Accepted Manuscript

Epigenetic modification by dietary factors: Implications in metabolic syndrome

Jae-Ho Park, Soon-Hee Kim, Myeong Soo Lee, Myung-Sunny Kim

PII: S0098-2997(16)30062-0
DOI: 10.1016/j.mam.2017.01.008
Reference: JMAM 688

To appear in: Molecular Aspects of Medicine

Received Date: 2 September 2016

Revised Date: 26 December 2016
Accepted Date: 3 January 2017

Please cite this article as: Park, J.-H., Kim, S.-H., Lee, M.S., Kim, M.-S., Epigenetic modification by
dietary factors: Implications in metabolic syndrome, Molecular Aspects of Medicine (2017), doi: 10.1016/
j.mam.2017.01.008.

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 ACCEPTED MANUSCRIPT
Epigenetic modification by dietary factors: implications in metabolic
syndrome

Jae-Ho Park1,2¶, Soon-Hee Kim1¶, Myeong Soo Lee3, Myung-Sunny Kim1,2*

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Division of Metabolism and Nutrition, Korea Food Research Institute, Gyeonggi-do 13539,

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Republic of Korea
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Department of Food Biotechnology, Korea University of Science & Technology, Gyeonggi-

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do 13539, Republic of Korea
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Clinical Research Division, Korea Institute of Oriental Medicine, Daejeon, 34054, Republic

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of Korea

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These authors contributed equally to this work.
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*
Corresponding author: Myung-Sunny Kim

Division of Metabolism and Nutrition,

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Korea Food Research Institute

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62 Anyangpangyo-ro 1201 beon-gil, Seongnam, Gyeonggi-do 13539, Republic of Korea.

E-mail: truka@kfri.re.kr; Fax: +82-31-780-9360
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Abstract

Dietary factors play a role in normal biological processes and are involved in the regulation

of pathological progression over a lifetime. Evidence has emerged indicating that dietary

factor-dependent epigenetic modifications can significantly affect genome stability and the

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expression of mRNA and proteins, which are involved in metabolic dysfunction. Since

metabolic syndrome is a progressive phenotype characterized by insulin resistance, obesity,

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hypertension, dyslipidemia, or type 2 diabetes, gene-diet interactions are important processes

involved in the initiation of particular symptoms of metabolic syndrome and their progression.

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Some epigenetic risk markers can be initiated or reversed by diet and environmental factors.

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In this review, we discuss recent advances in our understanding of the interactions between
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dietary factors and epigenetic changes in metabolic syndrome. We discuss the contribution of

nutritional factors in transgenerational inheritance of epigenetic markers and summarize the

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current knowledge of epigenetic modifications by dietary bioactive components in metabolic

diseases. The intake of dietary components that regulate epigenetic modifications can provide
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significant health effects and, as an epigenetic diet, may prevent various pathological
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processes in the development of metabolic disease.

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Keywords: Dietary factor; epigenetic marker; metabolic syndrome

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Contents

1. Introduction

2. Nutritional factors and epigenetic modifications

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2.1. Effects of undernutrition on epigenetic marks

2.1.1. Undernutrition causes fetal programming of MetS through epigenetic

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changes

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2.1.2. Can the effect of maternal undernutrition be overcome by full postnatal

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nutrition? AN
2.1.3. How can we manage the nutrition of offspring?

2.2. Overnutrition
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2.2.1. In utero overnutrition causes MetS in offspring

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2.2.2. Pregestational obesity is inherited to offspring through epigenetic changes

in sperm and egg

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2.2.3. Effect of gestational overnutrition on offspring

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2.2.4. Adverse effects of overnutrition are washed out in 3 generations

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2.2.5. Dietary intervention during the pregestation period can overcome the

adverse effects of overnutrition

2.2.6. Is it possible to predict the success of dietary intervention in MetS?

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2.2.7. Studies of biomarkers and miRNAs related with MetS

3. Nutritional components and epigenetic regulation

3.1. Methyl donors and cofactors

3.1.1. Effects of methyl donor deficiency on offspring

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3.1.2. Methyl donor supplemented diet in undernutrition

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3.1.3. Epidemiologic study of the correlation between methyl donor intake and

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MetS

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3.2. Protein AN
3.2.1. Protein deficiency and glucose metabolism in offspring

3.2.2. Protein deficiency and adiposity in offspring

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3.2.3. Protein deficiency on the regulation of cholesterol in offspring

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3.3. Polyunsaturated fatty acid (PUFA)

3.3.1. Genes altered by methylation associated with PUFA intake: EWAS study
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3.3.2. Effects of PUFA intake on genes involved in PUFA synthesis

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3.4. Sugar
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3.5. Bioactive food components and epigenetics

4. Summary and future perspectives

Acknowledgements

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References

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1. Introduction

Metabolic diseases linked to epigenetic modifications can be influenced by environmental

and dietary factors. It has become clear that the impact of dietary intake occurs over a

lifetime and epigenetic mechanisms provide the links between dietary factors and phenotypic

changes in metabolic syndrome (MetS). MetS is characterized by a number of metabolic risk

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factors that include obesity, insulin resistance, hypertension, and dyslipidemia. It represents a

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cluster of the risk factors related to an elevated risk of cardiovascular disease and type 2

diabetes (T2D) (Alberti et al., 2009). The global prevalence of MetS is increasing to epidemic

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proportions and a constellation of metabolic risk factors reflects overnutrition and sedentary

lifestyle.

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Nutrition has transgenerational epigenetic effects and scientific evidence shows that
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humans are sensitive to these effects, with dietary components likely to have the most
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profound impact. In the 1960s, it was suggested that not only genetic factors, but also

environmental conditions contribute to the development of diseases such as hypertension,

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obesity, cardiovascular disorder, and arthritis (Mayer and Thomas, 1967). In the 1970s, it was
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confirmed that food consumption affects offspring health in a rat model (Zamenhof et al.,

1971). This study showed that maternal dietary protein restriction leads to lower cerebral
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weight and total cerebral cell number in the second generation. Consistent with the previous

study, in another rat model, Benyshek et al. (2006) showed that malnourishment during
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gestation and perinatal life affects glucose metabolism in the adequately nourished grand-

offspring. In the 2000s, although environmental factors, including food, exercise, and stress,

are still considered as crucial risk factors for the development of many human diseases, few

studies have clearly demonstrated inherited traits affected by food and diet in humans.

However, many epidemiologic studies imply that the effects of environmental factors on one

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generation may affect its offspring. This is supported by reports showing that the incidence of

MetS in men is higher in Japanese immigrants in America and Brazil than in native Japanese

(Schwingel et al., 2007; Yoneda et al., 2008). It is important to note that second- and third-

generation Japanese-Brazilians present higher obesity, which may be, at least in part,

explained by inherited traits affected by the environment. In the analysis of individuals who

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were born during the Dutch Hunger Winter in 1944–1945, it was demonstrated that prenatal

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exposure to malnutrition and famine causes MetS phenotypes and persistent epigenetic

changes, including DNA hypomethylation (Heijmans et al., 2008). This suggests that

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maternal diet during critical periods of fetal development can affect the offspring’s

susceptibility for MetS later in life.

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Epigenomics is the study of epigenetic marks on a genome that captures the biological

influence of environmental and lifestyle factors. Epigenetic modifications may influence

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changes in the protein products of genes and can be transmitted to offspring without altering

the DNA sequence. These modifications are governed by several molecular processes. The
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main epigenetic mechanisms that regulate gene expression are DNA methylation, histone
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modifications, chromatin remodeling, and the influence of miRNA. DNA methylation at

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specific sites influences gene expression by directly interfering with transcription factor

complex binding or by inducing histone modifications mediated by methyl CpG-binding

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proteins (Milagro et al., 2013). Post-translational histone modifications, such as acetylation,

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methylation, and sumoylation, contribute to the regulation of gene expression through

chromatin modifications. Noncoding RNAs, such as miRNA, also contribute to epigenetic

regulation of gene expression.

The influence of nutritional or dietary components on epigenetic modifications and the

impact on metabolic dysfunction are assessed herein. We discuss the transgenerational

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inheritance of epigenetic traits in response to nutritional supply and epigenetic modifications

caused by dietary bioactive components, linking nutritional factors to metabolic function.

Because epigenetic alteration by dietary factors is important for the initiation of MetS and its

progression, it is reasonable to believe that investigating strategies that utilize dietary

components to target epigenetic modifications may be worthwhile in preventing and treating

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MetS.

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2. Nutritional factors and epigenetic modifications

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2.1. Effects of undernutrition on epigenetic marks

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2.1.1. Undernutrition causes fetal programming of MetS through epigenetic changes
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While undernutrition can be applied to all nutrients, here it will be used particularly to

describe reduced caloric intake. Growth-restricted fetuses exposed to undernutrition are more
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susceptible to MetS in adulthood, and this adverse effect is inherited by the next generation
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(F2) in a process involving epigenetic changes known as fetal programming (Salam et al.,
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2014). In the Dutch famine cohort, fetuses that experienced undernutrition during the third

trimester had lower birthweights than those of earlier stages; however, they also showed the
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lowest disease incidence (Painter et al., 2005; Roseboom et al., 2006). In contrast, fetuses that

experienced starvation during the early gestation stage, the most active developmental stage,
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showed higher incidences of glucose intolerance, dyslipidemia, and coronary heart disease
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(Painter et al., 2005; Roseboom et al., 2006) (Table 1).

Analysis of epigenetic changes in the cohort showed lower methylation levels of insulin-

like growth factor 2 (IGF2), a crucial protein during embryogenesis, in those exposed to

famine compared to non-exposed subjects. In particular, those exposed to starvation during

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the very early stages of gestation presented with significant IGF2 hypomethylation (Heijmans

et al., 2008; Livingstone and Borai, 2014). Together, these data strongly suggest that the

gestational stage at which malnutrition occurs is crucial for normal development and later

health conditions. Another study demonstrated significant changes in methylation of SMAD7,

CDH23, INSR, RFTN1, CPT1a, and KFL13 in mothers that were exposed to famine (Tobi et

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al., 2014). Since these genes are involved in developmental processes, the alteration of DNA

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methylation by prenatal malnutrition may be involved in fetal development and changes in

blood lipid levels (Tobi et al., 2014). This suggests that changes due to fetal nutritional

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exposure persist long-term via epigenetic changes, affecting metabolic health throughout the

entire lifetime.

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In utero, undernutrition also seems to affect feeding behavior as a consequence of
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epigenetic change. When sheep were exposed to undernutrition during the early gestation
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period, DNA methylation and histone H3 lysine K27 trimethylation (H3K27me3) in the

glucocorticoid receptor (GR) promoter were reduced, while histone H3 lysine K9 acetylation
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(H3K9ac) increased, in the hypothalamus of 5 year-old adult offspring (Begum et al., 2013).
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Consequently, mRNA and protein expression of GR, a neuropeptide involved in appetite, was

increased in the hypothalamus. In contrast, expression of proopiomelanocortin (POMC), an

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appetite-suppressing neuropeptide, decreased in male offspring (Begum et al., 2013). The

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increased fat-lean mass ratio in male offspring is explained by the possibility that epigenetic
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changes in appetite-related neuropeptide genes in the fetus after intrauterine undernutrition

may affect feeding behavior in adulthood through epigenetic memory, leading to the

impairment of energy balance; however, the sexual difference is not fully understood.

Although early gestation is a crucial period during which the fetus may become more

susceptible to metabolic disease in later life, this does not mean that undernutrition during

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other periods is not critical. In the Dutch famine cohort, those who were exposed to famine at

any gestational stage showed significantly higher glucose intolerance in adulthood than

unexposed siblings (Roseboom et al., 2006). In addition, when rat fetuses (F1) lost about 16%

of their bodyweight after induction of intrauterine growth restriction (IUGR) through bilateral

uterine artery ligation at E19 (corresponding to the ninth month of gestation in humans), the

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adult F2 generation showed significant weight gain and elevation of abdominal fat levels, as

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well as dyslipidemia and insulin resistance (Goodspeed et al., 2015). In the liver of offspring

F2 rats after IUGR, H19 expression was reduced by 39% compared to that of controls, and

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DNA methylation of certain CpG islands in the H19 promoter was increased by 40%

(Gonzalez-Rodriguez et al., 2016). H19, a long noncoding RNA (lncRNA), is an imprinted

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gene expressed only in maternally-originated alleles and is involved in placental growth
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(Bartolomei et al., 1991). In conclusion, these data indicate that undernutrition during the
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third trimester of gestation can also cause epigenetic changes leading to MetS-associated

problems, such as insulin resistance, body fat increment, and dyslipidemia, in offspring. They
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also show that epigenetic changes and altered phenotypes persist in the second generation.
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2.1.2. Can the effect of maternal undernutrition be overcome by full postnatal nutrition?
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The data described in section 2.1.1 raised the question of whether negative effects of fetal
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exposure to maternal undernutrition can be overcome by postnatal nutrition. This information

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will provide insights on how to manage nutrition to overcome the adverse effects of previous

undernutrition. When rats were exposed to undernutrition with only 50% of standard chow

(SC) for 50 generations, the weights of the pups from the line were lower than those of the

control rats at birth and throughout life (Hardikar et al., 2015). On the contrary, fat mass was

higher and bone mineral density (BMD) was lower in adulthood. When the offspring were

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fed ad libitum with SC, birthweights of the second generation recovered to normal levels and

the weight curve by age was similar to that of the control group. However, recuperation rats

showed heavier body mass and altered metabolic profiles, including elevated circulating

insulin, glucose, triglycerides (TG), and LDL-cholesterol. These rats also showed markedly

altered epigenetic H3K9me3 and H3K20me3 signatures, leading to the suppression of

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proinsulin gene transcription in the pancreas (Hardikar et al., 2015). These results suggest

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that metabolic adverse effects of in utero undernutrition were not recovered and persisted in

the second generation, even with normal postnatal nutrition.

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2.1.3. How can we manage the nutrition of offspring?

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Given that fetuses exposed to in utero undernutrition suffered from metabolic adverse
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effects with normal postnatal nutrition, what is the best nutritional intervention to minimize
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the adverse effects? According to Tosh et al. (2010), a period of nutritional restriction, rather

than a sudden ad libitum nutritional supply, may reduce MetS incidence. Most fetuses
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exposed to in utero undernutrition show a rapid postnatal growth to reach normal growth;
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however, this rapid growth is considered to cause obesity and MetS. Hence, the Tosh group

hypothesized that a delay in catch-up growth might prevent obesity and MetS. Thus, they
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provided rats with 50% food restriction in the late gestation period (10–21d) and offspring
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were provided either ad libitum diet or 50% nutritional restriction diet after weaning (Tosh et
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al., 2010). Male offspring with postnatal ad libitum diet gained weight, and levels of

H3K4me2 in IGF1 decreased while those of hepatic IGF1 increased, whereas, in adulthood,

offspring with continued postnatal restriction diet had weights and hepatic IGF1 expression

levels similar to those of the control group with continuous SC (Tosh et al., 2010). Although

continued 50% food restriction in humans is not desirable, it may be possible to reduce adult

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onset of MetS for those who were exposed to in utero undernutrition using postnatal

nutritional restriction. However, further studies on the proper extent and rate of nutritional

supply control are required.

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2.2. Overnutrition

In overnourished condition, parental obesity causes fetal epigenetic modifications and

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increases adult susceptibility to MetS. Maternal overnutrition is inherited to the next

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generations in the same way as maternal undernutrition, increasing the incidence of MetS in

offspring. Parental overnutrition also causes fetal outgrowth and increases incidence of MetS

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risk factors, such as postnatal obesity and diabetes, and these effects are passed onto offspring.
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2.2.1. In utero overnutrition causes MetS in offspring
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When female mice were fed a high-fat diet (HFD, 60% fat) from 4 weeks before pregnancy,
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a significantly higher caloric intake was observed in their offspring fed SC after weaning
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when compared to controls. They also showed higher body weights and blood TG levels,

along with increased insulin resistance in adulthood (24 weeks) (Masuyama and Hiramatsu,
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2012; Masuyama et al., 2015). In the adipose tissue of the offspring, adiponectin expression

significantly decreased, while leptin expression increased. In addition, H3K9 acetylation

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decreased and H3K9 methylation increased in the adiponectin promoter region. In contrast,
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the leptin promoter presented elevated methylation of H4K20. These results indicate that

maternal obesity or in utero exposure to a HFD might cause a risk of postnatal and adult

MetS in offspring, even when fed SC after weaning, through epigenetic modifications of

adipocytokines, adiponectin, and leptin. This is supported by another study showing that

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offspring of female rats fed a HFD from 80 days before gestation until weaning had

significantly higher body weights and leptin levels in adulthood than control mice, even with

continued SC after weaning (Marco et al., 2014). F0 rats fed with a HFD showed a lower

expression of POMC, an appetite suppressive neuropeptide, and its promoter was

hypermethylated. The hypermethylation was maintained in F1 females, although POMC

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expression was not reduced (Marco et al., 2014). It is also important to note that F1 offspring

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of the HFD female rats consumed more calories and gained more body weight after high fat

challenge (fed SC for 80 days after weaning then a HFD for 30 days). Therefore, it is

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considered that the change in POMC methylation induced by maternal HFD was stored as an

epigenetic memory and affected caloric consumption in adulthood; however, further studies

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are required on the correlation between POMC methylation and its expression.
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2.2.2. Pregestational obesity is inherited to offspring through epigenetic changes in

sperm and egg

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Since a significant amount of methylation is lost during fertilization, the question of

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whether parental epigenetic changes are inherited intact to offspring remains controversial. In

addition, it is disputed whether MetS in children whose parents present with obesity/MetS is
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mainly due to genetic or environmental factors. To investigate epigenetic changes via

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gametes in the absence of intrauterine and environmental effects, sperm and eggs from F0
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mice fed a HFD for 6 weeks were subjected to in vitro fertilization and transplantation into a

foster mother fed SC (Huypens et al., 2016). All offspring were initially fed SC and then

given a HFD. Incidence of obesity and insulin resistance was affected by the parental

condition. When one or both parents were obese, female offspring showed a tendency to

weight gain in an additive manner compared to control mice (weight gain rates of female

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offspring by parental obesity: paternal obesity, 8%; maternal obesity, 14%; parental obesity,

20%). In contrast, significant weight gain in male offspring was only observed when the

mother was obese (7–9%). Both male and female offspring showed glucose intolerance when

both parents, or only their mothers, were obese, demonstrating that HFD-induced insulin

resistance is inherited maternally. The weight of female offspring was more profoundly

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affected by parental obesity than that of male offspring, and maternal obesity also had a more

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significant effect (Huypens et al., 2016). These findings strongly suggest that diet-induced

epigenetic changes are inherited to offspring through gametes and can lead to elevated

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susceptibility to MetS.

It was also reported that diet-induced paternal obesity modulated sperm microRNA and

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DNA methylation of germ cells, which affected offspring health and transmitted obesity to
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future generations (Fullston et al., 2013). This is supported by a study showing that abnormal
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methylation status of F1 sperm, altering the expression of genes such as Igf2 and H19, is

transmitted to F2 offspring (Ding et al., 2012). In the Dutch famine cohort, the offspring of
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prenatally undernourished fathers were heavier and more obese than those of parents who
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were not exposed to prenatal undernourishment (Veenendaal et al., 2013). Consistent with the

previous results, de Castro Barbosa et al. (2016) demonstrated that miRNA expression and
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methylation changes induced by HFD in F0 spermatozoa showed the same pattern in F1.
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Increased Slc3a2 methylation and reduced methylation of Tbrg4 and Mfsd7 were observed
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both in F0 and F1 spermatozoa, and the expression of 10 miRNAs presented the same pattern

(de Castro Barbosa et al., 2016). Moreover, glucose intolerance was observed in F2 female

offspring, but not in males. Since the mother was fed SC, insulin resistance in female

offspring should be due to inheritance from F0 and F1 spermatozoa. These cases also showed

that paternal obesity/MetS affected only female offspring, indicating sex-specificity. In

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conclusion, these studies demonstrated that parental overnutrition delivers metabolic adverse

effects to offspring through epigenetic changes in sperm and eggs.

2.2.3. Effect of gestational overnutrition on offspring

What if mothers gained weight by excessive intake during the gestation period, although

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both parents were normal weight prior to conception? This situation is often observed in

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humans. Twenty-week-old adult mice whose mothers were fed a HFD during late gestation

presented a significant increase in caloric intake, body weight gain, and fat mass as well as

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insulin resistance and dyslipidemia (Khalyfa et al., 2013). Lower adiponectin expression and

higher leptin expression were observed in offspring adipose tissue, and the adiponectin

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promoter was hypermethylated (Khalyfa et al., 2013). Moreover, a report indicated that 7
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day-old pups whose mothers were fed a HFD during the entire gestational and breast feeding
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periods showed a significant increase in body weight, blood glucose levels, and liver TG

accumulation, while birthweights were not different (Yang et al., 2012a). Additionally,
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expression of Wnt1 and β-catenin, and acetylation of H4 and H3 (involved in the Wnt1
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promoter and coding region, respectively), was reduced, while H3K9 methylation was

increased in the liver of the offspring. It seems that maternal HFD suppressed Wnt/β-catenin
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signaling through histone modification in the liver of offspring, affecting liver development
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and incidence of MetS (Jin, 2008; Yang et al., 2012a).

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In addition, a high-lipid and high-energy diet (HLE) during the gestation and breast-

feeding periods induced global hypermethylation in the liver of the offspring. In particular,

methylation of Pparγ and Lxrα increased and their expression levels decreased (Yu et al.,

2015). These data indicate that HFD/HLE during gestation is sufficient to cause epigenetic

changes in the offspring, and the effects persist into adulthood. On the other hand, what if
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normal weight parents maintained their weight during the gestation period, but had postnatal

overnutrition? Neonatal overfeeding during the weaning period was enough to induce MetS

phenotypes, resulting in increased body weight and fat, and blood leptin, glucose, and insulin

levels (Plagemann et al., 2009; Plagemann et al., 2010). After weaning, methylation levels of

the insulin receptor (IR) and the POMC promoter in the hypothalamus significantly increased,

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and there was a positive correlation between the methylation level and blood glucose level

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(Plagemann et al., 2009; Plagemann et al., 2010). In the hypothalamus, insulin regulates food

intake by controlling the expression of neuropeptides. Thus, rapid weight gain by excessive

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postnatal overfeeding can cause diabetes and obesity disposition through methylation changes

in genes regulating food intake.

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2.2.4. Adverse effects of overnutrition are washed out in 3 generations
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So far, at least in animal studies, adverse effects of overnutrition are passed onto offspring,

and parental effects are not reset in offspring, but remain as a memory leading to MetS
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phenotypes, such as weight gain, insulin resistance, and dyslipidemia in adulthood (Khalyfa
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et al., 2013; Marco et al., 2014; Masuyama and Hiramatsu, 2012). The next question is: how

long do these effects last? Effects of a HFD arithmetically diminished over time and had
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almost disappeared from the third generation (Masuyama et al., 2015) based on body weight,
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HOMA-IR results, blood TG concentration, leptin level, and adiponectin mRNA expression
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in adipose tissue. Consistently, metabolically adverse effects of HFD almost disappeared in

the third generation in a murine model, assessed by determining body weight and insulin

sensitivity (Dunn and Bale, 2011).

Given that adverse effects of in utero exposure to maternal overnutrition persisted through

two generations, what are the effects of overnutrition in the neonatal stage, a relatively more

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developed stage than the fetus? Postnatal overnutrition during weaning produced metabolic

syndrome risk factors, such as obesity, insulin resistance, and glucose intolerance by the age

of 4 months (Pentinat et al., 2010). Follow up of the offspring indicated that adult F1

presented insulin resistance without obesity and F2 presented a weak insulin resistance

(Pentinat et al., 2010). In other words, overnutrition during the neonatal stage caused

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metabolically adverse effects in adulthood as well as in the next generation, and these effects

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almost disappeared in the second generation. Taken together, while infants in the neonatal

stage were also nutritionally sensitive, exposure to overnutrition during this stage presents a

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shorter wash out period than in utero exposure.

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2.2.5. Dietary intervention during the pregestation period can overcome the adverse
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effects of overnutrition
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In the case of parental overnutrition, offspring presented difficulties in recovering from

susceptibility to MetS even when continuously fed SC after weaning. If the offspring was
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also fed a HFD, the next generation presented worse metabolic phenotypes, due to the
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combined effects of parental and grandparental exposure to a HFD (Masuyama et al., 2015).

Hence, it is important to delete the epigenetic memory in our body through proper dietary
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intervention. In one study, epigenetic changes were ameliorated by diet or exercise

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intervention for 8 weeks in male obese mice fed a HFD (McPherson et al., 2015). Expression
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of mir-503 and mir-465b-5p decreased in mice fed a HFD compared to that of SC controls;

diet or exercise interventions restored mir-503 and mir-465b-5p expression to normal levels

(McPherson et al., 2015). In addition, diet or exercise intervention in obese fathers tended to

recover adiposity and insulin sensitivity in female offspring. These results suggest that

pregestational diet and exercise intervention by obese fathers could significantly prevent

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MetS predisposition in offspring.

Another study showed that weight reduction in obese adults could prevent metabolic

adverse effects through epigenetic changes, although the weight loss was not due to dietary

intervention, and its long-term effect was not examined (Marco et al., 2014). While obese rats

fed a HFD from 80 days before gestation until the breast feeding period showed decreased

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hypothalamic POMC expression and increased POMC promoter methylation, breast-feeding

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enhanced weight loss, reduced POMC methylation, and increased expression of Gadd45b, a

mediator of DNA demethylation.

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2.2.6. Is it possible to predict the success of dietary intervention in MetS?

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Discrepancy is often observed with a same diet intervention. Some subjects are successful
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in losing weight, while others respond weakly to the intervention. A study of an energy-
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restricted dietary program for patients with MetS showed that there were differences in

baseline methylation status in certain genes between high responders and low responders.
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Patients with obesity and MetS were subjected to 30% energy restricted dietary intervention
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for 8 weeks. Of these, those who lost over 8% of their weight after 8 weeks were classified as

high responders (39.6%) (Garcia-Lacarte et al., 2016). DNA methylation of long interspersed
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nuclear element 1 (LINE-1) in peripheral blood mononuclear cells (PBMCs) was

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significantly higher at basal LINE-1 in the high responder group (increased by 5.41%) than
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that of low responders, in which basal LINE-1 DNA methylation showed a positive

correlation with body weight loss. Therefore, these data suggest that LINE-1 methylation

level in PBMCs could be applied as a marker to predict the response to dietary restriction for

weight loss. In addition, baseline LINE-1 DNA methylation level was positively correlated

with baseline dietary total antioxidant capacity (TAC). It would be interesting to investigate

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whether sufficient intake of antioxidant substances in daily life can alter the LINE-1 DNA

methylation level, and alter the effects of hypocaloric diet intervention. In addition, people

with higher methylation levels of plasminogen activator inhibitor-1 (PAI-1, SERPINE1) in

white blood cells presented with a significantly higher reduction in weight, total fat mass,

blood total cholesterol level, and TG concentration after dietary intervention than those with

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lower levels (Lopez-Legarrea et al., 2013). In other words, a higher PAI-1 methylation level

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resulted in a greater response to intervention, and a higher total fat mass was correlated with a

lower PAI-1 methylation level (Lopez-Legarrea et al., 2013).

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On the other hand, arylesterase (ARE) and paraoxonase-1 (PON1) prevent the oxidation of

lipoprotein as an anti-atherosclerotic function. ARE activity showed a negative correlation

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with methylation level of certain CpG sites of PON1, while it positively correlated with
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dietary antioxidant levels (de la Iglesia et al., 2014). In addition, ARE activity was positively
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correlated with loss of body weight and fat, and reduced blood pressure and blood lipids after

dietary intervention (de la Iglesia et al., 2014). In other words, high responders to the
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intervention presented with a low basal PON1 methylation level and high ARE activity. This
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status could also be achieved by intake of antioxidants such as vitamin C or E (de la Iglesia et

al., 2014). Taken together, PON1 methylation and ARE activity may be considered as
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markers to predict response to diet. Milagro et al. (2012) demonstrated that methylation
C

levels of CpG sites in CLOCK and PER2 (clock genes) were positively correlated with the
AC

degree of weight loss in obese women.

In conclusion, it may be possible to predict the level of response to dietary intervention by

determining DNA methylation levels of LINE-1, PAI-1, PON1, CLOCK, and PER2 in the

blood. Otherwise, pattern analysis of methylation level among various candidate biomarkers

would be applicable to the prediction.

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2.2.7. Studies of biomarkers and miRNAs related with MetS

In a study of 200 obese subjects, Turcot et al. (2012) demonstrated that the methylation

level of LINE-1 in visceral adipose tissue (VAT) was negatively correlated with diastolic

blood pressure. It was also demonstrated that LINE-1 methylation level of VAT was

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correlated with MetS risk in obese subjects. However, since VAT is difficult to obtain, it

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would be necessary to further study the correlation of LINE-1 methylation levels in the blood

and VAT to be able to use it as a biomarker for MetS. Milagro et al. (2012) showed that

SC
certain epigenetic changes were correlated with phenotypes of obesity and diabetes in 60

normal weight or overweight subjects. Methylation levels of some CpG sites increased as

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factors such as BMI, adiposity, blood pressure, and MetS score became severe.
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In both diet-induced obesity (DIO) and db/db diabetic mouse models, Pparγ mRNA
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expression in VAT decreased and its promoter methylation level increased compared to those

of control mice (Fujiki et al., 2009). These results suggest that reduced expression of Pparγ
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due to DNA methylation in VAT may contribute to the pathogenesis of MetS, and DNA
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methylation level could be used as a biomarker in MetS.

Moreover, epigenome-wide methylation association studies (EWAS) can discriminate the

EP

phenotypic status of MetS. Two hundred fifty one CpG sites showed a significant difference
C

in methylation levels in control subjects with obesity and patients with T2D. Of these, 94%
AC

CpGs showed reduced methylation levels in patients with T2D. These CpG sites were located

on GRB10, ABCC3, MOGAT1, H19, and PRDM16 that are biologically relevant to the

development of T2D (Nilsson et al., 2015).

In a human study that comparatively analyzed miRNA expression in amnion from obese

and healthy weight mothers (n = 10, BMI > 30; n = 5, BMI < 25, respectively), 7 different

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miRNAs (miR-422b, miR-219, miR-575, miR-579, miR-523, miR-618, and miR-659) were

expressed only in obese mothers (Nardelli et al., 2014). Moreover, expression of 13 miRNAs

increased while 12 miRNAs were downregulated in obese mothers. These miRNAs are

involved with neurotrophin, cancer/ErbB, mammalian target of rapamycin, insulin,

adipocytokine, actin cytoskeleton, and mitogen-activated protein kinase signaling pathways.

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It is speculated that these miRNA profile changes affected placental growth and function. To

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determine how changes in methylation or expression of identified genes affects MetS,

functional validation of epigenetic changes on phenotypes must follow EWAS and miRNA

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analysis.

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3. Nutritional components and epigenetic regulation
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3.1. Methyl donors and cofactors
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In mammals, many dietary components containing folate, vitamin B6 (B6), vitamin B12
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(B12), betaine, choline, methionine, and serine are linked to epigenetic changes through the
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transfer of methyl groups from S-adenosyl methionine (SAM) to DNA and histone (Feil and

Fraga, 2011; Locasale, 2013; Milagro et al., 2012). Dietary sources of carbon units enter the
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one-carbon metabolism pathway consisting of folate and methionine cycles, and are

processed through these cycles affecting nucleotide metabolism, NADPH/NADP+ ratio,

C

protein translation, and redox balance as well as methylation levels of DNA and histones. For
AC

example, more folate is required to remethylate homocysteine in choline/betaine deprivation

conditions, and, conversely, requirements of choline/betaine increase in folate deprivation

conditions (Milagro et al., 2012). Epigenetic changes induced by methyl donors and cofactors

are well demonstrated in animals and cells. Supplementation with choline, betaine, and folic

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acid in pregnant mice stimulates DNA hypermethylation in the offspring, which prevents

MetS phenotypes in Avy/a mice (Wolff et al., 1998). Similarly, supplementation with choline,

betaine, methionine, folic acid, and B12 throughout pregnancy changes germline epigenetic

state in Avy/a mice (Cropley et al., 2006). Consistent with these, folic acid, B12, betaine, and

choline induce DNA hypermethylation and prevent intergenerational obesity in Avy/a mice

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(Waterland et al., 2008). In sheep, periconceptional deprivation of B12 and folate leads to

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increased fat, insulin resistance, and higher blood pressure in offspring through alteration of

DNA methylation (Sinclair et al., 2007). In humans, low maternal B12 and high folate levels

SC
during gestation lead to increased adiposity and insulin resistance in offspring (Yajnik et al.,

2008), while antenatal supplementation with folic acid reduced offspring MetS incidence in

U
Nepal (Stewart et al., 2009). In addition, moderate folate depletion causes DNA
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hypomethylation in healthy weight, postmenopausal women (Jacob et al., 1998; Rampersaud
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et al., 2000). Based on these observations, it can be hypothesized that dietary methyl donors,

or at least folate and folic acid, may affect MetS through regulation of DNA methylation.
D

In addition to DNA methylation, effects of methyl donors on histone modification play

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crucial roles in MetS. It has been demonstrated that a methyl-deficient diet (low methionine

without choline and folic acid), caused epigenetic alterations as well as pathomorphological
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changes in an animal model of nonalcoholic steatohepatitis (Pogribny et al., 2009). In the

C

liver of mice fed a methyl-deficient diet, hypomethylation of global and repeat sequences,
AC

such as LINE, SINE, and minor satellites was observed, while aberrant methylation of

histone H3 was evident. In postmenopausal women with nonalcoholic fatty liver disease,

deficient choline intake was associated with increased fibrosis (Guerrerio et al., 2012).

Although it is still unclear whether observations in animals can be reproduced in humans, due

to the paucity of intervention studies in humans, many data strongly suggest that dietary

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methyl donors and cofactors affecting the one-carbon metabolism pathway alter DNA

methylation and/or histone modification in MetS. They also suggest that reversible changes

of abnormal epigenetic status in diseases effected by dietary components may treat and

prevent further disease progression.

As noted above, SAM is synthesized in the one-carbon metabolism pathway and functions

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as a methyl donor in DNA methylation processes. Thus, it can be said that DNA methylation

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response is dependent on SAM level. For this reason, methionine, choline, folate, and B12

are classified as methyl donor nutrients and have a significant impact on DNA methylation

SC
either directly or indirectly. In particular, methionine can have the most direct effect as a

SAM precursor. Since SAM is converted to S-adenosyl homocysteine (SAH) after providing

U
a methyl group, SAM levels or tissue specific SAM:SAH ratios are sometimes used as a
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methylation index, although they are not absolute (Waterland, 2006). The most dramatic
M

example of the importance of methyl-rich nutrients for DNA methylation and epigenetics is

the markedly altered fur color and body fat mass of the Agouti mouse fed a methyl-rich diet.
D

The Agouti gene determines the fur color of mammals, and mice with the variable yellow
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Agouti (Avy) gene mutation have yellow, spotted fur and increased fat mass. When pregnant

Agouti mice were provided with a methyl-rich diet supplemented with folate, B12, betaine,
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and choline, high levels of SAM were synthesized leading to increased methylation in the
C

Agouti gene, which resulted in lean offspring with brown fur. Therefore, intake of methyl
AC

donors could directly affect DNA methylation levels and etiology (Cooney et al., 2002).

3.1.1. Effects of methyl donor deficiency on offspring

When rats were fed a methyl donor-deficient diet from 1 month before gestation until the

weaning period, their pups showed a significant reduction in body weight, and blood glucose,

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B12, and folate levels, while they had significantly higher levels of blood homocysteine, total

cholesterol, TG, FFA, and AST (Pooya et al., 2012). In the liver, SAM levels and SAM:SAH

ratios were significantly lower, whereas hepatic total lipid and TG concentrations were 5-7

fold higher. Methyl donor deficiency caused hypomethylation of PGC1, which significantly

reduced its binding with functional partners, including PPARα, ERRα, and HNF4α.

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Consequently, overall fatty acid oxidation was reduced (Pooya et al., 2012). These results

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indicated that a methyl donor-deficient diet during the gestation and breast-feeding periods

not only reduces the weight of offspring, but also causes hypomethylation of fatty acid

SC
oxidation-related genes in the liver, leading to liver steatosis.

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3.1.2. Methyl donor supplemented diet in undernutrition
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In an IUGR rat model, F2 offspring fed a methyl donor-supplemented diet showed
M

significant reductions in fat mass, and blood TG, VLDL, and FFA levels than F2 offspring fed

a control diet (Goodspeed et al., 2015). In addition, a significant improvement in insulin

D

resistance was observed (Goodspeed et al., 2015). Moreover, the F2 offspring of F1 with
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IUGR showed a 2.5-fold increased level of methylation in the IGF-1 promoter compared to

that of the offspring from the sham group, whereas F1 and F2 groups fed a methyl-rich diet
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showed a significant reduction in DNA methylation. A lower level of H19 expression was
C

observed in the liver of F2 rats born from F1 with IUGR, while H19 mRNA expression was
AC

increased by 6.6-fold. Expression of the H19 gene was lost before the weaning period in the

F2 offspring compared to the sham controls, whereas F2 rats fed a methyl-rich diet

maintained H19 expression longer and showed no MetS in adulthood (Gonzalez-Rodriguez et

al., 2016). This suggests that a methyl donor-supplemented diet will prevent predisposition to

MetS caused by undernutrition to some extent.

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3.1.3. Epidemiologic study of the correlation between methyl donor intake and MetS

In a study of about 500 mothers and infants with or without methyl donor intake, the group

with a high B12 intake (the highest quartile) showed a lower level of weight gain in 3-year-

old children than the group with a low B12 intake. As the maternal homocysteine level

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increased, male birth weight decreased. As mothers had a higher B6 (pyridoxal phosphate,

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PLP) intake, the methylation level at the MEG3DMR was higher (McCullough et al., 2016).

Therefore, it seems that maternal B vitamin intake affects children’s birth weight or neonatal

SC
weight gain, which may also affect later predisposition to metabolic disease.

3.2. Protein
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AN
3.2.1. Protein deficiency and glucose metabolism in offspring
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When pigs were fed a low protein (LP) diet during the full gestation period, male and

female offspring presented significantly lower birth weights, liver weights, and blood glucose
D

levels (Jia et al., 2012). However, male offspring presented significantly elevated levels of
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hepatic glucose-6-phosphatase (G6PC) activity and mRNA expression, and only male

offspring showed significant reduction in its promoter methylation. In addition, the G6PC
EP

promoter of LP-fed male presented lower levels of H3K9me3 and H3 and increased levels of
C

H3 acetylation and H3K4me3 (Jia et al., 2012). Based on these results, it is thought that
AC

maternal intake of LP inhibits fetal development and results in low glucose levels in offspring.

This results in epigenetic memory of G6PC hypomethylation, which might cause

hyperglycemia onset in adulthood, if it persists. On the other hand, the effect of maternal LP

intake on epigenetic change was sex-specific; the G6PC promoter of female offspring

showed high levels of H3K4me3, H3K9me3, and H3K27me3 (Jia et al., 2012). Meanwhile,

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when rats were provided with LP at the same 1/2 level (9%; control, 18%) during the full

gestation period, weights of both male and female pups were significantly lower, whereas

altered GLUT4 was found only in females (Zheng et al., 2012). The GLUT4 promoter of

skeletal muscle in female pups (D38) showed increased levels of H3ac, H4ac, and H3K4me2,

and GLUT4 expression levels were higher. Although glycogen content and expression level

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of glycogen synthase also increased, there was no significant difference in males. Thus,

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despite the fact that the effect of a maternal LP diet on glucose metabolism in offspring was

sex-specific (Zheng et al., 2012), this sex-specificity in rats was different from the results of

SC
the study using pigs. Taken together, a maternal LP diet inhibits fetal development,

particularly the development of the liver, and induces epigenetic change in genes regulating

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blood glucose leading to adverse effects in adult glucose metabolism.
AN
M

3.2.2. Protein deficiency and adiposity in offspring

The adult offspring of mice fed with an LP diet during the gestation had lower body weight
D

and less adipose tissue, even though SC was supplied (Jousse et al., 2011). In addition,
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fasting leptin levels were low, and leptin expression was stimulated several fold after feeding.

This rapid leptin expression in LP offspring may be due to the epigenetic memory of the
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nutritional stress in utero induced by the mother’s low protein intake (Jousse et al., 2011).
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Despite the low body weight and low adiposity, continued high food intake would cause an
AC

imbalance between food intake and energy expenditure contributing to MetS later in life.

3.2.3. Protein deficiency and the regulation of cholesterol in offspring

When rats were fed an LP diet during gestation, their offspring had a significantly lower

body weight at day 130 even with a normal postnatal diet, and only male offspring had high

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blood and liver cholesterol levels (Sohi et al., 2011). Expression levels of cholesterol 7α-

hydroxylase (Cyp7α1), involved in the maintenance of cholesterol homeostasis, were lower

in both male and female LP offspring at day 21. At day 130, the significant difference

disappeared in female adults, while male offspring maintained the downregulation. These

effects seem to be attributable to reduced acetylation of H3K9 and increased methylation of

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H3K9 at the Cyp7a1 promoter region in male LP offspring (Sohi et al., 2011).

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In conclusion, maternal protein restriction inhibits fetal development, particularly liver

development, and epigenetic changes occurring in utero can cause dysfunction in glucose

SC
metabolism, adiposity, and cholesterol metabolism in adulthood, leading to predisposition to

MetS.

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AN
3.3. Polyunsaturated fatty acid (PUFA)
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3.3.1. Genes altered by methylation associated with PUFA intake

In a study of 70 Greek children below 10 years of age, genes in which the methylation
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level of CpG sites/islands was correlated with the intake ratio of poly- and mono-UFA to
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saturated FA ([PUFA+MUFA]:SFA) were identified (Voisin et al., 2015). Representative

genes, selected based on coefficient and adjusted p-value, were NCOA1, PCED1A, and
EP

CCNA2 (Voisin et al., 2015). In a similar study, DNA from 185 Yup'ik Alaska Native adults
C

was subjected to EWAS analysis to identify CpG sites showing a correlation between
AC

methylation level and n-3 PUFA intake amount. Among them, 27 differentially methylated

CpG sites with p <1 × 10-7 were selected (Aslibekyan et al., 2014). These genes included

helicase-like transcription factor, actin α2 smooth muscle/Fas cell surface death receptor, and

protease serine 36/C16 open reading frame 67 (Aslibekyan et al., 2014). It was reported that

CpG 1 and 8 sites of CLOCK, related with circadian rhythm, also showed a negative
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correlation with MUFA intake and a positive correlation with PUFA intake (Milagro et al.,

2012). Although these studies revealed a statistically significant correlation between PUFA

intake and DNA methylation level in certain genes, it seems necessary to conduct a functional

validation study on how methylation levels and functions of these genes are actually altered

by fat/UFA intake.

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3.3.2. Effects of PUFA intake on genes involved in PUFA synthesis

Daily supplementation with olive oil or n-3 PUFA in people in their 50s and 60s reduced

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mRNA levels of fatty acid desaturase 2 (FADS2) and elongation of very long chain fatty acid

5 (ELOVL5), and increased the level of methylation at certain CpG sites in regulatory

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regions of these genes (Hoile et al., 2014). In particular, females showed greater differences
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between gene expression and methylation level, whereas there was no difference in the
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expression and methylation of FADS1, the rate-limiting enzyme for PUFA synthesis in the

liver. Consistently, adult offspring of rats fed with fish oil or butter from 2 weeks before
D

gestation to the weaning period showed a higher level of methylation in the FADS2 promoter
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and low expression, regardless of the fatty acid source (Hoile et al., 2013). In addition, both

20:4 n-6 and 22:6 n-3 contents in the liver and plasma decreased as maternal fat intake
EP

increased, regardless of the fatty acid type (Hoile et al., 2013). However, FADS1 expression
C

was not affected by fat intake. Despite the discrepancy between experimental models and
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subjects of analysis, the two studies clearly showed that higher fat intake increased

methylation of FADS2 and decreased its expression. In rats, maternal fat intake affected

PUFA synthesis by inducing epigenetic changes in enzymes involved in PUFA synthesis,

even in adult offspring.

However, an epidemiologic study reported that PUFA intake reduced the risk of

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cardiovascular MetS, and the group with high n-3 PUFA intake presented lower blood TG

and higher HDL-C levels than the group with low n-3 PUFA intake (Aslibekyan et al., 2014).

There were differences in phenotypes resulting from PUFA intake between the epidemiologic

study results and actual experimental results. Therefore, it appears necessary to further

investigate the effects of FADS2 expression reduction/methylation change by PUFA/MUFA

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intake on PUFA synthesis and metabolism.

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3.4. Sugar

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Frequent intake of simple sugars, such as fructose, raises blood glucose levels and causes

weight gain, negatively influencing MetS. Rats fed a 20% fructose solution for 14 weeks

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displayed weight gain, as well as dyslipidemia and hepatic steatosis. At the same time, it was
AN
observed that mtDNA content significantly increased, and transcription of mitochondrial
M

genes such as mt-ND3 and mt-CO1 also significantly increased along with global

hypomethylation of mtDNA. It seems that epigenetic change was involved in increases of

D

both hepatic mtDNA content and transcription by fructose (Yamazaki et al., 2016). On the
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other hand, fructose intake induced global DNA hypermethylation in the liver. Steatosis and

dyslipidemia by fructose intake seemed attributable to significant reduction of PPARα and

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CPT1α expression among lipid metabolism-related genes. The upstream regions of both
C

genes presented elevated DNA methylation, indicating that fructose caused epigenetic change
AC

in genes responsible for oxidation of fatty acid, leading to downregulation of gene expression

(Ohashi et al., 2015).

3.5. Bioactive food components and epigenetics

Not only methyl donors, but also food components consumed in daily life effect epigenetic
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modifications in offspring. Dolinoy et al. (2006) demonstrated that dietary genistein, the

major phytoestrogen in soy products, alters the epigenome of the offspring in utero and

consequently affects gene expression. The report also showed that genistein-induced DNA

hypermethylation persisted into adulthood, leading to changed coat color and protection from

obesity in Avy/a mouse offspring. Dolinoy et al. (2007) also showed that maternal

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supplementation with genistein negated the DNA hypomethylation induced by bisphenol A, a

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high-production-volume chemical associated with human health. These data suggest that

changes in DNA methylation, by environmental factors at least, are reversible, and regulation

SC
of DNA methylation is applicable to the treatment and prevention of diseases with which it is

associated. Genistein is a flavonoid mainly found in soybeans and known as a phytoestrogen

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interacting with estrogen receptors because its structure is similar to 17β-estradiol. Many
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human and animal studies have demonstrated various health beneficial effects of
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phytoestrogens in cardiovascular disease, hyperlipidemia, osteoporosis, and cancer (Bhathena

and Velasquez, 2002). Consistently, genistein is also beneficial in MetS, inflammation,

D

diabetes, and cancer (Guo et al., 2015; Nagaraju et al., 2013; Shay et al., 2015). Since the first
TE

identification of genistein as a regulator of DNA methylation in 2002 (Day et al., 2002),

many researchers have investigated genistein for the treatment of various cancers caused by
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impaired DNA methylation. In a study using nonhuman primates (Howard et al., 2011), it
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was demonstrated that soy proteins, including genistein and daidzein, improve body weight,
AC

insulin sensitivity, and lipid profiles, and change DNA methylation patterns in the liver and

muscle. Using HumanMethylation27 BeadChip, overall methylation increased in the group

fed a diet with soy proteins compared to the control group fed a diet with casein. Although it

was not clearly explained and further studies are needed, gender-specific effects of genistein

exposure were reported (Strakovsky et al., 2014). Administration of genistein to pups from

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postnatal day 1 to 22 increased fat mass, fat/lean mass, and adipocyte size and number, and

decreased muscle fiber perimeter in females, but not in males. Expression of adipogenic

factors and Wnt10b was decreased only in females. Genistein affects not only DNA

methylation, but also histone modification. Farhan et al. showed for the first time that

genistein abolishes the expression of cytochrome enzyme CYP27B1, possibly by inhibiting

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histone deacetylase (HDAC) in prostate cancer cells (Farhan and Cross, 2002; Farhan et al.,

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2003). Since chromatin immunoprecipitation confirmed the role of genistein as an inhibitor

of HDAC in human bladder cancer cells (Pong et al., 2006), many studies supported the

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inhibitory role of genistein, especially in cancer. Although no report demonstrated that

genistein affects histone acetylation in MetS, due to its structural similarity to estrogen,

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studies should be conducted to treat menopausal symptoms such as obesity and osteoporosis.
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Catechins, including catechin, epicatechin, their oligomers (proanthocyanidin), and
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epigallocatechin-3-gallate (EGCG) are natural phenolic compounds mainly found in cocoa,

green tea, prunes, apples, and grape seeds. Although their various physiological roles in
D

cancer, Alzheimer’s disease, and aging are known (Afzal et al., 2015), they are considered
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biological modulators of MetS (Milagro et al., 2013). Supplementation with catechins in diet-

induced obese mice significantly reduces body weight gain, fat accumulation, and levels of
EP

insulin and leptin in the blood, and stimulates lipid catabolism by regulating the expression of
C

acyl-CoA oxidase and acyl-CoA dehydrogenase (Murase et al., 2002). In addition, EGCG
AC

reduces body weight gain, fat accumulation, blood glucose levels, TG levels, liver damage,

and insulin resistance, and attenuates inflammatory cytokines such as monocyte

chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interleukin-6 (IL-6), and

granulocyte colony-stimulating factor (G-CSF) in high-fat/Western-style diet-induced obese

mice (Chen et al., 2011). Catechin attenuates dyslipidemia and insulin resistance and reduces

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inflammatory cytokines such as adiponectin, resistin, visfatin, TNF-α, and MCP-1 in adipose

tissue of high-fructose-fed rats (Vazquez Prieto et al., 2015). Yun et al. (2010) demonstrated

that some of these effects are mediated by epigenetic regulation in normal-weight and obese

human subjects. They showed that EGCG treatment increases HDAC activity and HDAC-2

expression in regulatory T cells (Tregs) and decreases NF-κB expression. EGCG also

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increases IL-10 production and the number of Tregs expressing Foxp3 in human subjects.

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Epigenetic regulation by EGCG is also supported by a report showing that it inhibits histone

acetyltransferase (HAT) activity and NF-κB activation (Choi et al., 2009). Another epigenetic

SC
mechanism of catechins appears to be the inhibition of DNA methylation since they inhibit

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DNA methyltransferases in different human cancer cell lines and in vitro assay systems (Fang
AN
et al., 2003; Lee et al., 2005).

Curcumin is a polyphenol mainly found in turmeric. Curcumin has anticancer, anti-

M

inflammatory, and antioxidant effects in animals and cells (Sharma et al., 2005). In addition,

curcumin is considered as an anti-obesity molecule since it suppresses the differentiation of

D

3T3-L1 adipocytes and reduces body weight gain and adiposity, probably by regulating
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oxidation and fatty acid esterification in high-fat-fed mice (Ejaz et al., 2009). The anti-obesity

effects of curcumin are also supported by reports demonstrating that curcumin treatment
EP

improves obesity-related inflammation and diabetes in high-fat-fed mice by regulating

C

glucose levels, adiponectin production, and NF-κB activity (Weisberg et al., 2008). In
AC

addition, curcumin attenuates hyperlipidemia and insulin resistance in high-fat-fed hamsters

by lowering levels of hepatic cholesterol and TG, free fatty acid, and leptin (Jang et al., 2008).

Consistent with the widely appreciated crucial roles of inflammation in MetS such as obesity,

diabetes, and atherosclerosis (Baker et al., 2011), Shanmugam et al. (2003) demonstrated that

increased blood glucose can stimulate inflammatory signaling cascade in monocytes through
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the NF-κB pathway in diabetes. Curcumin inhibits NF-κB activity in human lymphoma cells

(Shishodia et al., 2005). It is noteworthy that high-glucose-induced changes in pro-

inflammatory cytokines such as TNF-α, and IL-6 are mediated by HAT and HDAC activities,

which are inhibited by curcumin (Yun et al., 2010). In addition, since curcumin was

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suggested as a DNA hypomethylating agent (Liu et al., 2009), many reports have

demonstrated that curcumin demethylates promoter regions of genes in different cancer cell

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lines (Jiang et al., 2015; Khor et al., 2011; Link et al., 2013; Liu et al., 2011; Parashar et al.,

SC
2012; Shu et al., 2011). However, there is no direct evidence that curcumin affects DNA

methylation in MetS.

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Fisetin is a dietary flavonoid mainly found in strawberries, apples, onions, and cucumbers
AN
and is known as an anti-aging, anticancer, and antiviral agent. In relation to adipogenesis,

fisetin inhibits early adipogenesis by facilitating Sirt1-mediated deacetylation of PPARγ and

M

Foxo1 in 3T3-L1 cells (Kim et al., 2015). Fisetin also inhibits hyperglycemia-induced
D

production of inflammatory cytokines, such as IL-6 and TNF-α, by reducing expression and
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HAT activity of CBP/p300, a NF-κB coactivator, in monocyte cells (Kim et al., 2012).

Although fisetin can regulate DNA methylation by inhibiting DNA methyltransferase in in

EP

vitro systems, no study has investigated the effects of fisetin on DNA methylation in MetS

(Lee et al., 2005; Yin et al., 2013).

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Sulforaphane (SFN), an organosulfur compound, is an isothiocyanate found in cruciferous

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vegetables such as broccoli, mustard, radish, kale, and cabbages. Many researchers have

investigated SFN for its beneficial effects on oxidative stress, neurodegeneration, and human

colon and prostate cancers (Clarke et al., 2008; Tarozzi et al., 2013). The chemopreventive

effect of SFN was demonstrated by reports showing that it increases acetylation of histones

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by inhibiting histone deacetylase (HDAC), leading to the activation of cell cycle arrest and

apoptosis of prostate and colorectal cancer cell lines (Myzak et al., 2006; Myzak et al., 2004).

The inhibitory effect of SFN on HDAC was reproduced in xenograft animal models (Myzak

et al., 2007). In addition, SFN inhibits HDAC activity in cervical and lung cancer cells (Ali

Khan et al., 2015; Jiang et al., 2016), and can reduce DNMT1 activity in human colon Caco-2

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and prostate cancer cells, although whether SFN stimulates demethylation of specific CpG

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sites remains controversial (Traka et al., 2005). Recently, it has been demonstrated that SFN

plays a role as an anti-obesity molecule by inhibiting adipogenesis and lipogenesis in an

SC
HFD-induced obese mouse model (Choi et al., 2014). In these mice, SFN decreased the

expression of PPARγ, C/EBPα, and leptin, which led to the suppression of adipocyte

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differentiation. In addition, SFN increases the phosphorylation of AMP-activated protein
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kinase and acetyl-CoA carboxylase, which results in the inhibition of lipogenesis and the
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promotion of fatty acid oxidation. Consistent with the animal model, SFN inhibits adipocyte

differentiation in cell-based systems through cell cycle regulation (Choi et al., 2012). In
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relation to diabetes, SFN effectively reverses various deficits in experimental diabetic

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neuropathy by regulating nuclear erythroid 2-related factor and NF-κB (Negi et al., 2011).

However, no report has demonstrated epigenetic regulation by SFN in obese and diabetes
EP

models; thus, further studies must be carried out.

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Resveratrol is a stilbenoid contained in grapes, blueberries, and mulberries. It has various

AC

health beneficial roles in obesity, T2D, cardiovascular diseases, aging, and nonalcoholic fatty

liver disease (Baile et al., 2011; Bujanda et al., 2008; Camins et al., 2010). Although many

studies in cancers demonstrate that resveratrol regulates DNA methylation and histone

modification (Gao and Tollefsbol, 2015; Kala et al., 2015; Mirza et al., 2013), few studies

have focused on its role in MetS. In a recent report, it was demonstrated that resveratrol
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inhibited the expression of proinflammatory cytokines, such as IL-1β, IL-6, TNF-α, and IFN-

γ, through DNA hypomethylation at promoter regions; conversely, anti-inflammatory IL-10

expression was decreased by DNA hypermethylation in diabetic rat aortas (Lou et al., 2014).

However, the reason resveratrol has different effects on DNA methylation located at different

genomic regions remains poorly understood. It was recently reported that resveratrol and its

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structurally similar chemical, pterostilbene, inhibit the increase of adipose tissue weight and

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reduce Fasn expression in adipose tissue derived from diet-induced obese rats (Gracia et al.,

2014). However, only pterostilbene can alter the DNA methylation status of the Fasn

SC
promoter induced by obesogenic diet; resveratrol had no effect. Further studies, including

investigation of additional DNA methylation regions, must be conducted to clarify whether

U
resveratrol can affect DNA methylation. Resveratrol also affects histone modification. Han et
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al. (2015) recently demonstrated that resveratrol prevented deoxycorticosterone acetate-
M

induced hypertension in rats by altering histone methylation of the aorta and renal artery.

Since resveratrol also inhibits proliferation and adipogenesis in a SIRT1-dependent manner

D

(Rayalam et al., 2008), while it induces apoptosis of adipocytes in a SIRT1-independent

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manner (Mader et al., 2010), more studies must be conducted.

Quercetin, a flavonoid found in radish, onion, kale, and cranberry, has various biological
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properties that improve human health and diseases such as cancer, inflammation, and viral
C

infection (Li et al., 2016). Although quercetin effectively reduces adipogenesis in

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preadipocytes (Moon et al., 2013; Yang et al., 2008; Yang et al., 2012b), the underlying

mechanisms are not fully understood. One of the putative mechanisms is the regulation of

histone acetylation, which is supported by reports demonstrating that quercetin regulates

histone acetylation in cancer cells (Jia and Chen, 2008; Lee et al., 2011; Yoshida et al., 1992).

A recent report clearly showed that quercetin attenuates MetS by modulating the expression

35
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of histone deacetylases SIRT1 and SIRT2 in white adipose tissue, although further studies

will be carried out to understand the effect of combination with resveratrol on adipogenesis

(Peredo-Escarcega et al., 2015). Quercetin also regulates DNA methyltransferase and may

affect DNA methylation in vitro (Lee et al., 2005). A study using an animal model showed

that quercetin decreases DNA methyltransferase activity and DNA methylation of the NF-E2

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related factor 2 (Nrf2) promoter and consequently protects against inflammation (Liu et al.,

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2015). DNA hypomethylation by quercetin is also supported by reports showing decreased

DNA methylation in the p16INK4a promoter in colon cancer and esophageal cancer cells

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(Tan et al., 2009; Zheng et al., 2014). Since it is evident that global and site-specific

methylation status is affected by quercetin, further studies are required to demonstrate the

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roles of quercetin on DNA methylation in MetS.
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Diallyl disulfide (DADS) is an organosulfur compound found in garlic and onion.
M

Although the roles of DADS in DNA methylation have not been extensively studied, its roles

in histone modification are well known. DADS increases the acetylation of histones H3 and
D

H4 and expression of adipogenesis-related genes such as LPL, FAS, and SREBP1c, not only
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in cancer, but also in adipogenesis (Lee et al., 2007), confirmed by oil red O staining.

Together, these data suggest that DADS regulates adipocyte differentiation through histone
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acetylation.
C
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4. Summary and future perspectives

In terms of the relationship between nutrition and MetS, undernutrition and overnutrition

are the most studied topics. Focus on those areas is attributable to the primary interest in

energy balance, a particular issue with MetS. It is clear that parental nutrition is a critical

factor determining the health of descendants that continuously affects offspring throughout
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life. Proper nutritional supply to mother is critical during gestational stages with the most

active developmental progress. As the abnormal methylation status of F1 sperm and the MetS

phenotypes under malnutrition is transmitted to F2 offspring, paternal nutrition is also critical

factor to understand it.

Given that these were first-generation studies on establishing the relationship between food

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and nutrition, MetS, and epigenetics, second-generation studies should focus on finding

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appropriate nutritional interventions to restore deleterious epigenetic changes. In addition, as

the expected lifespan increases, people will have much longer lives after childbirth. Thus,

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studying the effect of current nutritional status in childhood/adolescence/adulthood on the

delayed onset or prevention of MetS within one generation and beyond transgenerational

U
inheritance is needed. Although previous studies showed that environmental factors, such as
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malnutrition, methyl donors, and phytochemicals, affect epigenetic modification and health in
M

offspring, further studies are needed to investigate their underlying mechanisms and to

develop clinical applications.

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Acknowledgements
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This project was supported by the Nutritional Epigenomics Study of the Korea Food

Research Institute (KFRI, E0150302). Authors do not have any conflict of interest. We thank
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Drs. Sang Woon Choi (Chaum Life Center, CHA University), Dae Young Kwon (KFRI), and
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MoonJu Hong (KFRI/UST) for fruitful discussions.

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Figure legend

Figure 1. Summary on transgenerational inheritance of metabolic phenotypes by nutritional

factor

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Table 1. Example of epigenetic marks influenced by nutritional factors
Nutritional exposed
Adult offspring (F1) F2 Species Ref
Factors subject/time
Undernutrition Pregnant/ (Heijmans,
IGF2 me ↓ human
1st trimester 2008)
SMAD7, CDH23, INSR, RFTN1,
Pregnant human (Tobi, 2014)
CPT1 , KFL13
hypothalamic GR me↓, H3K27
Pregnant/ 1st half sheep (Begum, 2013)
me↓, H3K9Ac
(Gonzalez-

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Pregnant/ E19- liver H19 me↑ rat Rodriguez,
2016)
Insulin H3K9 me,
Pregnant rat (Hardikar, 2015)
H3K20 me↑
(offspring: ad libitum SC) IGF1
Pregnant/ 2nd half rat (Tosh, 2010)

RI
H3K4me ↓
Overnutrition (Masuyama,
Before-during adiponectin H3K9ac↓, H3K9me↑ 2012;
mouse
pregnancy leptin H4K20me ↑ Masuyama,
2015)

SC
Before-during
POMC me↑ rat (Marco, 2014)
pregnancy
Pregnant(2nd adiponectin ↑
mouse (Khalyfa, 2013)
half)-weaning leptin↓
Full-term

U
(D7) Wnt1 H4ac↓, H3ac↓,
pregnancy- rat (Yang, 2012)
H3K9me↑
weaning
Full-term
AN
global hypermethylation in liver
pregnancy- mouse (Yu, 2015)
PPARγ me↑, LXR  me↑
weaning
(Plagemann,
(D21) insulin receptor me↑, 2009;
neonatal-weaning rat
POMC me↑ Plagemann,
M

2010)
Before Slc3a2 me↑, Tbrg4 me↓, Mfsd7
(de Castro
pregnancy(paternal me↓, miRNA let-7c↑ in rat
Barbosa, 2016)
only) spermatozoa
D

CLOCK, BMAL1, PER2 me in

human (Milagro, 2012)
blood
PPARγ me↑ mouse (Fujiki, 2009)
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Methyl-rich Pregnant Avy me↑ mouse (Cooney, 2002)

diet
(Goodspeed,
IUGR offspring IGF2 me↓ rat
2015)
(McCullough,
Vitamin B6 (neonate) MEG3 me↑ human
2016)
EP

Before-during
Methyl donor-
pregnancy- (D21) PGC1- me↓ rat (Pooya, 2012)
deficiency
weaning
Low protein
C

(neonate) G6PC me↓, H3K9me↓,

Pregnant pig (Jia, 2012)
H3 ac↑, H3K4me↑ in male liver
AC

(D38) GLUT4 H3 ac, H4ac,

Pregnant H3K4me↑in female skeletal rat (Zheng, 2012)
muscle
Pregnant Leptin me↓ mouse (Jousse, 2011)
Pregnant Cyp7 1 H3K9 ac↓, H3K9me↑ rat (Sohi, 2011)
Unsaturated Adult FADS2 me↑, ELOVL me↑ human (Hoile, 2014)
fatty acid Before-during
pregnancy- FADS2 me↑ rat (Hoile, 2013)
weaning

59