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ABSTRACT 3,3′,4′,5,7-Pentahydroxyflavone-3-rhamnoglucoside (rutin) is a flavonoid with a wide range of pharmacological activities. Dietary rutin is hardly absorbed because the microflora in

the large intestine metabolize rutin into a variety of compounds including quercetin and phenol derivatives such as 3,4-dihydroxyphenolacetic acid (DHPAA), 3,4-dihydroxytoluene (DHT),

3,4-hydroxyphenylacetic acid (HPAA) and homovanillic acid (HVA). We examined the potential of rutin and its metabolites as novel histone acetyltransferase (HAT) inhibitors. DHPAA, HPAA and

DHT at the concentration of 25 μM significantly inhibited in vitro HAT activity with DHT having the strongest inhibitory activity. Furthermore, DHT was shown to be a highly efficient

inhibitor of p300 HAT activity, which corresponded with its high degree of inhibition on intracellular lipid accumulation in HepG2 cells. Docking simulation revealed that DHT was bound to

the p300 catalytic pocket, bromodomain. Drug affinity responsive target stability (DARTS) analysis further supported the possibility of direct binding between DHT and p300. In HepG2 cells,

DHT concentration-dependently abrogated p300-histone binding and induced hypoacetylation of histone subunits H3K9, H3K36, H4K8 and H4K16, eventually leading to the downregulation of

lipogenesis-related genes and attenuating lipid accumulation. In _ob/ob_ mice, administration of DHT (10, 20 mg/kg, iv, every other day for 6 weeks) dose-dependently improved the NAFLD

pathogenic features including body weight, liver mass, fat mass, lipid accumulation in the liver, and biochemical blood parameters, accompanied by the decreased mRNA expression of lipogenic

genes in the liver. Our results demonstrate that DHT, a novel p300 histone acetyltransferase inhibitor, may be a potential preventive or therapeutic agent for NAFLD. You have full access to

this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS SELECTIVE INHIBITION OF CBP/P300 HAT BY A-485 RESULTS IN SUPPRESSION OF LIPOGENESIS AND HEPATIC

GLUCONEOGENESIS Article Open access 11 September 2020 SL010110, A LEAD COMPOUND, INHIBITS GLUCONEOGENESIS VIA SIRT2-P300-MEDIATED PEPCK1 DEGRADATION AND IMPROVES GLUCOSE HOMEOSTASIS IN

DIABETIC MICE Article 11 February 2021 H3K9ME3 DEMETHYLATION BY JMJD2B IS REGULATED BY PIRFENIDONE RESULTING IN IMPROVED NASH Article Open access 21 October 2024 INTRODUCTION Nonalcoholic

fatty liver disease (NAFLD), ranging from steatosis to cirrhosis to hepatocarcinoma, is characterized by an abnormal accumulation of triglycerides (TG) in the liver without alcohol

consumption [1]. NAFLD has been well established to not only be confined to liver-related morbidity and mortality but is also a multisystemic disease affecting several extrahepatic organs

and regulatory pathways, such as the kidney, colon, bone, endocrine and cardiovascular systems [2,3,4]. Liver steatosis, marked as the first stage of NAFLD, is closely associated with

obesity, type 2 diabetes mellitus, insulin resistance, and drug-induced liver injury [5]. Recently, several examples of dynamic changes in epigenetic markers due to environmental stimuli,

such as stress and nutritional interventions, have been reported, including histone acetylation as a gene expression marker [6,7,8]. Few studies have provided evidence that the dysregulation

of hepatic function is orchestrated by epigenetic mechanisms in NAFLD [9]. Among the phenomena of epigenetic modulation, histone acetyltransferase (HAT)-mediated acetylation of histone tail

lysine residues is usually associated with the activation of gene transcription [10]. Indeed, altered expression and activity of certain histone acetylation modifying enzymes influence gene

expression in NAFLD [11]. This may lead to the alteration of hepatic metabolism and cellular transformation through the regulation of various signaling pathways, such as hepatic lipid

metabolism, insulin resistance, mitochondrial damage, oxidative stress, and inflammation. These pathways are implicated in the development and progression of NAFLD [12,13,14,15,16].

Recently, beneficial effects of natural dietary compounds on the prevention and treatment of NAFLD have been reported, but little research has been conducted to analyze the molecular

mechanisms related to epigenetic modulation. 3,3′,4′,5,7-Pentahydroxyflavone-3-rhamnoglucoside (rutin) is a flavonoid under the flavonol subgroup that has a wide range of pharmacological

properties, such as antioxidative, antimicrobial, antifungal, and anti-allergic properties [17]. In addition, current research has shown that its multispectrum pharmacological effects

benefit the treatment of various chronic diseases, such as cancer, diabetes mellitus, hypertension, and hypercholesterolemia [18]. Dietary rutin is hardly absorbed because the microflora in

the large intestine metabolize rutin into a variety of compounds, including quercetin and phenol derivatives such as 3,4-dihydroxyphenolacetic acid (DHPAA), 3,4-dihydroxytoluene (DHT),

3,4-hydroxyphenylacetic acid (HPAA), and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, HVA) [19, 20]. Recent studies have shown the suppressive effects of rutin metabolites on

oxidative stress, inflammation, and advanced glycation end product (AGE) formation [20,21,22]; however, to date, no in-depth explanations have been published regarding the molecular

mechanisms of rutin metabolites in NAFLD. This study is the first attempt to demonstrate the efficacy of rutin metabolites in preventing NAFLD and to explore their roles in epigenetic

modulation. In the present study, we investigated the potential of rutin metabolites as HAT inhibitors (HATis) and demonstrated that DHT is a potent HATi that successfully ameliorated NAFLD

in both in vitro and in vivo NAFLD models. DHT suppressed HAT activity, notably p300, lipid accumulation, and lipogenic gene expression induced by a mixture of oleic and palmitic acids in

HepG2 cells. In silico docking simulation and the DARTS assay revealed that DHT directly binds to the bromodomain of p300. In an obese mouse model, administration of DHT reduced body weight,

liver mass, plasma TG, plasma LDL cholesterol, and lipogenic-related gene expression in the liver. MATERIALS AND METHODS CHEMICALS AND REAGENTS Rutin (Cat. No. R5143), DHPAA (Cat. No.

850217), DHT (Cat. No. M34200), HPAA (Cat. No. H49901), and HVA (Cat. No. H1252) in powder form were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMSO for the

experiments. Oleic acid, palmitic acid, and oil red O staining solution were also purchased from Sigma-Aldrich. Water-soluble tetrazolium salt (WST-1) used to measure cytotoxicity was

obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). CELL CULTURE HepG2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in a

humidified atmosphere with 5% CO2 at 37 °C in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (Coring, NY, USA). A nonfat bovine

serum albumin-conjugated combination of oleic acid (Sigma-Aldrich, St. Louis, MO, USA) and palmitic acid (Sigma) at a ratio of 4:1 (OPA) was used to establish an NAFLD model using HepG2

cells. To evaluate the effect of rutin and its metabolites on hepatic lipid accumulation, HepG2 cells were concurrently treated with the indicated concentrations of OPA for 18 h.

CYTOTOXICITY HepG2 cells were seeded in 24-well plates at a density of 5 × 104 cells/well. Next, at ~70% confluency, cells were treated with the indicated concentrations of rutin metabolites

in each experiment in the presence or absence of OPA. After a 24-h incubation, WST-1 solution (Enzo Life Sciences) was added to the wells, and the cells were incubated for 3 h. Thereafter,

the absorbance values were measured at 450 nm (Molecular Devices, Sunnyvale, CA, USA). LIPID CONTENT HepG2 cells were seeded in 24-well plates at a density of 5 × 104 cells/well. After

reaching ~70% confluency, the cells were treated with OPA in the presence or absence of rutin metabolites. After a 24-h incubation, the cells were fixed with 4% paraformaldehyde for 15 min

at room temperature and were then incubated with 60% isopropanol for 5 min, followed by staining with 0.1% oil red O staining solution (Sigma-Aldrich) for 15 min. The cells were then washed

with water, and images were captured under a light microscope (Olympus IX51; Olympus Corporation, Central Valley, PA, USA). For lipid quantification, isopropanol was added to each well to

dissolve the red dye. After 10 min, the absorbance values were measured at 510 nm (Molecular Devices). HISTONE EXTRACTION HepG2 cells were seeded at a concentration of 5 × 106 in 100-mm

dishes. After the cells reached 70% confluency (~2 × 108 cells), the cells were treated for 18 h with the indicated concentrations of OPA, with or without metabolites of rutin. The histone

proteins from HepG2 cells were extracted using a Histone Extraction Kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol. IN VITRO HAT AND HISTONE DEACETYLASE (HDAC)

ASSAYS HAT and HDAC activities in the HeLa nuclear extracts (BioVision, Milpitas, CA, USA) were assessed using a commercially available kit according to the manufacturer’s protocol

(BioVision). To examine the inhibitory effect of DHT on p300, 100 ng of p300 recombinant protein (Enzo Life Science) was used as the enzyme source instead of the nuclear extracts. For

autoradiography-based in vitro HAT activity assays, either the HeLa cell nuclear extract or the p300 recombinant protein was incubated with HAT assay buffer (50 mM HEPES, pH 8.0; 10%

glycerol; 1 mM dithiothreitol [DTT]; 1 mM phenylmethylsulfonyl fluoride; and 10 mM sodium butyrate), 1 µL of [3H] acetyl-CoA, and 5 µg of biotinylated-H4 peptide (Millipore, Billerica, MA,

USA) along with metabolites of rutin at the indicated concentrations at 30 °C for 1 h. The reactions were stopped by adding 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS–PAGE) sample buffer. Proteins from each sample were then separated on 15% SDS–PAGE gels and visualized via autoradiographic analysis. QUANTITATIVE REVERSE-TRANSCRIPTION POLYMERASE CHAIN

REACTION (QRT-PCR) Cells were seeded in 24-well plates at 5 × 104 cells/well. At ~70% confluency, the cells were treated with the indicated concentrations of DHT in the presence or absence

of OPA. After an 18-h incubation, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). qRT-PCR was performed using an I Cycler iQ system (Bio-Rad Laboratories,

Hercules, CA, USA) with SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA). PCR amplification was carried out in triplicate using the primers listed in Supplementary

Table 1. mRNA levels were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (_GAPDH_) mRNA, and relative expression levels were calculated using the comparative ΔΔCT method.

WESTERN BLOT ANALYSIS Cells were grown and treated in a similar fashion as those used for qRT-PCR. Cell extracts were then prepared using lysis buffer (Cell Signaling, Danvers, MA, USA)

containing protease and phosphatase inhibitors (Roche, Basel, Switzerland) and were incubated on ice for 30 min. The lysates were centrifuged at 20,000 × _g_ for 20 min at 4 °C. The cell

lysates were separated on SDS–PAGE gels and transferred to cellulose membranes (Whatman, Dassel, Germany). The membranes were blocked with 5% (_w_/_v_) skimmed milk (BD Biosciences, Sparks,

MD, USA) solution in 1× phosphate buffered saline (PBS) containing Tween-20 (PBST) for 1 h. The blocked membranes were incubated overnight with the indicated primary antibodies at 4 °C

(Supplementary Table S2). The membranes were then washed with 1× PBST, incubated with the appropriate secondary anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibody (Thermo

Scientific, Rockford, IL, USA) for 1 h, and visualized using Fusion Solo 6 S (Vilber Lourmat, Collegien, France) with an enhanced chemiluminescence detection reagent (Thermo Scientific).

HISTONE TAIL BINDING ASSAY In the biotinylated histone peptide-binding assay, 1 µg of biotin-labeled Lys acetylated histone H3 (AcH3) tail peptide (Anaspec, Fermont, CA, USA) was bound to

NeutrAvidin (Pierce); pull-down assays were performed with flag-tagged recombinant p300 (1041–1161 amino acid) containing the bromodomain (Active Motif, Carlsbad, CA, USA). The binding assay

was performed at 4 °C for 2 h in binding buffer [20 mM Tris-HCl (pH 7.1), 120 mM KCl, 1 mM DTT, 1 mM ethylenediaminetetraacetic acid, 0.1% Nonidet P-40 (NP-40), 10% glycerol, and 1 mM

PMSF]. The histone tail and protein complexes were separated via SDS–PAGE and detected using a flag antibody (Sigma). The biotinylated AcH3 tail was stained with Ponceau S solution (Sigma)

to demonstrate the loading control. DRUG AFFINITY RESPONSIVE TARGET STABILITY (DARTS) ASSAY The DARTS assay was performed according to a previously described protocol [23]. HepG2 cells were

incubated in 10-cm dishes until 80% confluency, followed by lysis in M-PER buffer (Pierce, Rockford, IL, USA), which contains 30 mM NaCl, protease, and phosphatase inhibitors. After

centrifugation (14,000 × _g_, 10 min, 4 °C), 10× TNC buffer [500 mM Tris-HCl (pH 8.0), 500 mM NaCl, 100 mM CaCl2] was added to the lysates prior to the determination of protein concentration

using a DC protein assay kit (Bio-Rad Laboratories), according to the manufacturer’s instructions. The proteins in the supernatant (2 μg/μL) were digested with pronase (1:1,000,000 protein

to pronase ratio) for 30 min. Digestion was stopped by adding 5× Laemmli sample buffer, followed by denaturation at 95 °C for 5 min. CELL AND LIVER TISSUE FRACTIONS Cells were seeded at a

concentration of 5 × 106 in 100-mm dishes. After reaching ~70% confluency (~2 × 108 cells), the cells were treated with the indicated concentrations of OPA, with or without the metabolites

of rutin, for 18 h. Harvested cells were lysed in lysis buffer [10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, and protease inhibitor cocktail (Roche)] at 3000 r/min and 4 °C for 3

 min, and the resulting supernatants were used as cytosol fractions. Nuclear fractions from HepG2 cells were obtained by following the manufacturer’s protocol (Abcam). Liver tissues obtained

at the end of the animal experiments were washed with ice-cold PBS and lysed according to the manufacturer’s protocol (Abcam) to obtain the cytosolic and nuclear fractions. Briefly, tissues

were weighed and cut into small pieces. To obtain the cytosolic fractions, tissue pieces were homogenized in pre-extraction buffer containing DTT using a glass tissue homogenizer (Thomas

Scientific, NJ, USA). Extraction buffer was added to the pellets, and the resulting suspensions were incubated on ice for 15 min and were then further homogenized using a Teflon pestle

(Thomas Scientific). The suspensions were then centrifuged at 14,000 r/min at 4 °C for 10 min, and the supernatants were used as nuclear fractions. Each nuclear fraction was used for the HAT

assays. ANIMAL EXPERIMENTS Seven-week-old male _ob/ob_ mice were purchased from Orient Bio (Seongnam, Gyeonggi, Korea) and housed at the College of Medicine of Ulsan University. All animal

experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ulsan University Guide for the

Animal Care and Use Committee (2017-02-148). A total of 20 male _ob/ob_ mice were divided into three groups (2 or 3 mice/cage) and housed in a temperature- and humidity-controlled room with

a 12-h light/dark cycle, with free access to food and water. After a 1-week acclimation period, saline (control group), 10 mg, or 20 mg/kg DHT was injected into the lateral tail vein of each

mouse once every alternate day. Body weights were measured at the beginning of the experiment and subsequently every week for 6 weeks. At the end of the experiment, mice were sacrificed by

cervical dislocation under anesthesia (100 mg/kg ketamine + 5 mg/kg xylazine), and blood samples were collected for serum isolation via abdominal heart puncture. After laparotomy, the liver,

retroperitoneal fat, and epididymal fat were weighed, and a portion of the liver from each mouse was fixed in 4% formalin solution for hematoxylin and eosin (H&E) staining. The

remaining livers were used for the experiments as indicated. H&E STAINING Liver specimens were fixed in 4% buffered formalin, embedded in paraffin, and cut into 4–5-μm-thick sections,

which were stained with H&E. Lipid accumulation in each liver specimen was assessed through microscopic observation using an Eclipse 80i microscope (Nikon Instruments, Inc., Melville,

NY, USA). MEASUREMENT OF TRIGLYCERIDES (TGS), TOTAL CHOLESTEROL, AND LOW-DENSITY LIPOPROTEIN CHOLESTEROL (LDL-C) The serum levels of TGs, total cholesterol, and LDL-C were measured

enzymatically using a commercial kit (Asan Pharm, Seoul, Korea). PROTEIN–LIGAND DOCKING SIMULATIONS FOR THE E1A BINDING PROTEIN P300 (EP300) To identify the active site in EP300, the

three-dimensional X-ray crystallographic structure of EP300 was downloaded from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) (PDB ID: 4BHW) [24]. The three-dimensional

structure of DHT used as a ligand for docking was downloaded from the PubChem site (CID: 9958) (https://pubchem.ncbi.nlm.nih.gov) [25]. The specific simulation methods were described in

detail in a previous study by Chung et al. [11]. STATISTICAL ANALYSIS Data were analyzed using Student’s _t_-test for comparing two groups or one-way analysis of variance (ANOVA) with

Tukey’s multiple comparison test for comparing multiple groups, and values are expressed as the means ± standard deviations (SD) or means ± SEM. Statistical analyses were conducted using

SPSS ver. 20 (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at _P_ < 0.05, _P_ < 0.01, and _P_ < 0.001. RESULTS DHT HAS THE MOST SIGNIFICANT

INHIBITORY EFFECT ON HAT ACTIVITY Initially, to test whether rutin and its metabolites (Fig. 1a) can modulate HAT activity, we measured HAT activity using a cell-free system. Rutin (100 µM)

or one of its metabolites, including DHPAA, HPAA, DHT, and HVA, was incubated with HeLa nuclear extract. The results indicated that DHT had the most significant inhibitory effect on HAT

activity compared to that of the other metabolites (Fig. 1b). To confirm the anti-HAT activity of DHT, an autoradiography assay was performed using radiolabeled H4 tail peptides. Consistent

with the results of the in vitro HAT activity assay, DHT exhibited the most powerful HATi effect (Fig. 1c), although the effects of DHPAA and HPAA in the cell-free system and the

autoradiography assay were slightly different from the in vitro HAT activity. To ensure that the activities of rutin and its metabolites, including DHT, were specifically directed against

HATs, we assessed their effects on HDAC activities. Trichostatin A (TSA), used as a positive control, efficiently inhibited nuclear HDAC activities, whereas rutin and its metabolites did not

affect HDAC activity (Fig. 1d). Together, these data showed that DHT, among the rutin metabolites, most efficiently abrogated HAT activity without affecting HDAC activity. DHT SELECTIVELY

INHIBITS P300 ACETYLTRANSFERASE ACTIVITY To further confirm the effect of DHT as a HATi, the changes in both HAT and HDAC activities were measured with DHT at various concentrations in a

cell-free system. DHT diminished HAT activity in a dose-dependent manner (Fig. 2a), whereas HDAC activity was not affected by DHT (Fig. 2b). Next, we examined the enzyme specificity of DHT.

The HAT activities of p300, CREB-binding protein (CBP), and p300/CBP-associated factor (pCAF) were measured either in the absence or presence of increasing concentrations of DHT. DHT was

shown to be a highly efficient inhibitor of p300 acetyltransferase activity (Fig. 2c). The p300 inhibitory effect of DHT was supported by the results of an autoradiography assay using a

radiolabeled H4 tail peptide (Fig. 2d). Collectively, these results showed that DHT specifically inhibited p300 acetyltransferase activity. THE ANTILIPOGENIC EFFECT OF DHT IS ASSOCIATED WITH

ITS HAT INHIBITORY CAPACITY We previously demonstrated that HAT activity increases in NAFLD in vitro and in vivo [11]. To determine whether the rutin metabolites attenuated the increased

HAT activity found in HepG2 cells undergoing lipid accumulation, we measured the change in HAT activity after exposing an OPA-induced NAFLD model to rutin metabolites. HepG2 cells with or

without various rutin metabolites were treated with OPA for 24 h. Nuclear extracts were then used to evaluate HAT enzyme activity. OPA-induced HAT activity was significantly inhibited by

DHPAA, HPAA, DHT, and HVA (Fig. 3a). Interestingly, similar to the previous result (Fig. 1b), DHT showed the strongest HATi effect. To observe the correlation between HATi activity and

intracellular lipid accumulation in HepG2 cells, oil red O staining was performed under the same conditions as above. The inhibition of lipid accumulation by rutin metabolites was also

similar to that of the observed HATi activity (Fig. 3b). The results showed that the metabolites were not cytotoxic at the concentration used (Fig. 3c). To check the effect of different

concentrations of DHT on HAT activity and lipid accumulation, we treated cells with 25–50 μM DHT and remeasured their corresponding HATi effects and degrees of inhibition of lipid

accumulation. DHT showed both HATi and antilipogenic effects in a dose-dependent manner (Fig. 3d, e). Next, RT-qPCR was conducted to determine whether DHT was implicated in the expression of

acetyl-CoA carboxylase (_ACC_), ATP-citrate lyase (_ACLY_), fatty acid synthase (_FASN_), and sterol regulatory element-binding protein 1c (_SREBP1c_). As expected, there was a significant

increase in the mRNA expression of these genes in the OPA-treated group; mRNA expression significantly decreased in OPA-treated groups that were exposed to DHT (Fig. 3f). These data suggest

that DHT decreased lipid accumulation in hepatic cells by abrogating the transcription of lipogenesis-related genes via its HATi capacity. DHT BINDS TO THE BROMODOMAIN OF P300 AND INHIBITS

P300–HISTONE COMPLEX FORMATION Previously, we demonstrated the selective p300 inhibitory effect of DHT. We then examined the binding affinity of DHT and p300 through docking simulation. The

most likely binding site on p300 was the catalytic pocket for the bromodomain. We identified the top five docking structures with free energy values between −7.181 kcal/mol and −6.547 

kcal/mol (Fig. 4a). In particular, the most likely binding residues were PRO1074, TYR1089, MET1124, ASN1127, ALA1128, and TYR1131 (Fig. 4b). To confirm DHT-p300 binding at the molecular

level, we conducted a DARTS assay using HepG2 cells. The binding of chemicals to specific proteins increases protein stability and resistance to proteases. The cells were treated with DHT,

and the lysates were then digested with pronase, a mixture of proteases, as indicated. DARTS analysis revealed that DHT directly bound to p300, and the resistance of p300 to pronase

increased proportionately to the DHT concentration (Fig. 4c). It is well established that the bromodomain of p300 is responsible for its histone binding [26]. Based on this, we hypothesized

that docking of DHT in the p300 bromodomain would inhibit histone binding with p300 and would consequently suppress histone acetylation. To support our hypothesis, we performed a histone

tail binding assay. Biotinylated AcH3 and flag-tagged p300 recombinant protein containing the bromodomain were incubated with or without DHT (Fig. 4d). Then, biotinylated AcH3 was

immunoprecipitated with avidin beads, and the bound p300 protein was detected using an anti-flag antibody. Remarkably, our results demonstrated that the binding of p300 and histone H3

decreased as the concentration of DHT increased (Fig. 4d). To observe whether a decrease in the binding of p300 and histone protein would actually lead to a reduction in histone acetylation,

we detected the histone acetylation status following DHT treatment. DHT effectively obstructed the OPA-induced hyperacetylation of the histone subunits H3K8, H3K36, H4K8, and H4K16 (Fig. 

4e). Together, these data indicate that DHT interrupted p300-histone protein binding by docking at the histone binding site of p300 and consequentially inhibiting hyperacetylation of histone

proteins caused by OPA-induced lipid accumulation. DHT ADMINISTRATION IMPROVES THE NAFLD PATHOGENIC FEATURES IN VIVO To strengthen the previously gathered data, we examined the effect of

DHT on significant NAFLD features. A dose of 10 or 20 mg/kg DHT was injected into the lateral tail vein once every alternate day for 6 weeks in _ob/ob_ mice (Fig. 5a). The DHT-injected mouse

groups demonstrated a lower average body weight than the control mouse group (Fig. 5b). The average weight gain was also significantly lower in the DHT-injected groups than in the control

group (Fig. 5c), and there were no significant differences in daily food intake between the DHT-injected mice and the control mice (_P_ < 0.05) (Fig. 5d). Enlarged tissue sizes of the

liver and retroperitoneal and epididymal fat and increased tissue mass were observed in the control group but not in the DHT-injected group (Fig. 5e, f). In H&E-stained liver tissue,

less lipid accumulation was detected in DHT-injected mice than in control mice (Fig. 5g). In the blood test, TG, LDL-C, alkaline phosphatase (ALP), and alanine aminotransferase (ALT) levels

effectively decreased following DHT treatment (Fig. 5h). Moreover, the mRNA expression of lipogenic genes, such as _FASN_, _ACLY_, and _PPARγ_, decreased in the DHT-injected groups (Fig. 

5i). These results established that DHT administration ameliorated NAFLD peculiarities in _ob/ob_ mice. DISCUSSION NAFLD is one of the major health concerns worldwide. Its development and

progression are affected by various factors, such as genetics, epigenetics, and environmental factors. A large number of studies have provided evidence to support that the critical

components implicated in the progression of NAFLD pathogenesis are epigenetically regulated [27,28,29]. In addition, recent studies have shown that epigenetic modifications are reprogrammed

for NAFLD [30, 31]. For this reason, there is increasing interest in developing new preventive or therapeutic applications that target NAFLD based on epigenetics owing to its reversible

properties. In particular, epigenetic regulation using natural compounds with favorable safety profiles is expected to be a novel preventive or therapeutic strategy for metabolic syndromes,

including NAFLD [7, 32, 33]. In a preliminary study, we observed that sprouts containing a large amount of rutin, which has known beneficial effects against metabolic diseases, show anti-HAT

capacity. Based on our preliminary observations, in this study, we elucidated the in-depth epigenetic mechanism underlying NAFLD control by rutin and its metabolites at the molecular level.

Initially, we investigated the anti-HAT efficacy of rutin and its metabolites, namely, DHPAA, HPAA, DHT, and HVA. Our data showed that DHPAA, HPAA, and DHT showed potential in effectively

inhibiting HAT activity. In particular, DHT, a phenol derivative of rutin, demonstrated the most potent HAT inhibitory effect among all the rutin metabolites. Several small molecules

targeting HAT that have been derived from natural products, such as anacardic acid, curcumin, and garcinol [34, 35], contain phenolic structures that are prone to oxidation; thus, it is

occasionally difficult to distinguish whether their efficacy in disease models is due to the antioxidant effect or the anti-HAT effect. Chen et al. reported that DHT had the most significant

inhibitory effect on LPS-induced NO production and inflammatory cytokine production among rutin metabolites [20]. Recently, a study indicated that DHT is a plausible rutin colonic

metabolite that suppresses oxidative stress by enhancing peroxiredoxin-6 protein expression, a protein responsible for the cellular antioxidant defense system [21]. These studies allow us to

indirectly or directly infer that the beneficial effects of DHT are mediated by its antioxidant effect. However, to the best of our knowledge, no studies have shown the efficacy of DHT as a

HAT inhibitor. Our data clearly demonstrate that HAT activity is regulated in a DHT dose-dependent manner. Based on the above facts, we believe that it is justifiable to identify the

bioactive effects of DHT based on its HAT inhibitory capacity. The acetylation modification of proteins in the liver is associated with a broad range of cellular activities. This is

supported by a study showing that ~1000 proteins in human liver tissue are subjected to lysine acetylation modification [36]. The development and progression of NAFLD is no exception. A

pregnant woman’s high-fat diet was shown to trigger the development of NAFLD in the fetal liver; statistically significant hyperacetylations and gross alterations of acetylation at the

H3K14, H3K9, and H3K18 subunits have also been observed [37]. Global histone H3 acetylation also increases in a serotonin reuptake inhibitor-induced NAFLD model [38]. We recently reported

that increased HAT activity following treatment with a mixture of oleic and palmitic acids (OPA) induces histone hyperacetylation and promotes NAFLD phenotypes such as steatosis and

lipogenic gene expression in HepG2 cells [39]. Our data showed that histones H3 and H4 were hypoacetylated in an OPA-induced NAFLD model in HepG2 cells. This result was attributed to the

increased HAT activity in the model. Interestingly, DHT effectively blocked the phenomena through its anti-HAT activity, especially by selectively inhibiting p300. According to our data, the

effect of HATs appeared to be tissue-specific; in the differentiation of preadipocytes (3T3-L1) to adipocytes, pCAF activity was more significant than p300 activity (data not shown). P300

dynamically upregulates many transcription factors, including _SREBP1c_ and _PPARγ_, which are responsible for lipogenesis [40, 41], and consequently enhances the expression of lipogenic

genes in NAFLD [42]. Thus, regardless of its molecular mechanism, these results show that inhibition of p300 activity can serve as a key to prevent the development and progression of NAFLD.

In the present study, we provided a plausible mechanism underlying p300 inhibition caused by the derivative of a natural compound. To explain this, we proved the mode of action of DHT as a

p300 inhibitor through the DARTS assay and docking simulations. The results demonstrated that DHT bound to p300, and the docking site was predicted to be a bromodomain located in the p300

catalytic pocket. The domain not only increases p300 activity by providing a docking surface for acetyl-CoA, the intracellular acetyl donor [43], but also plays an important role in

activating the target level of gene transcription through binding with histone H3 [44]. The bromodomain has emerged as a novel therapeutic target for cancer, cardiovascular disease, and type

2 diabetes, and its inhibitors, such as apabetalone and JQ-1, disrupt the interaction of the domain with acetylated histones [45]. We elucidated that DHT directly interrupted the access of

the p300 bromodomain to histone H3 using the histone tail binding assay. Therefore, the results shown in our study suggested that DHT inhibited p300 activity by obstructing the binding of

both acetyl-CoA and the histone tail to the p300 bromodomain. Physically, acetylation of the histone tail changes the positive charge of lysine residues to negative, thereby weakening its

electrostatic affinity to DNA, resulting in conformational changes in euchromatin and enhancing gene transcription [46, 47]. Thus, from an epigenetic point of view, our hypothesis that DHT

prevents the onset and progression of NAFLD through its p300 inhibitory capacity is firmly supported by the compelling results obtained in this study. Our data showed that DHT induced

inhibition of the hyperacetylation of histone proteins, including H3K9, H3K36, H4K8, and H4K16, and decreased expression of lipogenic genes, such as _ACC1_, _ACLY_, _FASN_, and _SREBP1c_.

Consistent with our results, we recently reported that tannic acid, a plant-derived polyphenol, suppresses HAT, notably p300 activity, hyperacetylation of histone marks, and lipogenic gene

expression and ameliorates NAFLD [11]. Based on the present findings and the previous literature, it can be postulated that the beneficial effects of DHT on the development and progression

of NAFLD are closely associated with DHT-dependent epigenetic regulation of the lipid metabolic pathway. In conclusion, we have not only provided powerful evidence that DHT is a new p300

inhibitor but also elucidated a plausible scenario describing the underlying molecular mechanism controlling NAFLD through DHT (Fig. 6). However, we did not observe whether DHT affected the

occupancy of p300 on the promoter region of the lipogenic genes. Considering that this study provides strong evidence to support DHT-mediated epigenetic regulation in controlling NAFLD,

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Scholar  Download references ACKNOWLEDGEMENTS This study was supported by the Main Research Program (E-0150301) of the Korea Food Research Institute (KFRI), funded by the Ministry of

Science, ICT & Future Planning. AUTHOR INFORMATION Author notes * These authors contributed equally: Jangho Lee, Ji-Hye Song. AUTHORS AND AFFILIATIONS * Korea Food Research Institute,

Jeollabuk-do, 55365, Republic of Korea Jangho Lee, Min-Yu Chung, Jae Ho Park, Jin-Taek Hwang & Hyo-Kyoung Choi * Department of Biomedical Sciences, Asan Medical Center, University of

Ulsan College of Medicine, Seoul, 05505, Republic of Korea Ji-Hye Song * Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea Jin-Hyuk Lee * Department

of Bioinformatics, Korea University of Science and Technology, Daejeon, 34113, Republic of Korea Jin-Hyuk Lee * Major of Food Science and Biotechnology, Division of Bio-convergence, Kyonggi

University, Suwon, 16227, Republic of Korea Tae-Gyu Nam * Department of Food Biotechnology, Korea University of Science and Technology, Daejeon, 34113, Republic of Korea Jin-Taek Hwang

Authors * Jangho Lee View author publications You can also search for this author inPubMed Google Scholar * Ji-Hye Song View author publications You can also search for this author inPubMed 

Google Scholar * Min-Yu Chung View author publications You can also search for this author inPubMed Google Scholar * Jin-Hyuk Lee View author publications You can also search for this author

inPubMed Google Scholar * Tae-Gyu Nam View author publications You can also search for this author inPubMed Google Scholar * Jae Ho Park View author publications You can also search for

this author inPubMed Google Scholar * Jin-Taek Hwang View author publications You can also search for this author inPubMed Google Scholar * Hyo-Kyoung Choi View author publications You can

also search for this author inPubMed Google Scholar CONTRIBUTIONS HKC and JTH designed the research. JL, JHS, MYC, and HKC performed the research. JL, JHL, TGN, and JHP contributed to the

data analysis. JL, HKC, and JTH wrote the paper. CORRESPONDING AUTHORS Correspondence to Jin-Taek Hwang or Hyo-Kyoung Choi. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLES RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Lee, J., Song, JH., Chung, MY. _et

al._ 3,4-dihydroxytoluene, a metabolite of rutin, suppresses the progression of nonalcoholic fatty liver disease in mice by inhibiting p300 histone acetyltransferase activity. _Acta

Pharmacol Sin_ 42, 1449–1460 (2021). https://doi.org/10.1038/s41401-020-00571-7 Download citation * Received: 24 August 2020 * Accepted: 02 November 2020 * Published: 10 December 2020 *

Issue Date: September 2021 * DOI: https://doi.org/10.1038/s41401-020-00571-7 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link

Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * non-alcoholic fatty

liver disease * rutin metabolites * 3,4-dihydroxytoluene * p300 histone acetyltransferase * epigenetic regulation * OPA-induced NAFLD model * _ob/ob_ mice