**9. Energy states and epigenetics**

The energy states of organisms, such as fasting and caloric restriction, are memorized in cells, and can lead to the development of future diseases. Recently, epigenetic mechanisms have received much attention in the field of metabolism. The epigenome is a mechanism for regulation of gene expression without changing the genome sequence. Due to its high plasticity, epigenetic regulation is suitable for the memory of metabolic states. Such epigenetic regulations include covalent modifications of DNA, histone, and RNA. More than 100 modifications have been identified, among which methylation, phosphorylation, ubiquitination, and acetylation are well studied, while glycosylation, crotonylation, and succinylation, although functional, are poorly understood [37]. These modifications influence spatial chromatin structure and recruitment of transcription factors and enzymes that are involved in chromatin remodeling. A number of studies have revealed that fasting and obesity-associated diseases that induce a fasting-like metabolic state have a functional link to the epigenome.

Accumulating evidence indicates that diet is one of the most important environmental factors that cause epigenetic changes in the growth process of organisms. One striking evidence of dietary effects on early development was found in honey bees [38]. Female honey bees have two distinct phenotypes, and female larvae that receive royal jelly exclusively develop into fertile queen bees while the other females develop into sterile workers. These phenotypic changes are accompanied by differential DNA methylation patterns and gene expressions between queens and workers through DNA methyltransferase 3 (DNMT3). Such epigenetic alterations during development in response to nutritional states are also found in mammals. A well-known example is the nutritional effect on coat color of the mouse by DNA methylation of the Agouti viable yellow (Avy) locus [38]. Insertion of an endogenous retrovirus (ERV) upstream of this gene locus causes constitutive expression of the Agouti gene as the ERV functions as an alternative promoter, resulting in yellow coat color and adult-onset of obesity. The DNA methylation level at the ERV locus increased in offspring but not in the mother in response to gestational intake of compounds related to the methionine cycle including folate. In rats, the locus-specific change in DNA methylation of the Ppara gene was also observed in offspring of the father or mother fed with a low protein diet [38]. It is also reported that feeding high-fat diet to pregnant Japanese macaques led to global hyperacetylation of histone H3 in their offspring [38]. In humans, epidemiological studies developed the important concept of 'Developmental Origins of Health and Disease' (DOHaD), which proposes that unfavorable adaptation to nutritional stress at the embryonic stage is a risk for abnormal growth and development and subsequent health disorders later in life [38]. Cohort studies on the Dutch Famine of 1944–1945 revealed that the severe maternal starvation during the peri-conceptional period induced mental and metabolic abnormalities such as obesity and type 2 diabetes along with changes in DNA methylation in the next generation.

Although epigenetic profiles are more prone to be altered by nutritional states during peri-conceptional, gestational, and early postnatal periods, dietary effects on the epigenetic state are also found in adult animals. Several lines of evidence have revealed that overnutrition induces epigenetic changes in adult organs. One study revealed 232 differentially methylated regions (DMRs) on the genomic DNA in the adipose tissue between mice fed a normal chow diet and those fed a high fat diet [39]. Importantly, the DMRs were also found in humans when comparing lean and obese subjects. Histone modifications are also influenced by diet. It is reported that mice fed a high-fat diet show the increased acetylation levels of histone H3K9 and H3K18 in the genomic regions encoding TNFα and CCL2 in the liver [40]. A mass spectrometry-based approach showed that high-fat diet increased the level of di-methylated histone H3K36 (H3K36me2) and other histone modifications in the mouse liver [41]. In human urine-derived podocyte-like cells, palmitate treatment induced sustained activation of FOXO1 even in the presence of insulin, which was accompanied by the increased H3K36me2 level in the promoter region of the FoxO1 gene [42]. The H3K27me3 in the FoxO1 promoter region decreased in the kidney of rat fed a high-fat diet. In addition to the DNA and histone modifications, RNA methylation is an emerging regulatory mechanism of epigenetics/epitranscriptomics. For example, there reported that a high-fat diet enhanced the expression of fat mass and obesity-associated protein (FTO)/alkB homolog 9 (ALKBH9), an RNA demethylase, and suppressed the N6-methyladenosine (m6A) levels in the mouse adipose tissue, which resulted in obesity [43]. The beneficial effects of fasting on health have also attracted attention in the field of epigenetics. Fasting is implicated in histone modifications via ketone body production as described below. In addition, a clinical human study showed that fasting stress increased methylated CpG sites of the Leptin and Adiponectin genes only in the group born with normal body weight but not in the group born with low body weight [44]. Collectively, epigenetic profiles reflect nutritional states not only in the fetus and infants but also in adults.

## **10. Metabolite and co-factor-mediated regulation of epigenetic enzymes**

Although it is not well understood which component in the diets is responsible for epigenetic changes in organisms, it should be noted that the activity of epigenetic enzymes depends on metabolites and co-factors such as metals. These enzymes include acetyltransferases, deacetylases, methyltransferases, and demethylases for DNA, histone, and RNA. In reactions that add or remove epigenetic modifications, these enzymes utilize metabolites derived from metabolic pathways including glycolysis, fatty acid oxidation, the methionine cycle, and the TCA cycle. These metabolic pathways supply metabolites, such as acetyl-CoA, NAD+ , S-adenosylmethionine (SAM), and α-KG as substrates to the enzymes (**Figure 7**). Ferrous iron is also an essential co-factor for epigenetic demethylases. Considering that the kinetic and thermodynamic properties of the interaction between an epigenetic enzyme and a metabolite are in a similar range as the physiological concentrations of metabolites [37], it is possible that dynamic changes in the availability of metabolites and cofactors may affect epigenetic outcomes. Here, we discuss the potential for regulation of epigenetic modifications through metabolites and co-factors.

HATs, such as CBP/p300, are enzymes that transfer an acetyl group from acetyl-CoA to lysine residues on histone proteins (**Figure 7B**). Acetyl-CoA is supplied from various nutrients through metabolic pathways such as glycolysis, the TCA cycle, and fatty acid oxidation (**Figure 7A**). In a study examining which nutrient-derived acetyl-CoA alters histone acetylation, only lipids, among various nutrients, induced direct acetylation of histones via fatty acid oxidation in mammalian cells [45]. Another *Metabolic Responses to Energy-Depleted Conditions DOI: http://dx.doi.org/10.5772/intechopen.100391*

#### **Figure 7.**

*Metabolite-mediated regulation of epigenetic enzymes. A. Production of substrates and co-factors of epigenetic enzymes in the metabolic process, B. Substrates, co-factors, and inhibitory metabolites of epigenetic enzymes.*

study showed that the treatment of pancreatic β cells with palmitate increased HAT activity and histone acetylation [44]. However, a mass spectrometry-based study demonstrated that a high-fat diet rather decreased acetyl-CoA levels in the mouse white adipose tissue, which correlated with histone acetylation [45], suggesting that the regulation of histone acetylation by metabolites is more complex in obesity.

Acetyl groups on histones are removed by the action of HDACs. SIRTs (SIRT1–7), a class III HDACs, are NAD+ -dependent deacetylases that sense the energy state in cells (**Figure 7**). They have been shown to be involved in longevity in a variety of species, including yeast, *C. elegans*, and Drosophila, although epigenetic mechanisms are not fully understood [32, 33]. Importantly, a decrease in NAD+ levels due to activated glycolysis was sufficient to inhibit the activity of NAD+ -dependent deacetylases and promoted histone H4K16 acetylation during differentiation of murine muscle cells [37], suggesting that fasting can be involved in the regulation of epigenetic enzymes. Butylate, a short-chain fatty acid produced by intestinal fermentation, is known to inhibit histone deacetylases [45]. Butyrate and a class I HDAC inhibitor have been reported to suppress obesity-associated phenotype in a mouse model of high-fat diet induced obesity (**Figure 7B**) [45]. Similarly, β-hydroxybutyrate, one of the ketone bodies, inhibits class I HDACs (HDAC1, 2, 3, and 8) (**Figure 7B**) [6]. Considering that the inhibition potency (IC50) of β-hydroxybutyrate on these HDACs is around 2–5 mM in an in vitro assay, and that the concentration of β-hydroxybutyrate in humans during long-term fasting is 6–8 mM, β-hydroxybutyrate is a potential physiological inhibitor of HDACs [6]. In the mouse kidney, fasting induced hyperacetylation of histone H3K9 and H3K14, and the HDAC-mediated expression of FoxO3 enhanced the expression of oxidative stress resistance genes [46].

DNMTs, histone methyltransferases (e.g., enhancer of zeste homolog 2 (EZH2), SET domain-containing methyltransferases (SETs), and mixed-lineage leukemias

(MLLs)), and RNA methyltransferase (e.g., methyltransferase like 3 (METTL3) and METTL14) require SAM as a methyl donor for methylation of DNA, histone, and RNA, respectively (**Figure 7B**). SAM is provided by the methionine cycle from dietary components such as methionine and folate (**Figure 7A**). Decreased folate levels in the circulation was reported in patients with type 2 diabetes, and the folate levels were correlated with DNA methylation levels in the liver [44]. Additionally, administration of folate to mice fed a high-fat diet altered the DNA methylation patterns of genes in the adipose tissue and improved obesity-associated phenotype [44].

Among epigenetic demethylases, DNA demethylases (ten-eleven translocation methylcytosine dioxygenases (TETs)), histone lysine demethylases with a JmjC domain (e.g., KDMs), and RNA demethylases (ALKBH5 and FTO/ALKBH9 require oxygen and α-KG as substrates and ferrous iron as a cofactor (**Figure 7B**). α-KG is an intermediate metabolite of the TCA cycle and is also supplied by a flux of amino acids such as glutamate (**Figure 7A**), while ferrous iron is taken up from outside the cell via transferrin receptors or supplied internally by ferritin-selective autophagy [47]. Interestingly, these demethylases are classified into the 2-oxoglutaratedependent dioxygenase (2OGD) family, and the structure of their catalytic domain is highly conserved among all the enzymes [48]. Notably, the iron-binding site of the enzymes is composed of a highly conserved amino acid sequence, that is, histidine, and aspartate or glutamate located two amino acids away from the histidine, followed by histidine located around a hundred amino acids away from the two amino acids (HXD/G…H). A ferrous iron molecule bound to these amino acids serve as a catalytic center of the demethylase, which oxidizes the methyl group of the substrates and removes the methyl group from DNA, histone, and RNA. Several studies suggested that both α-KG and iron are critical regulators of 2OGDs. α-KG has been shown to increase during adipocyte differentiation and to promote differentiation through demethylation of H3K9 at the Pparg locus [49]. Similarly, adipocyte differentiation in 3T3-L1 cells, which involves dynamic changes in the epigenome, is inhibited by iron depletion, although how the ferrous iron level is altered during differentiation has not been explored [50, 51]. It is also noteworthy that some types of cancer cells harboring mutations on IDH1 and IDH2 produce an inhibitor of 2OGDs, (R)-2-hydroxyglutarate (R-2HG) (**Figure 7**) and show characteristic DNA and histone hypermethylation [37]. Additionally, fumarate and succinate can inhibit 2OGDs (**Figure 7**), and deletion of fumarate dehydrogenase and succinate dehydrogenase induced histone and DNA hypermethylation [37]. Considering the recent findings that KDM5A/JARID1A and KDM6A/UTX function as oxygen sensors whose demethylase activity is inhibited under hypoxia [52, 53], it is possible that change in the concentrations of metabolites and cofactors may also affect the demethylase activity of 2OGDs, and thus control epigenetic consequences of cellular processes.

Therefore, it is conceivable that energy states could regulate epigenetic mechanisms and be memorized, subsequently influencing the onset of a variety of diseases. Thus, understanding the relationship between energy states and the epigenome is essential for establishment of an appropriate diet-based therapy. As future challenges, it is necessary to elucidate how concentrations of metabolites and cofactors change during biological processes, and which epigenetic enzymes are responsible for the metabolite- and/or cofactor-mediated epigenetic alterations. However, measuring local concentrations of metabolites and cofactors, especially in the nucleus, has been difficult due to technical barriers. We have recently developed a fluorescence resonance energy transfer (FRET)-based biosensor to measure nuclear α-KG concentrations and have found that nuclear α-KG concentrations increase with adipocyte differentiation [54]. The development of such tools will shed light on the regulatory mechanisms of the epigenome by biomolecules in the future.
