldehyde generation.
Both events cause vicious cycles.
TCA cycle of mitochondria requires NAD + at threepoints through reactions. Insufficient NAD + hinders asteady cycle and reduces the formation of ATP ( Fig. 6 ).

ALDH
Acetaldehyde

Acetic acid

Glycolaldehyde

Glycolic acid

Methylglyoxal

Pyruvic acid

Glyceraldehyde

Glyceric acid

Phosphorylation

NAD +NAD H + H +GAPDHGlyceraldehyde-3-phosphate

1, 3-bisphosphoglyceric acidNAD +NAD H + H +Fig. 5. Aldehyde metabolizing enzyme that utilizes the cofactor NAD +.

NAD + is consumed as a cofactor, glycative stress with aldehyde excess condition leads to NAD + deficiency. NAD +,nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced form).

_ 84 _Glycative Stress Research

Fig. 6. TCA cycle.

Under the condition with NAD + deficiency, the circuit does not run smoothly and ATP production decreases. Subsequently, fumaricacid increases. When fumaric acid binds to GAPDH (succinylation), its activity is attenuated. AcetylCoA. acetyl coenzyme A; NAD +,nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced form); FAD +, flavin adenine dinucleotideadenine
dinucleotide; FADH2, flavin adenine dinucleotideadenine dinucleotide (reduced form); ATP, adenosine triphosphate; ADP, adenosine
diphosphate; GTP, guanosine triphosphate; GDP, guanosine diphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ALDH,
aldehyde dehydrogenase; SDH, sorbitol dehydrogenase; LDH ( H ), lactate dehydrogenase ( H type); 2(SC), S- (2-succinyl)cysteine; GA,
glyceraldehyde.

Dysfunctions in the TCA cycle reduce the formation of
NADH (nicotinamide adenine dinucleotide, reduced form)and FADH 2 (flavin adenine dinucleotideadenine dinucleotide,
reduced form), which results in the decrease of supply to theelectron transfer systems. Therefore, the formation of ATP
decreases in the electron transfer system. As a result, ashortage of energy production is caused in mitochondria anddiverse functions decrease in hepatocyte.
・ Glyoxalase (GLO)
There are two types of GLO, glyoxalase I (GLO1)
and glyoxalase II (GLO2), where the GLO system iscomposed 55). They express in multiple cells such as theliver and the kidney. Methylglyoxal (MGO) is converted
into Sd-lactoylglutathione by GLO1, which is bound by
glutathione (GSH) through a catalytic role like a cofactor.
Furthermore, it is hydrolyzed to D-lactic acid via GLO2.
Finally, D-lactic acid is metabolized to pyruvic acid. As
MGO is formed both in carbohydrates and fatty-acid-derived
manners, MGO is associated with diverse glycative-stressrelated diseases in the onset and progression. Therefore, thisenzyme which metabolizes MGO plays a significant role indefense functions for glycative stress.
The increased MGO production and the excessive
burden on GLO lead to the state of deficient GSH. Thisfinding is frequently observed in progressed fatty livers
and steatohepatitis. On the contrary, the depletion of GSH
induces severe liver disorders. For example, acetaminophen(APAP) overdose induces a liver dysfunction 56) caused by thedepletion of GSH which inhibits the activities of GLO, andconsequently, MGO markedly increases.
・ Oxidized protein hydrolase (OPH)
OPH is a type of serine protease and is the same as
acylpeptide hydrolase (ACPH) with free protein N-terminal
acylation amino acid 57). Furthermore, it is designated as an
acylamino-acid-releasing enzyme (AARE) 58). OPH, which
exists in diversified tissues such as human red blood cells,
livers, kidneys, brains and hearts, degrades above-mentioned
oxidized proteins and glycated proteins on a preferential
basis 59).

_ 85 _

From Fatty Liver to Steatohepatitis: Involvement of Aldehydes

3. Epigenetic changes and glycative stress
Epigenetic changes refer to a chemical modification of
DNA with a purpose of turning gene control on/off. In a caseof controlled physiological modification, a base sequence
does not alter and is remembered as cell characteristics.
The base sequence is continued as genetic inherence.
Physiological DNA methylation, via methyltransferase (DNAmethyltransferase), has the addition of a carbon to the methylgroup, the fifth position of cytosine base with CpG sequence.
Consequently, it is converted into 5-methylcytosine. As aresult, gene expressions are affected and the inhibition of
expression could occur. Via demethylating enzymes, themethyl group is released and it returns to cytosine. Then,gene expression returns to the original state.
Methylation age, a measurement system of biologicalage, is an attempt where aging degrees of cells are assessed
based on assessments of methylation degrees of DNA.
Overview of the measurement method is as follows: first, thebisulfite processing is conducted. Unmethylated cytosine in
DNA is converted to uracil 60, 61). Contrarily, cytosine which is
not methylated and not modified in other manners remains
as cytosine without any conversions. Therefore, after bisulfite
processing, types of DNA with different base sequences
are expressed. When analyzing differences with the use of
a DNA sequencer, where and how frequently methylations
have occurred were detected. However, this method of
methylation age does not provide accurate examinationsof pure methylated cytosine. Other than methylation,
multiple modifications are included, such as dimethylation,
trimethylation, carbonylation and alkylation.
Methylation age measurements have provided multiple
findings. Along with aging, methylation age is aging. It has
been suggested that methylation age is accelerated in thestate of intensive glycative stress, including the presence ofdiabetes 62, 63). It is notable that repeated spikes of glucose
and hyperlipidemia, inducing excessive aldehyde generation,
promote DNA methylation 64). The reason is that the presenceof excessive aldehydes induces cytosine carbonylation in a
non-physiological aspect. Glycative stress definitely affects
epigenetic changes.

4. Process from fatty liver to steatohepatitis
Healthy livers, which have glycogen granules and lipid
droplets in the cytoplasm, play the role of energy storage.
Lipid droplets are organelles, which exist in not only theliver and adipose cells but also cells throughout the body.
Lipid droplets store lipids and toxic lipids, having functions
to protect the body from intracellular lipotoxicity 65). Healthy
persons have a limited amount of lipid droplets in cytoplasm.
However, lipid droplets are observed in intranuclear areasof patients with steatohepatitis. Its causative factors areunidentified. We speculate that lipid droplets are rich in
Amadori-PE in the steatohepatitis.
Simple fatty liver refers to the state of stored(macrovesicular) lipid droplets in hepatocyte. Lipid
droplets consist of a phospholipid monolayer (membrane ofendoplasmic reticulum) wrapping nucleus of triacylglyceroland cholesterol ester 66). Simple fatty livers carry a benign
prognosis. Even in a case of considerable lipid storage,

cirrhosis and liver cancer do not typically develop.
Steatohepatitis refers to a pathological condition, where
cytopathy, necrosis, inflammation and fibrosis are added
to fatty liver ( Fig. 7 ). Alcohol drinking (ethanol ingestion)induces NAD + consumption, due to ethanol/acetaldehyde
metabolization via alcohol dehydrogenase and sequentially,
ALDH. This leads to the formation of reduced form
NADH. Excessive NADH stimulates fatty acid synthesis in
hepatocyte. An excess of NADH (an increase in the NADH/
NAD + ratio) stimulates fatty acid synthesis in hepatocyte
and reduces fatty acid β-oxidation, resulting in fat deposition
and the progression of fatty liver 67). NAD + functions as a
cofactor for multiple enzymatic reactions, many of which
are significant reactions. Therefore, the disturbance ofequilibrium caused by glycative stress (excessive aldehydes)
could be a boundary to distinguish traveling directions
between remaining as fatty livers and progressing tosteatohepatitis. Steatohepatitis is developed when infiltrationof inflammatory cells, mainly neutrophil, macrophages andKupffer cells, as well as inflammatory changes are added tofatty liver in this condition. The progression of steatohepatitis
leads to liver cirrhosis due to the aggravation of fibrosis. Thismay be a pathogenesis of cancers in the liver. At the timeof diagnosis, the range of steatohepatitis is extensive from acase with only little fibrosis to hepatic cirrhosis. How fibrosis
developments progress decides the resulting prognosis. Thereare cases of fatty liver/inflammatory liver cirrhosis where
only a slight lipid droplet remains or lipid droplet disappears
in hepatocyte.
・ Inflammation
Neutrophil is dominant in inflammatory cells observedin steatohepatitis. In addition, macrophage and Kupffer cells
are recognized. RAGE, which expresses in these cells, binds
with AGEs and then, inflammatory cytokines (i.e., TNFα[tumor necrosis factor α], IL6 [interleukin-6]) are secreted.
Thus, inflammation is induced. Consequently, cellular
injuries are induced.
It was experimentally suggested by Leung C et al.
that glycative stress aggravates steatohepatitis 68, 69). The
experiments, where rats were fed a methionine/choline
deficient diet for six weeks and then, the methionine choline
deficient (MCD) diet containing high AGEs for anothersix weeks, suggested that steatohepatitis progressed 68). In
comparison with an MCD diet alone, the MCD diet containing
high AGEs increased the content of liver AGEs. This elevated
the triglycerides level, the formation of NADPH-dependenceROS, 4HNE addition, fatty degeneration, and inflammatory
cytokines, and fibrosis index (α-SMA [α-smooth muscle
actin], CTGF [connective tissue growth factor], COL1A andpicrosirius). These findings suggest the possibility that
inflammations are developed via the secretion of inflammatory
cytokines when AGEs bind with RAGE on macrophage andKupffer cells of liver tissues.
Study with an experiment suggested that steatohepatitis
with fibrillization occurred after 33 weeks of feeding mice ahigh fat, high fructose and high cholesterol (HFHC) diet 69),
induced an increase of fatty-acid-derived aldehyde, 4HNE,
and increased gene expression of α-SMA and type 1 collagenas a fibrosis index. This model suggests that the formation ofcarbohydrate-derived aldehyde due to the excessive ingestion_ 86 _Glycative Stress Research

Fig. 7. From fatty liver to steatohepatitis.

Excess aldehydes cause proteins, lipids, and DNA to undergo glycation modifications. This places a strain on aldehyde metabolic
enzymes, which consume and decrease NAD + and GSH. Fibrosis progresses in fibroblasts and Ito cells. RAGE in immune response cells
is stimulated, releasing inflammatory cytokines. Amadori-PE increases in lipid droplets. The combined effects of these changes lead tosteatohepatitis. The involvement of LRP1 remains to be determined. HFD, high fat diet; ROS, reactive oxygen species; GA, glutaraldehyde;
4HNE, 4 - hydroxynonenal; AGEs, advanced glycation endproducts; RAGE, receptor for AGEs; Amadori-PE, Amadori-glycatedphosphatidylethanolamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NAD +, nicotinamide adenine dinucleotide; GSH,
glutathione; LRP1, low-density lipoprotein receptor related protein 1; 2(SC), S-(2-succinyl) cysteine; NADPH, nicotinamide adenine
dinucleotide phosphate (reduced form); NADH, nicotinamide adenine dinucleotide (reduced form); NFκB, nuclear factor κB; Ito cell,
hepatic stellate cell.

of fructose antecedently occurs followed by chain reactions.
Enhanced ROS production via the RAGE/NADPHoxidase signaling pathway, combined with the oxidation ofincreased fatty acids by a high-fat diet, further enhances
the production of fatty acid-derived aldehydes (i.e., MGO),
forming a vicious cycle in the mechanism leading from fatty
liver to steatohepatitis. The consumption of GSH during
the metabolism of MGO by GLO increases oxidative stress,
further adding to the vicious cycle.
This experiment concluded that the diet containing
AGEs alone did not induce fatty liver symptoms or
steatohepatitis in normal livers of animals which werenormally fed 69).
・ Fibrosis
It has been known that aldehydes promote liver fibrosis,
which is a cause of alcoholic liver disease. Typical collagenformation in the liver is a fibroblast recognized as Ito cell,which is also designated as hepatic stellate cell. Ito cells store
vitamin A and exist in the space of Disse, which a small area
between the sinusoids and hepatocytes.
Ito cells are activated during liver injury and change
to a myofibroblast-like morphology, resulting in increased
collagen production. They are also closely involved in thepathogenesis of liver cirrhosis. AGEs promote the activationand proliferation of Ito cells, and said to be leading to theformation of ROS 68).
Intact procollagen inhibit the collagen synthesis via Itocells in a concentration-dependent manner. However, whencarboxyl-terminal telopeptides of procollagen are modified
by acetaldehyde, Ito cells are not able to distinguish fromnormal procollagen. The collagen production increases due
to negative -feedback-inhibition. In livers of patients withalcoholism, procollagen and procollagen-related substances
are formed and fibrosis progresses. The content of GSH
in Ito cells decreases, and consequently, the expression ofcollagen type 1 mRNA and the formation of collagen proteinincreases 70). Other than acetaldehyde, fatty-acid-derived
aldehydes, including 4 -hydroxynonenal, stimulate thecollagen formation in liver fibroblasts, which leads to thepromotion of fibrosis 67).
・ Insulin resistance
Steatohepatitis is invariably accompanied by insulin
resistance. Among the factors that increase insulin resistancein I RI (immuno-reactive insulin) that we measure, themost important contributor is aldehyde excess. The reason
is that aldehydes have an impact from the stage of insulin_ 87 _

From Fatty Liver to Steatohepatitis: Involvement of Aldehydes

biosynthesis in pancreatic β cells. β cells undergo abiosynthesis of insulin via pre-proinsulin and proinsulin( Fig. 8 ) 4).
When Arg 31-Arg 32, which connect insulin B-chain
and C-chain, and Lys 64-Arg 65, which connect C peptide
and A-chain, undertake glycative modifications, it is difficult
to cleave by protease at C-chain terminal. Therefore,
ratios of proinsulin/insulin in cells elevate. Proinsulin andinsulin are secreted maintaining this ratio with responses to
glucose stimuli. However, IRI, which we measure, includes
both proinsulin and insulin. In patients with diabetes, 9 %of insulin is modified with glycation 71). Glycated insulin
does not have insulin resistance. In the state of excessive
aldehydes, the composition ratio of active intact insulin
is notably decreased in I RI ( Fig. 8 ). This is the reality of
insulin resistance.
・Abnormality in mitochondrial functions
Hepatocyte plays the key role of defense system forexcessive aldehydes. GAPDH, ALDH and GLO are forced
to exert their full capacities consuming NAD + and GSH.
Insufficient GHS inefficiently eliminates ROS. Fatty acid
which is contained in lipid droplets inside hepatocytes areoxidated at a rapid pace. This leads to a vicious cycle where
further fatty-acid-derived aldehydes increase. Moreover, theshortage of NAD + affects the mitochondrial TCA cycle,
which hinders a smooth cycle. Consequently, in addition to
the reduction of ATP production, the volume of fumaric acidincreases (Fig. 6 ) 72-74).

Fumaric acid reacts with cysteine ​​residue (-SH) ofprotein/peptide and S-(2-succinyl) cysteine (2SC) is formed.
This is another type of post-translational modification ofproteins. Fumaric acid, which is produced in mitochondria,
reacts with GAPDH as well. This induces 2SC-GAPDH
generation. As a result, the enzyme activities of GAPDH
decrease 72). This results in the state where metabolic
capacities significantly decrease for GA, which is formed
as carbohydrates-derived aldehyde, GA, which is formed inglycolysis, and GA, which is formed in polyol pathway.
It is observed that model mice created by feeding ahigh fat diet exhibit a reduction of protein level in liver tissue
GAPDH as well as the elevation of GA and MGO in theplasma 7, 75). The mechanism is inferred as follows: GAPDH
and ALDH exert full capacities to respond to the excessive
fatty-acid-derived aldehydes via the induction of a high fat
diet. The enzyme activities decrease due to the 2SC-GAPDH
formation, which is induced by fumaric acid via the functional
abnormality of the TCA cycle. Consequently, plasma GA
increases. As the reactivity of GA is significantly high,GA readily reacts with monosaccharides and fatty acids.
Consequently, diverse short-chain aldehydes are generated
in a chain reaction manner. Plasma concentration of MGO,
which is also generated from carbohydrate and fatty acid, is
300 ~ 400 nmol/L at fasting and increases by 30- 40 % after
30 minutes of oral ingestion of 75 g of glucose 8).
・ Non-physiological epigenetic alteration of LRP1
Low-Density Lipoprotein Receptor-Related Protein 1

Fig. 8. Glycative modification in the stage of insulin biosynthesis.

The process of insulin biosynthesis in pancreatic β cells is affected by glycation stress (excess aldehydes). When the residues of Arg or
Lys of the proinsulin B chain are glycated, it becomes protease resistant and the C chain cannot be cleaved. This increases the rate ofproinsulin secretion and increases the I RI value, which cross-recognizes proinsulin and insulin. Glycated insulin has no insulin activity,
so the rate of active insulin in the I RI decreases. AL, aldehyde; I RI. immuno-reactive insulin.

_ 88 _Glycative Stress Research(LRP1) takes on a well-rounded role in plasma clearance ofchylomicron and VLDL remnant lipoprotein. Its dysfunctions
are related to obesity, diabetes and fatty liver diseases 76, 77).
LRP1, which expresses abundantly in the liver, functions
in multiple physiological processes; the removal of diverse
circulatory molecules, such as proteinase inhibitor complex,
serpin enzyme complex, chylomicron remnant and activatedclotting factor. It is reported that LRP1 polymorphism isrelated to the elevation of triglycerides, the reduction of HDLlevel 78, 79), and the onset risk of metabolic syndrome 80). There
are multiple experiments regarding LRP1, in vitro researchof cell culture, and in vivo research of conditional knockout
mouse lacking tissue-specific LRP1. Findings from thesestudies suggest a strong possibility that the malfunction of
LRP1 is related to a mechanism of alternations from simple
fatty liver to steatohepatitis.
LRP1 undergoes non-physiological epigenetic alterations.
Occurrence frequency of epigenetic alternation is different,
depending on location and the surroundings of gene promoters,
the area of initial transcription of DNA. The promoter ofthe LRP1 gene, in particular, is expected to easily provide
epigenetic alternations. It is reported by Dr. Nikola Barić that
LRP1 is more sensitive to epigenome factors than RAGE 81).
The increase of DNA methylation inhibits the expressionof the gene ( localization: 12q13.3) with the decrease of
the transcription, which leads to the damage in most of its
functions, including the function which is related to thedischarge of harmful and toxic macromolecules from thebrain. The determinative factor for weakening the multiple
processes is that the DNA methylation continues progression
through its life cycle.
Here, we must be reminded of “what is called thephenomenon of DNA methylation, in varied research reports,
includes diverse modifications such as carbonylation andalkylation”. LRP1 gene promoter abundantly contains CpG
island and is highly sensitive to DNA methylation. Thismeans that LRP1 is a region where epigenetic events, such as
glycative stress or excessive aldehydes, are highly expected
to induce non-physiological carbonylation of DNA. With
the relation to the elevation of levels in DNA methylation
as well as DNA carbonylation, the transcriptive activity of
LRP1 decreases. When CpG sites are completely methylated,transcriptive activity thoroughly disappears. Thus, theexpression of LRP1 ceases 82).
A high-lipid diet is a primary risk factor of not solely
steatohepatitis but cirrhosis and hepatoma 83). The expressionof LRP1 in hepatocyte is significant for the defense systemagainst high-lipid-diet-related fatty liver and steatohepatitis
84-86). On the other hand, the reduction of LRP1 expression
raises the risk of steatohepatitis. Moreover, the reductionof LRP1 level is related to unfavorable prognosis ofhepatocellular carcinoma 87). The LRP1 inactivation in
hepatocyte aggravates HF-diet-induced obesity, impaired
glucose tolerance, insulin tolerance and fatty liver 84). The
inactivation of hepatic L RP1, with synergistic effect of
dietary cholesterol, accelerates development from liverdiseases to steatohepatitis 85, 86). Intensive reduction of LRP1
expression induces the hepatocellular degeneration 88).
The mechanism of the LRP1 inactivation is supposed
as follows: Fat-acid-derived aldehydes, which is induced bya high-fat diet, evoke the carbonylation of cytosine in the

L RP1 gene promoter domain. This leads to the decreaseof LRP1 expression. This is the reason for the LRP1
inactivation.
Epigenetic alteration occurs for histone as well 11).
Acetylation/deacetylation of histone protein, in particular,
lysine residue and arginine residue, are physiologicalmodification sites. Histone acetyltransferase (HAT) activates
the transcription of acetylation. Histone deacetylase
inhibits the transcription via deacetylation. Furthermore,this site of histone protein undergoes modifications ofmethylation, phosphorylation, ubiquitylation, citrullinationand crotonylation. Aldehydes induce phosphorylation andglycative modification (carbonylation) in a non-physiologicalmanner.
Histone deacetylase (HDAC) and sterol regulatory
element-binding protein (SREBP) play significant roles inmaintaining the homeostasis of hepatic lipid metabolism.
HDAC3 activates estrogen-related receptor alpha, induces
the transcription of fatty acid transporter gene Cd36, andconsequently leads to the lipid absorption and obesity 89).
Furthermore, HDAC3 knockout mouse shows the accelerationof lipid generation, the accumulation of binding substancefat-acid-derived aldehydes, and the reduction of ATP
formation. Lethal phenotype is observed due to hepatic
disorders 90). Thus, the acetylation and deacetylation ofhistone must be strictly controlled. When histone protein ismodified by excessive aldehydes, HDAC is disabled. Lipid
metabolism abnormalities and hepatocellular dysfunction are
expected to occur.
Rats fed a high-fat and high-fructose diet havesteatohepatitis with the increased expression of HDAC3 91).
The reason for this phenomenon is the excessive aldehydes
which are fatty acid- and carbohydrate-derived. The glycative
modification of histone increases and the transcriptive
activity is accelerated. It is supposed that the expression of
HDAC3 increases as a responsive-reaction.

5. Involvement of oxidative stress

One of significant mechanism for the development NASH
is that AGEs induce ROS on a cellular level 92, 93), activating
downstream NADPH oxidase via RAGE. AGEs activate
hepatic stellate cells and increase the ROS formation 68).
Additionally, Kupffer cells increase the ROS formation via
RAGE with the addition of AGEs 69). These findings indicatethat glycation stress (aldehyde excess) results in increased
ROS production secondary to increased oxidative stress frombaseline conditions. Oxidation of fatty acids by ROS induces
fatty acid-derived aldehyde production, which further
enhances glycation stress.
Athletes, who have been recognized to have no
relationship with fatty liver, develop steatohepatitis insome cases. Exercise load is extremely heavy and oxidative
damage is conspicuous in these cases. Results of a clinicaltest is shown regarding the healthy individuals, and theathletes, who are university student club members of Ekiden,Long Distance Relay, having heavy exercise load. ( Table 2 :unpublished data) The index of 8-hydroxy-deoxyguanosine
is employed for the oxidative damage of DNA (constituent
base guanine) and isoprostane for the oxidative damage offatty acid. Index of 8-OHdG in the group of athletes is 10 ~_ 89 _

From Fatty Liver to Steatohepatitis: Involvement of Aldehydes

Table 2. Urinary oxidative stress indicators: Comparison between healthy subjects and hard-training athletes.
Subjcets

Healthy subjects
23

19

M: 11, F :12

M : 1944.7 ± 7.9
23.0 ± 2.1
11.3 ± 6.5± 71.021.8 ± 7.0
119.0 ± 54.0
85.6 ± 56.2

NumberSex
Age (year)(year)BMI
8-OHdG generation rate

(ng/kg/hr）

15-Isoprostane generation rate

(ng/kg/hr)

8- OHdG/Cre(ng/mg Cre)

15-Isoprostane/Cre(ng/mg Cre)

Athletes with hard training

4.3 ± 2.8
11.0 ± 3.8
4.1 ± 1.9

19.8

206.6 ± 57.8
1675.9 ± 1246.9

Results are expressed as mean ± standard deviation. Note that oxidative stress markers are extremely high in the athletes (members of university EkidenClub) in season. Ekiden, long distance relay. Unpublished data.

20 times as high as that of healthy individual and isoprostanein athletes is 20 ~ 40 times as high as healthy individuals.
The tests on athlete group were measured on the first dayof a training camp. The values of the date are almost sameas those of the day after four weeks. It is clarified that theathlete group has intensively high level of oxidative stressdamage and their active oxygen and free radicals are in theexcessive state. It can be said that oxidative stress contributes
greatly to the development of fatty liver disease in athletes.
In hepatocyte of the athletes, lipid droplets for the
storage of energy are observed. They are significant forenergy source after intracellular glycogen is deleted.
However, when intensive exercise load induces the excessive
formation of ROS, fatty-acid-derived aldehydes form at anaccelerated pace from hepatic lipid droplets. The increased
MGO raises the load on GLO, and a large quantity of GSH
is consumed. Thus, the function of antioxidative defense
is lowered. A large quantity of AGEs, which form due toprotein modification, stimulates RAGE in macrophage andKupffer cells. Inflammatory cytokine is released, which
induces inflammation. Simultaneously, the formation ofcollagen increases via fibroblast cells, which are stimulated
glycated collagen, and Ito cells, which alters myofibroblastlike cells due to the stimulation of hepatic damage. Therefore,
fibrosis progresses. It is considered that the combination of
inflammation and fibrosis leads to the alternation from fatty
liver to steatohepatitis.
Athletes have unique circumstances. It is known that
athletes exercise at high intensity, which increases glucose
use by muscles and enhances gluconeogenesis in the liver.
For example, alanine is converted to pyruvate by alanine
aminotransferase, which then produces glucose 6-phosphate(G6P). However, at the Anti-Aging Medicine General Meetingin May 2024, it was reported that “rapid gluconeogenesis
in mice increases MGO, a short-chain aldehyde, in theliver ” 94). Although verification in humans is necessary in
the future, the production of short- chain aldehydes throughgluconeogenesis will require attention in the future.
Another reason is the post-exercise beverage. In
addition to minerals, so-called sports drinks often containisomerized sugar (70 % or more fructose) as an energy
source, rather than glucose alone. Drinking large amountsof these beverages can lead to excessive intake of fructose.
In order for fructose to enter the glycolysis pathway, it must

pass through fructose-1-phosphate (F 1P) produced byfructokinase or fructose - 6 -phosphate ( F6P) produced byhexokinase, and GA, a short- chain aldehyde, is produced
from F1P. In fact, the aforementioned publication reported
that a considerable amount of glyceraldehyde is produced in
the small intestine when mice are administered fructose 94).
It has been reported that hepatitis occurs when short-chain
aldehydes increase in the liver and cysteine, which reacts
with aldehydes and is involved in detoxifying aldehydes, isdepleted 6).
The combination of the above habits intensifies thealdehyde load and further increases the risk of fatty hepatitis
in athletes.

Conclusion

Prevention and treatment for steatohepatitis

There are varied arguments regarding treatment
methods. However, treatment methods for the inhibition ofsteatohepatitis have not been established. It is necessary
to prevent progression, not neglecting it, when you are toldthat you have a fatty liver. Basic methods are alimentary
therapy and exercise therapy. If you have obesity, diabetes,
dyslipidemia and/or hyperpiesia in your background, theymust be cured in every way possible.
Glycative stress (excess aldehydes) contributes greatly
to the onset and progression of steatohepatitis. It is unclear
at what point fatty liver and steatohepatitis become distinct.
We speculate that the determining factor is whether or notthere is a lack/depletion of NAD + due to excess aldehydes,
and whether or not there is a TCA cycle disorder. In
particular, a malfunction of the TCA cycle causes fumaricacid modification of GAPDH, which leads to a vicious cycleof increasing aldehydes. Another key may be epigenetic
glycation. In this current situation, we focused on the LTP1
gene. This issue is a future challenge.
It is important to prevent excess aldehydes throughglycation care. The first step is to control aldehyde
generation derived from carbohydrates, fatty acids, andluxury items, i.e., alcohol, or smoking. The second step is toinhibit the glycative modification of substances in the body(i.e., proteins, lipids, DNA), and the third step is to promote_ 90 _Glycative Stress Research

the decomposition and excretion of glycation products (i.e.,AGEs). It is essential to fully understand the impact ofglycative stress on the development of steatohepatitis and topractice glycation care.

Acknowledgments
The essential part of this paper was published in the
28 th Society for Glycative Stress Research held in Tokyo in
2024.

Research Grant
This work was supported by a scientific research grantfrom the Ministry of Education, Culture, Sports, Science andTechnology (JSPS Kakenhi 23K10882).

Conflict of interest declaration
Not applicable.

References
1) Argo CK, Caldwell SH. Epidemiology and natural history

of non-alcoholic steatohepatitis. Clin Liver Dis. 2009; 13:
511-531.
2) Starley BQ, Calcagno CJ, Harrison SA. Nonalcoholic fatty
liver disease and hepatocellular carcinoma: A weighty
connection. Hepatology. 2010; 51: 1820-1832.
3) Yagi M, Yonei Y. Glycative stress and anti-aging: 1. Whatis glycative stress? Glycative Stress Res. 2016; 3: 152-155.
4) Yonei Y, Yagi M, Sato K, et al Glycative stress: Molecularimpacts and defense mechanisms. Glycative Stress Res.
2023; 10: 145-158.
5) Sookwong P, Nakagawa K, Fujita I, et al. Amadoriglycated phosphatidylethanolamine, a potential marker for
hyperglycemia, in streptozotocin-induced diabetic rats.
Lipids. 2011; 46: 943-952.
6) Zheng Y, Martin-Morales A, Wang J, et al. Phenethylamine
in chlorella alleviates high-fat diet-induced mouse liver
damage by regulating generation of methylglyoxal. NPJ
Sci Food. 2021; 5: 22.
7) Sato K, Zheng Y, Martin-Morales A, et al. Generation ofshort chain aldehydes and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH). Glycative Stress Res, 2022; 9:129-134.
8) Maessen DE, Hanssen NM, Scheijen JL, et al. Post-glucose
load plasma α-dicarbonyl concentrations are increased inindividuals with Impaired glucose metabolism and type2 diabetes: The CODAM study. Diabetes Care. 2015; 38:
913-920.
9) Takeuchi M, Sakasai-Sakai A, Takata T, et al. Intracellular
toxic AGEs ( TAGE ) triggers numerous types of cell
damage. Biomolecules. 2021; 11: 387.
10) Takeuchi M, Sakasai-Sakai A, Takata T, et al. Effects of
toxic AGEs (TAGE) on human health. Cells, 2022; 11:
2178.
11) Tsukada Y, Fang J, Erdjument-Bromage H, et al. Histone
demethylation by a family of JmjC domain-containing
proteins. Nature. 2006; 439(7078): 811- 816.
12) Kalász H. Biological role of formaldehyde, and cycles
related to methylation, demethylation, and formaldehyde
production. Mini Rev Med Chem. 2003; 3: 175-192.
13) He R, Lu J, Miao J. Formaldehyde stress. Sci China Life
Sci. 2010; 53: 1399 -1404.
14) Zhang Y, Yang Y, He X, et al. The cellular function andmolecular mechanism of formaldehyde in cardiovasculardisease and heart development. J Cell Mol Med. 2021; 25:5358-5371.

15) Uchikawa T, Kanno T, Maruyama I, et al. Demethylationof methylmercury and the enhanced production offormaldehyde in mouse liver. J Toxicol Sci. 2016; 41: 479487.
16) Esterbauer H, Schaur RJ, Zollner H. Chemistry andbiochemistry of 4 -hydroxynonenal, malonaldehyde andrelated aldehydes. Free Radic Biol Med. 1991; 11: 81-128.
17) Dey A, Cederbaum AI. Alcohol and oxidative liver injury.
Hepatology. 2006; 43(2 Suppl 1): S63-S74.
18) Fraser DA, Wang X, Lund J, et al. A structurally
engineered fatty acid, icosabutate, suppresses liver
inflammation and fibrosis in NASH. J Hepatol. 2022; 76:
800- 811.
19) Inagi R. Glycative stress and glyoxalase in kidney disease
and aging. Biochem Soc Trans. 2014; 42: 457- 460.
20) Hong Y, Sheng L, Zhong J, et al. Desulfovibrio vulgaris,
a potent acetic acid-producing bacterium, attenuates
nonalcoholic fatty liver disease in mice. Gut Microbes.
2021; 13: 1-20.
21) Ramos Meyers G, Samouda H, Bohn T. Short chain fatty
acid metabolism in relation to gut microbiota and genetic
variability. Nutrients. 2022; 14: 5361.
22) Leung C, Rivera L, Furness JB, et al. The role of the gut
microbiota in NAFLD. Nat Rev Gastroenterol Hepatol.
2016; 13: 412- 425.
23) Coppola S, Avagliano C, Calignano A, et al. The protectiverole of butyrate against obesity and obesity-relateddiseases. Molecules. 2021; 26: 682.
24) Shimizu H, Ohue-Kitano R, Kimura I. Regulation of hostenergy metabolism by gut microbiota-derived short-chain
fatty acids. Glycative Stress Res. 2019; 6: 181-191.
25) Shashni B, Tajika Y, Ikeda Y, et al. Self-assembling
polymer-based short chain fatty acid prodrugs ameliorate
non-alcoholic steatohepatitis and liver fibrosis. Biomaterials.
2023; 295: 122047.
26) Nomoto K, Yagi M, Arita S, et al. Skin accumulation ofadvanced glycation end products and lifestyle behaviors inJapanese. Anti-Aging Medicine. 2012; 9: 165-173.
27) Wijarnpreecha K, Scribani M, Kim D, et al. The interactionof nonalcoholic fatty liver disease and smoking on mortality
among adults in the United States. Liver Int. 2019; 39:1202 -1206.
28) Ou H, Fu Y, Liao W, et al. Association between smoking
and liver fibrosis among patients with nonalcoholic fatty
liver disease. Can J Gastroenterol Hepatol. 2019; 2019:6028952.

_ 91 _

From Fatty Liver to Steatohepatitis: Involvement of Aldehydes29) Mumtaz H, Hameed M, Sangah AB, et al. Association

between smoking and non-alcoholic fatty liver disease inSoutheast Asia. Front Public Health. 2022; 10: 1008878.
30) Butterfield DA, Hardas SS, Lange ML. Oxidatively
modified glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and Alzheimer's disease: Many pathways toneurodegeneration. J Alzheimers Dis. 2010; 20: 369-393.
31) Harada N, Mitani R, Yamachi R. Multifunctional
moonlighting proteins. Journal of Japanese Biochemical
Society. 2015; 87: 279-285. (in Japanese)32) Veneziale CM. Regulation of d-triokinase and NADlinked glycerol-dehydrogenase activities in rat liver.
European Journal of Biochemistry. 1972; 31: 59-62.
33) Feraudi M, Kohlmeier M, Glaser W, et al. Mechanism ofaction of D-glyceraldehyde-3-phosphate dehydrogenase.
Acta Biochim Biophys Acad Sci Hung. 1977; 12: 317-327.
34) Meyer-Siegler K, Mauro DJ, Seal G, et al. A humannuclear uracil DNA glycosylase is the 37-kDa subunit ofglyceraldehyde -3 - phosphate dehydrogenase. Proc Natl
Acad Sci US A. 1991; 88: 8460-8464.
35) Sirover MA. New insights into an old protein: Thefunctional diversity of mammalian glyceraldehyde-3 phosphate dehydrogenase. Biochim Biophys Acta. 1999;
1432: 159 -184.
36) Chuang DM, Hough C, Senatorov VV. Glyceraldehyde -3phosphate dehydrogenase, apoptosis, and neurodegenerative
diseases. Annu Rev Pharmacol Toxicol. 2005; 45: 269-290.
37) Tarze A, Deniaud A, Le Bras M, et al. GAPDH, a novel
regulator of the pro-apoptotic mitochondrial membrane
permeabilization. Oncogene. 2007; 26: 2606-2620.
38) Ryzlak MT, Pietruszko R. Heterogeneity of glyceraldehyde-3-phosphate dehydrogenase from human brain. Biochim
Biophys Acta. 1988; 954: 309-324.
39) Ishitani R, Chuang DM. Glyceraldehyde-3 -phosphate
dehydrogenase antisense oligodeoxynucleotides protect
against cytosine arabinonucleoside-induced apoptosis incultured cerebellar neurons. Proc Natl Acad Sci U S A.
1996; 93: 9937-9941.
40) Ishitani R, Kimura M, Sunaga K, et al. An antisense
oligodeoxynucleotide to glyceraldehyde-3 -phosphate
dehydrogenase blocks age-induced apoptosis of mature
cerebrocortical neurons in culture. J Pharmacol Exp Ther.
1996; 278: 447- 454.
41) Sawa A, Khan AA, Hester LD, et al. Glyceraldehyde3-phosphate dehydrogenase: Nuclear translocation
participates in neuronal and non-neuronal cell death. Proc
Natl Acad Sci U S A. 1997; 94: 11669-11674.
42) Patterson RL, van Rossum DB, Kaplin AI, et al. Inositol
1, 4, 5-trisphosphate receptor/GAPDH complex augments
Ca 2 + release via locally derived NADH. Proc Natl Acad
Sci U S A. 2005; 102: 1357-1359.
43) Xu KY, Becker L C. Ultrastructural localization of
glycolytic enzymes on sarcoplasmic reticulum vesicles.
J Histochem Cytochem. 1998; 46: 419-427.
44) Vasiliou V, Bairoch A, Tipton KF, et al. Eukaryotic
aldehyde dehydrogenase (ALDH) genes: Human polymor
phisms, and recommended nomenclature based on divergent
evolution and chromosomal mapping. Pharmacogenetics.
1999; 9: 421-434.
45) Jackson B, Brocker C, Thompson DC, et al. Update on thealdehyde dehydrogenase gene (ALDH) superfamily. Hum
Genomics. 2011; 5: 283-303.

46) Yoshida A, Hsu LC, Yasunami M. Genetics of humanalcohol-metabolizing enzymes. Prog Nucleic Acid Res
Mol Biol. 1991; 40: 255-287.
47) Wang MF, Han C L, Yin SJ. Substrate specificity ofhuman and yeast aldehyde dehydrogenases. Chem Biol
Interact. 2009; 178: 36-39.
48) Wang RS, Nakajima T, Kawamoto T, et al. Effects ofaldehyde dehydrogenase-2 genetic polymorphisms on
metabolism of structurally different aldehydes in humanliver. Drug Metab Dispos. 2002; 30: 69-73.
49) Lassen N, Estey T, Tanguay RL, et al. Molecular cloning,baculovirus expression, and tissue distribution of thezebrafish aldehyde dehydrogenase 2. Drug Metab Dispos.
2005; 33: 649-656.
50) Kitagawa K, Kawamoto T, Kunugita N, et al. Aldehyde
dehydrogenase (ALDH) 2 associates with oxidation of
methoxyacetaldehyde; In vitro analysis with liver subcellular
fraction derived from human and Aldh2 gene targetingmouse. FEBS Lett. 2000; 476: 306-311.
51) Esterbauer H, Schaur R J, Zollner H. Chemistry andbiochemistry of 4-hydroxynonenal, malonaldehyde andrelated aldehydes. Free Radic Biol Med. 1991; 11: 81-128.
52) Zhang H, Gong DX, Zhang YJ, et al. Effect of
mitochondrial aldehyde dehydrogenase-2 genotype on
cardioprotection in patients with congenital heart disease.
Eur Heart J. 2012; 33: 1606-1614.
53) Markesbery WR, Lovell MA. Four-hydroxynonenal, aproduct of lipid peroxidation, is increased in the brain in
Alzheimer’s disease. Neurobiol Aging. 1998; 19: 33-36.
54) Kwon HJ, Won YS, Park O, et al. Aldehyde dehydrogenase
2 deficiency ameliorates alcoholic fatty liver but worsens
liver inflammation and fibrosis in mice. Hepatology.
2014; 60: 146-157.
55) He Y, Zhou C, Huang M, et al. Glyoxalase system: A
systematic review of its biological activity, relateddiseases, screening methods and small molecule
regulators. Biomed Pharmacother. 2020; 131: 110663.
56) Yamada N, Karasawa T, Kimura H, et al. Ferroptosis
driven by radical oxidation of n-6 polyunsaturated fatty
acids mediates acetaminophen-induced acute liver failure.
Cell Death Dis. 2020; 11: 144.
57) Fujino T, Tada T, Beppu M, et al. Purification andcharacterization of a serine protease in erythrocyte cytosolthat is adherent to oxidized membranes and preferentiallydegrades proteins modified by oxidation and glycation.
J Biochem. 1998; 124: 1077-1085.
58) Yagi M, Ishigami M, Mori R, et al. Reduction effect ofoxidized protein hydrolase (OPH) on advanced glycation
end products and OPH-like activity in human stratum
corneum. Glycative Stress Res. 2017; 4: 184-191.
59) Goto S, Takahashi R. Environmental factors and cellular
proteins in aging. Experimental Medicine. 1998; 16: 23242331. (in Japanese)60) Krueger F, Kreck B, Franke A, et al. DNA methylome
analysis using short bisulfite sequencing data. Nat Methods.
2012; 9: 145-151.
61) Emantoko Dwi Putra S, Martriano Humardani F,
Antonius Y, et al. Epigenetics of diabetes: A bioinformatic
approach. Clin Chim Acta. 2024; 557: 117856.
62) Kho M, Wang YZ, Chaar D, et al. Accelerated DNA
methylation age and medication use among African
Americans. Aging (Albany NY). 2021; 13: 14604-14629.

_ 92 _Glycative Stress Research63) Vetter VM, Spieker J, Sommerer Y, et al. DNA

methylation age acceleration is associated with risk of
diabetes complications. Commun Med (Lond). 2023; 3: 21.
64) Horitani K, Iwasaki M, Kishimoto H, et al. Repetitive
spikes of glucose and lipid induce senescence-likephenotypes of bone marrow stem cells through H3K27me3
demethylase-mediated epigenetic regulation. Am J Physiol
Heart Circ Physiol. 2021; 321: H920-H932.
65) Imai N, Ohsaki Y, Cheng J, et al. Distinct features of two
lipid droplets types in cell nuclei from patients with liverdiseases. Sci Rep. 2023; 13: 6851.
66) Penno A, Hackenbroich G, Thiele, C. Phospholipids andlipid droplets. Biochim Biophys Acta. 2013; 1831, 589-594.
67) Lieber CS. Alcoholic fatty liver: Its pathogenesis andmechanism of progression to inflammation and fibrosis.
Alcohol. 2004; 34: 9-19.
68) Leung C, Herath CB, Jia Z, et al. Dietary glycotoxins
exacerbate progression of experimental fatty liver disease.
J Hepatol. 2014; 60: 832-838.
69) Leung C, Herath CB, Jia Z, et al. Dietary advanced
glycation end-products aggravate non-alcoholic fatty liverdisease. World J Gastroenterol. 2016; 22: 8026-8040.
70) Liu RM, Liu Y, Forman HJ, et al. Glutathione regulates
transforming growth factor-beta-stimulated collagenproduction in fibroblasts. Am J Physiol Lung Cell Mol
Physiol. 2004; 286: L121-L128.
71) Hunter SJ, Boyd AC, O'Harte FP, et al. Demonstrationof glycated insulin in human diabetic plasma anddecreased biological activity assessed by euglycemichyperinsulinemic clamp technique in humans. Diabetes.
2003; 52: 492-498.
72) Alderson NL, Wang Y, Blatnik M, et al. S-(2-Succinyl)
cysteine: A novel chemical modification of tissue proteinsby a Krebs cycle intermediate. Arch Biochem Biophys.
2006; 450: 1-8.
73) 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.
74) Frizzell N, Rajesh M, Jepson MJ, et al. Succinationof thiol groups in adipose tissue proteins in diabetes:
Succination inhibits polymerization and secretion of
adiponectin. J Biol Chem. 2009; 284: 25772-25781.
75) Martin-Morales A, Arakawa T, Sato M, et al. Developmentof a method for quantitation of glyceraldehyde in various
body compartments of rodents and humans. J Agric Food
Chem. 2021; 69: 13246 -13254.
76) van de Sluis B, Wijers M, Herz J. News on the molecularregulation and function of hepatic low density lipoprotein
receptor and LDLR-related protein 1. Curr Opin Lipidol.
2017; 28: 241-247.
77) Jaeschke A, Haller A, Cash JG, et al. Mutation in the
distal NPxY motif of LRP1 alleviates dietary cholesterolinduced dyslipidemia and tissue inflammation. J Lipid
Res. 2021; 62: 100012.
78) Teslovich TM, Musunuru K, Smith AV, et al. Biological,
clinical and population relevance of 95 loci for blood
lipids. Nature. 2010; 466: 707-713.
79) Q i Q, Liang L, Doria A, et al. Genetic predisposition todyslipidemia and Type 2 diabetes risk in two prospective
cohorts. Diabetes. 2012; 61: 745-752.

80) Delgado-Lista J, Perez-Martinez P, Solivera J, et al. Top

single nucleotide polymorphisms affecting carbohydrate
metabolism in metabolic syndrome: From the LIPGENE
study. J Clin Endocrinol Metab. 2013; 99: E384-E389.
81) Barić N. The role of the low-density lipoprotein receptor
related protein 1 (LRP1) and the receptor for advanced
glycation end products (RAGE) in the ageing process.
Glycative Stress Res. 2024; 11: 7-20.
82) Xing P, Liao Z, Ren Z, et al. Roles of low-density
lipoprotein receptor-related protein 1 in tumors. Chin J
Cancer. 2016; 35: 6.
83) Ioannou GN, Morrow OB, Connole M, et al. Association
between dietary nutrient composition and the incidence of
cirrhosis or liver cancer in the United States population.
Hepatology. 2009; 50: 175-184.
84) Ding Y, Xian X, Holland WL, et al. Low-density lipoprotein
receptor related protein-1 protects against hepatic insulin
resistance and hepatic steatosis. EBioMedicine. 2016; 7:
135-145.
85) Hamlin AN, Basford JE, Jaeschke A, et al. LRP1 protein
deficiency exacerbates palmitate-induced steatosis andtoxicity in hepatocytes. J Biol Chem. 2016; 291: 1661016619.
86) Hamlin AN, Chinnarasu S, Ding Y, et al. Low-density
lipoprotein receptor-related protein -1 dysfunctionsynergizes with dietary cholesterol to accelerate
steatohepatitis progression. J Biol Chem. 2018; 293: 96749684.
87) Huang XY, Shi GM, Devbhandari RP, et al. Low level of
low-density lipoprotein receptor related protein 1 predicts
an unfavorable prognosis of hepatocellular carcinoma
after curative resection. PLoS One. 2012; 7.
88) Herz J, Clouthier DE, Hammer RE. LDL receptor related
protein internalizes and degrades uPA-PAI-1 complexes
and is essential for embryo implantation. Cell. 1992; 71:
411- 421.
89) Kuang Z, Wang Y, Li Y, et al. The intestinal microbiota
programs diurnal rhythms in host metabolism throughhistone deacetylase 3. Science. 2019; 365: 1428-1434.
90) Papazyan R, Sun Z, Kim YH, et al. Physiologicalsuppression of lipotoxic liver damage by complementaryactions of HDAC3 and SCAP/SREBP. Cell Metab. 2016;24; 863-874.
91) Witayavanitkul N, Werawatganon D, Chayanupatkul M,
et al. Genistein and exercise treatment reduced NASH
related HDAC3, IL-13 and MMP-12 expressions in
ovariectomized rats fed with high fat high fructose diet.
Tradit Complement Med. 2021; 11: 503-512.
92) Basta G, Schmidt AM, De Caterina R. Advanced glycation
end products and vascular inflammation: Implications foraccelerated atherosclerosis in diabetes. Cardiovasc Res.
2004; 63: 582-592.
93) Santos JC, Valentim IB, de Araújo OR, et al. Developmentof nonalcoholic hepatopathy: Contributions of oxidative
stress and advanced glycation end products. Int J Mol Sci.
2013; 14: 19846-19866.
94) Sato K, Ujihara M. Short-chain aldehydes produced byoral administration of fructose. The 24 th Meeting of theJapanese Society of Anti-Aing Medicine (May 31 to Jun 2,
2024, Kumamoto, Japan). (abstract in Japanese)_ 93 _