Obesity, a complex and multifactorial disorder, has emerged as a global epidemic with a considerable impact on public health, quality of life, and healthcare expenditure. The incidence of obesity has significantly increased worldwide over the past several decades.¹ While genetic factors play an important role in the development of obesity, the rapid increase in the number of people with obesity is believed to be because of dramatic changes in environmental factors, such as dietary shifts toward high-fat and high-sugar foods, reduced physical activity, and reduced energy expenditure.^1,2 However, little is known regarding how environmental factors lead to obesity.

Epigenetic modifications, such as DNA methylation, can lead to adaptive and temporal changes in gene expression in response to environmental factors. Studies have demonstrated that DNA methylation plays a role in the programming of body size^3,4 and is associated with body weight and obesity.^5-7 Therefore, understanding DNA methylation may provide insights into the development of obesity caused by environmental factors and the increase in the obesity rate.

Effective anti-obesity drugs, such as glucagon-like peptide-1 analogs, have been recently developed, but the risk of weight rebound after drug withdrawal has not been eliminated. Future therapeutic applications targeting DNA methylation may lead to an obesity treatment that does not suffer rebound because DNA methylation affects the trend of gene expression. The hypothalamic feeding center is a key region in the systemic energy balance, which can be influenced by epigenetic factors. In the present review, we explore the role of DNA methylation in the development of obesity, focusing on the hypothalamic feeding center.

The addition of a methyl group to the fifth-position carbon of cytosine (5-methylated cytosine [5mC]) followed by a guanine in a sequence called CpG is a major methyl modification of DNA.⁸ Recent studies have identified methyl modifications other than 5mC on DNA, such as N6-methyldeoxyadenosine (m6dA);⁹ however, the rate of these other modifications is much lower than that of 5mC⁹ and their physiological roles remain relatively unknown. Therefore, in this review, we focus on 5mC.

Hypermethylation of CpG islands in promoter regions represses gene expression.⁴ DNA methylation involves the addition of methyl groups by DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, and the removal of methyl groups by DNA demethylases, including the ten–eleven translocation methylcytosine dioxygenases (TETs) TET1, TET2, and TET3.⁸ DNA methylation directly affects gene expression and contributes to various biological processes, including cell differentiation, genome imprinting, epigenetic inheritance, and environmental adaptation.^10,11

Notably, DNA methylation has been strongly associated with body size. In honeybees, the genetic composition of worker and queen bees is the same; however, when a young bee is fed royal jelly during the larval stage, it becomes sexually mature and increases in body size to develop into a queen bee. Knockdown of DNMT3 during the larval stage induces queen bee development without royal jelly feeding,¹² suggesting that the downregulation of DNMT3-induced DNA methylation mediates nutrition and sexual maturation/growth. Indeed, DNA methylation patterns in the brain differ between queens and workers.¹³ These findings indicate that DNMT3-induced DNA methylation plays an important role in body size programming in honeybees.

DNMT3A has been reported to be associated with growth and obesity in humans. Overgrowth syndromes are a group of disorders caused by several different genes that cause excessive weight gain and enlargement of the head and/or extremities at or after birth.¹⁴ One type of overgrowth syndrome, Tatton–Brown–Rahman syndrome (TBRS), is caused by mutations in DNMT3A.⁴ In this syndrome, partial or total loss of DNMT3A function is presumed to be because of heterozygous missense mutations, nonsense mutations, frameshift mutations, and deletions in DNMT3A.^6,15 In patients with TBRS, more than 80% exhibit overgrowth, in which the height and/or head circumference are at least two standard deviations higher than the average. In addition, more than 80% of patients have intellectual disabilities and 67% are obese.^6,7 These findings indicate that DNMT3A is associated with human obesity. Acute myeloid leukemia is also caused by mutations in DNMT3A, which seem to be involved in both TBRS and acute myeloid leukemia;^6,16 additionally, a few patients with both acute myelogenous leukemia and TBRS have been reported.⁶ These results suggest that DNMT3A-induced DNA methylation plays a critical role in programming body size and obesity in humans. Heterozygous Dnmt3a-deficient mice also exhibit overeating, obesity, and increased body length, similar to patients with TBRS,¹⁷ highlighting the importance of DNMT3A in the regulation of body weight and growth in mammals.

TETs are also involved in body size, particularly in the fetal and offspring stages. Approximately 75% of Tet1 knockout mice are born with a remarkably small size.¹⁸ Tet1 heterozygous mice that were the progeny of a Tet1-deficient father and a wild-type mother manifested variations in body size, with more than 70% having an extraordinarily small or large body size. Abnormal DNA methylation and gene expression of imprinted genes, including Peg10 and Igf2r, are related to these phenotypes.¹⁹ Notably, the rate of reduction in size at E10.5–E18.5 is higher in Tet1 and Tet2 double-knockout mice than in Tet1 knockout/Tet2 heterozygous mice or Tet1 heterozygous/Tet2 knockout mice, indicating that TET2 also contributes to body size.²⁰ These data suggest that demethylating enzymes, at least TET1 and TET2, are indispensable for maintaining a normal body size.

Genomic imprinting may mediate body size phenotypes induced by aberrant DNA methylation. Genomic imprinting is highly dependent on epigenetic modifications, especially DNA methylation.²¹ Most animals have two sets of chromosomes, and many autosomal genes are expressed on both chromosomes. In contrast, imprinted genes are expressed only on one side of the chromosome, and the other side is silenced by DNA methylation.²¹ Therefore, the expression of imprinted genes is highly dependent on DNA methylation and might experience strong influence of aberrant DNA methylation genes. Some imprinted genes, such as Igf2-H19, Dlk1, and Grb10, are associated with fetal growth and body size.²²

Nutrients and metabolites are involved in DNA methylation (Fig. 1), during which the methyl group is provided by S-adenosylmethionine (SAM) synthesized from methionine and adenosine triphosphate (ATP). As methionine synthesis involves the folate cycle, betaine, vitamins, and zinc, the levels of these nutrients and metabolites influence DNA methylation and demethylation. TETs are α-ketoglutarate-dependent dioxygenases that convert α-ketoglutarate to succinate during the demethylation reaction.²³ Isocitrate dehydrogenase (IDH), an enzyme that produces α-ketoglutarate from isocitrate in the tricarboxylic acid cycle, is upregulated by adenosine monophosphate (AMP)-activated protein kinase, the activity of which is regulated by the ratio of AMP to ATP levels.^24-26 Thus, DNA methylation is influenced by numerous metabolites derived from the diet and systemic and intracellular metabolism. Indeed, the amount of methyl donors, such as choline, betaine, folic acid, vitamin B12, methionine, and zinc, in the diet of pregnant mice alters the level of DNA methylation in their pups, which manifests in the coat color of agouti mice.^27,28 Obesity is largely caused by an imbalance between energy intake and metabolism, and intracellular metabolism may be altered in individuals with obesity. It remains unclear how the DNA methylation metabolic pathway is altered in neurons of the hypothalamic feeding center in obesity.

The hypothalamic feeding center plays a central role in the energy balance by regulating food intake, heat production, and glucose metabolism in response to nutritional, hormonal, and neural signals that control systemic energy homeostasis.^29,30 The arcuate nucleus (ARC) of the hypothalamus contains the first-order neurons that sense these systemic energy signals. Two important types of first-order neurons, neuropeptide Y and agouti-related protein (NPY/AgRP) neurons and proopiomelanocortin (POMC) neurons, are orexigenic and anorexigenic, respectively, and modulate food intake and energy expenditure.^31,32 NPY/AgRP and POMC neurons project to second-order neurons, particularly to the paraventricular hypothalamus (PVH).³⁰ DNA methylation levels in the hypothalamic feeding center are influenced by diet (Table 1). Several studies have reported that high-fat diet (HFD) and overnutrition induce the DNA methylation of Pomc and Npy in the hypothalamus.^33-36 The cafeteria diet, a highly variable, palatable, and energy-dense diet that mimics the modern western diet, also decreased DNA methylation levels at the Pomc promoter in the ARC and the Npy promoter in the PVH of female rats.^37,38 These findings indicate that diet affects the DNA methylation levels of key genes in the hypothalamic feeding center. DNA methylation levels in the Pomc promoter region are correlated with susceptibility to obesity. Rats that are resistant to obesity have lower DNA methylation levels in the Pomc promoter region in the hypothalamus than rats that develop obesity after 21 weeks of an HFD.³⁹ In addition, the expression level of Pomc was significantly higher in the obesity-resistant rat group than in the obesity-developing rat group.³⁹ Therefore, the tendency of Pomc expression to be regulated by DNA methylation may affect the susceptibility to HFD-induced obesity.

The DNA demethylation pathway is also potentially affected by diet. 5-Hydroxymethylated cytosine (5hmC) is the first form of 5mC that is oxidized by TETs to initiate demethylation. In addition, 5hmC serves as a stable epigenetic marker and induces physiological functions.⁴⁰ Acute exposure to an HFD decreases hypothalamic 5hmC level in young adult male mice but not in females. The decrease in hypothalamic 5hmC level is partially reversed to normal by switching from an HFD to a low-fat diet,⁴¹ suggesting that the fat percentage in foods affects 5hmC level in the hypothalamus.

Most DNA methylation patterns are lost after fertilization,⁸ and methyl modifications occur dynamically under the influence of environmental factors. In the mouse brain, Dnmt3a expression increases during the postnatal 3 weeks and then declines to a relatively low level in adulthood, whereas Dnmt3b is expressed from E10.5 to E13.5.⁴² In the postnatal mouse ARC, Dnmt3a expression peaks at P12.⁴³ In the hypothalamus, most neuron-specific DNA methylation is established postnatally between P0 and P21.⁴⁴ Brain development in early postnatal rodents is comparable to that in third-trimester human embryos.⁴⁵ Therefore, early life, such as the late fetal stage in humans and the early postnatal period in rodents, is an important period for the generation of DNA methylation patterns in the hypothalamic feeding center, which is presumably induced by DNMT3A.

Studies in rodents showed that maternal overfeeding and overnutrition increase the methylation levels of the Pomc promoter and reduce Pomc expression in offspring (Table 1).^35,36,46 In addition, overfeeding and overnutrition during the neonatal period lead to hypermethylation of Pomc promoter and reduction of Pomc gene expression.^47,48 Furthermore, neonatal dietary supplementation with conjugated linoleic acids does not alter Agrp and Npy mRNA expression but significantly reduces Pomc mRNA expression via hypermethylation of the proximal specificity protein 1 (Sp1) binding site in the Pomc promoter.⁴⁹ HFD feeding in pregnant mice alters the DNA methylation level of tyrosine hydroxylase (Th), the rate-limiting enzyme for dopamine synthesis, in the hypothalamus but not in the ventral tegmental area of their offspring at the adult stage.⁵⁰

Together, these results indicate that environmental exposure, especially diet and nutrition during pre- and postnatal development, may alter the DNA methylation patterns of feeding center genes that are involved in the development of obesity in adulthood. A methyl-balanced diet, in which the composition of the methyl donor is controlled, prevents prenatal stress-triggered abnormal DNA methylation patterns in the hypothalamus.⁵¹ A methyl-balanced diet is key support of the health of pregnant mothers and the fetus.

Emerging evidence from hypothalamic feeding neuron-specific loss-of-function studies of DNA methylation-related proteins suggests that DNA methylation plays a role in energy homeostasis (Table 2).

DNMT3A is highly expressed in AgRP neurons during the early postnatal period.⁴³ Surprisingly, deletion of Dnmt3a in AgRP neurons did not alter body weight or food intake and instead resulted in reduced energy expenditure and an increase in body fat owing to a reduction in locomotor activity and voluntary exercise.⁴³ At the molecular level, there was a notable change in bone morphogenetic protein 7 (Bmp7), which is an upstream component of the transforming growth factor β signaling pathway, with a decrease in DNA methylation level and an increase in expression level in AgRP neuron-specific Dnmt3a deletion mice.⁴³ Interestingly, DNMT3A in AgRP neurons, which has a robust effect on feeding and body control, affects voluntary movement but not food intake and metabolism. The contribution of hypothalamic BMP7 to energy homeostasis has been reported,⁵² although its precise role in AgRP neurons remains unclear.

TET3 is present in AgRP neurons and negatively regulates these neurons. Tet3 expression in mouse AgRP neurons is elevated under fed conditions compared with fasted conditions; furthermore, TET3 plays an indispensable role in leptin-induced repression of Agrp expression by inducing 5hmC modifications in the Agrp promoter, which stabilizes the association of signal transducer and activator of transcription 3 (STAT3) and the corepressor complex with the Agrp promoter.⁵³ Tet3 deletion in AgRP neurons increases body weight through increased food intake and reduction in energy expenditure.⁵³ Bobcat339, a synthetic small molecule that decreases TET3 in AgRP neurons, prevents weight loss in food-restricted conditions by increasing food intake, and attenuating compulsive wheel running. However, bobcat339 does not alter body weight, food intake, or wheel running under normal conditions.⁵⁴ These data indicate that TET3 plays an important role in energy homeostasis in AgRP neurons.

The DNA methylation reader methyl-CpG-binding protein 2 (MECP2) binds to 5mC to regulate gene expression.⁵⁵ POMC neuron-specific deletion of Mecp2 decreases Pomc expression and increases body weight, body fat percentage, food intake, and respiratory exchange ratio,⁵⁶ suggesting that DNA methylation and its downstream pathways play a crucial role in Pomc expression and energy homeostasis in POMC neurons.

Our previous studies revealed the role of DNMT3A in Sim1-Cre neurons that express Cre mainly in the PVH. Dnmt3a expression level was decreased in the PVH of HFD-induced obese mice, and Sim1-Cre-specific deletion of Dnmt3a induced obesity owing to increased food intake and decreased energy expenditure.⁵ Genome-wide analysis of mRNA expression levels in the PVH revealed that Th expression level was highest in the deletion mice. Th is the rate-limiting enzyme in catecholamine synthesis, and the synthesis of dopamine, the major catecholamine synthesized in the hypothalamus, is increased in these mice. Therefore, upregulation of dopamine synthesis in the PVH may play an important role in the epigenetic induction of obesity. In addition, Th is a target of DNMT3A in pancreatic progenitor cells. The number of Th-expressing β-cells is greatly increased in pancreatic endocrine cell–specific Dnmt3a deletion mice.⁵⁷ Th is located in the genomic region adjacent to a cluster of imprinted genes, such as Ins2, Igf2, and H19;⁵⁸ additionally, Th is a non-canonically imprinted gene that is imprinted only in certain specific brain areas, including the hypothalamus,⁵⁹ affecting food intake and preferences.^59,60 As genome imprinting is highly dependent on epigenetic modifications, especially DNA methylation, it may play a key role in Th regulation by DNA methylation. Deletion of Mecp2 in Sim1-Cre neurons induces increases in body weight and food intake, with a reduction in PVH brain-derived neurotrophic factor (Bdnf) and corticotropin-releasing hormone (Crh) expression,⁶¹ highlighting the importance of DNA methylation and its downstream pathway in the PVH for body weight control.

Epigenome editing is a powerful technology that allows the manipulation of epigenetic markers, such as DNA methylation, in a specific genomic region.^62,63 Using a modified version of CRISPR-associated protein 9 (Cas9), dCas9, which lacks endonuclease activity, and the placement of DNMT or TET around guide RNA (gRNA), DNA methylation levels can be increased or decreased in a specific genomic region.^64-66 Injection of the CRISPR-dCas9-VP64 transcriptional activator system in combination with gRNA against the Tet1, Tet2, and Tet3 loci in the ventromedial hypothalamus (VMH) in male mice increased Tet2 and Tet3 expression levels in the VMH and slowed weight gain during exposure to an HFD.⁴¹ Therefore, DNA methylation patterns in specific genomic regions may influence the development of obesity. DNA methylation levels in the Pomc promoter region were manipulated using the CRISPR dCas9-TET1 and dCas9-DNMT3a system.⁶⁷ The dCas9-TET1 system increased 5hmC level in the Pomc promoter region, whereas the dCas9-DNMT3a system did not alter weight gain during HFD exposure.⁶⁷ Epigenome editing has been applied to the feeding center in only a few prior studies. The large size of the dCas9 gene, which exceeds the size limitation of the adeno-associated virus (AAV) vector, hinders use of AAV technology for delivery of the epigenome editing system. Thus, introducing an epigenome editing system into specific neurons remains challenging. The development of Cre-dependent epigenome editing mice combined with gRNA AAV injection, similar to the Cre-dependent genome editing system using the Rosa-CRISPR mouse,⁶⁸ may greatly improve the ability to conduct epigenetic studies in the feeding center.

The present review highlights current findings on the role of DNA methylation in the hypothalamic feeding center, focusing on the relationships between nutrition, obesity, and epigenome editing. These studies indicate that DNA methylation is related to energy homeostasis and development of obesity. However, some questions remain. For example, it is unclear how the metabolic control of DNA methylation changes when systemic metabolism is altered. Additionally, it has not been elucidated whether genomic imprinting plays a role in body weight control induced by DNA methylation. Epigenetics studies in the hypothalamic feeding center are in their infancy. Further research is required to better understand the mechanisms underlying the development of obesity caused by environmental factors and the increase in the incidence of obesity.

The authors declare no conflict of interest.

Study concept and design: CY, WA, and DK; drafting of the manuscript: CY, WA, and DK; critical revision of the manuscript: DK; and study supervision: DK.