Different fermentation strategies by Schizochytrium mangrovei strain pq6 to produce feedstock for exploitation of squalene and omega-3 fatty acids.

Schizochytrium mangrovei strain PQ6 was investigated for coproduction of docosahexaenoic acid (C22: 6ω-3, DHA) and squalene using a 30-L bioreactor with a working volume of 15 L under various batch and fed-batch fermentation process regimes. The fed-batch process was a more efficient cultivation strategy for achieving higher biomass production rich in DHA and squalene. The final biomass, total lipid, unsaponifiable lipid content, and DHA productivity were 105.25 g · L-1 , 43.40% of dry cell weight, 8.58% total lipid, and 61.66 mg · g-1  · L-1 , respectively, after a 96 h fed-batch fermentation. The squalene content was highest at 48 h after feeding glucose (98.07 mg · g-1 of lipid). Differences in lipid accumulation during fermentation were correlated with changes in ultrastructure using transmission electron microscopy and Nile Red staining of cells. The results may be of relevance to industrial-scale coproduction of DHA and squalene in heterotrophic marine microalgae such as Schizochytrium.

Transcriptome sequencing and comparative analysis of Schizochytrium mangrovei PQ6 at different cultivation times.

OBJECTIVE: The heterotrophic marine microalga, Schizochytrium mangrovei PQ6, synthesizes large amounts of polyunsaturated fatty acids (PUFAs) with possible nutritional applications. We characterized the transcriptome of S. mangrovei PQ6, focusing on lipid metabolism pathways throughout growth. RESULT: Cell growth, total lipid, and docosahexaenoic acid (DHA, 22:6n-3) contents of S. mangrovei PQ6 in 500 ml batch cultures rapidly increased on day 1 in cultivation and reached their maximum levels on day 5. Maximum lipid accumulation in 500 ml batch cultures occurred on day 5, with total lipid and DHA contents reaching 33.2 ± 1.25% of dry cell weight (DCW) and 136 mg/g DCW, respectively. 11,025 unigenes, 28,617 unigenes and 18,480 unigenes from the transcriptomes of samples collected on day 1, 3, and 5 in cultivation were identified, respectively. These unigenes of the three samples were further assembled into 30,782 unigenes with an average size of 673 bp and N50 of 950 bp, and a total of 9,980 unigenes were annotated in public protein databases. 93 unigenes involved in lipid metabolism in which expression patterns corresponded with total lipid and DHA accumulation patterns were identified. CONCLUSION: The possible roles of PUFAs pathways, such as those mediated by fatty acid synthase, polyketide synthase, and desaturase/elongase, co-exist in S. mangrovei PQ6.

Cyanidin is an agonistic ligand for peroxisome proliferator-activated receptor-alpha reducing hepatic lipid.

To investigate the underlying mechanism of targets of cyanidin, a flavonoid, which exhibits potent anti-atherogenic activities in vitro and in vivo, a natural chemical library that identified potent agonistic activity between cyanidin and peroxisome proliferator-activated receptors (PPAR) was performed. Cyanidin induced transactivation activity in all three PPAR subtypes in a reporter gene assay and time-resolved fluorescence energy transfer analyses. Cyanidin also bound directly to all three subtypes, as assessed by surface plasmon resonance experiments, and showed the greatest affinity to PPARα. These effects were confirmed by measuring the expression of unique genes of each PPAR subtype. Cyanidin significantly reduced cellular lipid concentrations in lipid-loaded steatotic hepatocytes. In addition, transcriptome profiling in lipid-loaded primary hepatocytes revealed that the net effects of stimulation with cyanidin on lipid metabolic pathways were similar to those elicited by hypolipidemic drugs. Cyanidin likely acts as a physiological PPARα agonist and potentially for PPARß/δ and γ, and reduces hepatic lipid concentrations by rewiring the expression of genes involved in lipid metabolic pathways.

Ombuin-3-O-ß-D-glucopyranoside from Gynostemma pentaphyllum is a dual agonistic ligand of peroxisome proliferator-activated receptors α and δ/ß.

We demonstrated that ombuin-3-O-ß-D-glucopyranoside (ombuine), a flavonoid from Gynostemma pentaphyllum, is a dual agonist for peroxisome proliferator-activated receptors (PPARs) α and δ/ß. Using surface plasmon resonance (SPR), time-resolved fluorescence resonance energy transfer (FRET) analyses, and reporter gene assays, we showed that ombuine bound directly to PPARα and δ/ß but not to PPARγ or liver X receptors (LXRs). Cultured HepG2 hepatocytes stimulated with ombuine significantly reduced intracellular concentrations of triglyceride and cholesterol and downregulated the expression of lipogenic genes, including sterol regulatory element binding protein-1c (SREBP1c) and stearoyl-CoA desaturase-1 (SCD-1), with activation of PPARα and δ/ß. Activation of LXRs by ombuine was confirmed by reporter gene assays, however, SPR and cell-based FRET assays showed no direct binding of ombuine to either of the LXRs suggesting LXR activation by ombuine may be operated via PPARα stimulation. Ombuine-stimulated macrophages showed significantly induced transcription of ATP binding cassette cholesterol transporter A1 (ABCA1) and G1 (ABCG1), the key genes in reverse cholesterol transport, which led to reduced cellular cholesterol concentrations. These results suggest that ombuine is a dual PPAR ligand for PPARα and δ/ß with the ability to decrease lipid concentrations by reducing lipogenic gene expression in hepatocytes and inducing genes involved in cholesterol efflux in macrophages.

Induction of ABCA1 and ABCG1 expression by the liver X receptor modulator cineole in macrophages.

We investigated the effect of cineole on the expression of genes related to reverse cholesterol transport and hepatic fatty acid metabolism. Cineole, a small aroma compound in teas and herbs, significantly stimulated the transactivation of liver X receptor modulator (LXR)-α and LXR-ß. The mRNA and protein expression of LXRs and their target genes, including ABCA1 and ABCG1, was significantly increased in macrophages stimulated with cineole. This led to the subsequent removal of cholesterol from the cells. Interestingly, cineole showed tissue-selective LXR induction: hepatocytes stimulated with cineole showed significantly reduced expression of LXR-α and LXR-α-responsive genes, including FAS and SCD-1 (P <0.05). Accordingly, hepatocytes treated with cineole displayed reduced cellular lipid accumulation compared with control cells, as assessed by Oil Red O lipid staining and cholesterol quantification. These results suggest that cineole is a selective LXR modulator that regulates the expression of key genes in reverse cholesterol transport in macrophages without inducing lipogenesis in hepatocytes. This selective LXR modulator may have practical implications for the development of hypocholesterolemic or anti-atherosclerotic agents and also suggests.

Cyanidin, a natural flavonoid, is an agonistic ligand for liver X receptor alpha and beta and reduces cellular lipid accumulation in macrophages and hepatocytes.

Cyanidin, a natural flavonoid abundant in fruits and vegetables, is known to regulate cellular lipid metabolism; however, its underlying mechanism of action and protein targets remain unknown. Here, the ligand binding activity of cyanidin on liver X receptors (LXRs) was investigated utilizing surface plasmon resonance and time-resolved fluorescence energy transfer (TR-FRET) analyses. LXRs are nuclear receptors which function as critical transcription factors in the regulation of cellular lipid and glucose metabolism. This includes the stimulation of high-density-lipoprotein synthesis and activation of reverse cholesterol transport. The present findings show that cyanidin induces the transactivation of LXRs and binds directly to the ligand-binding domain of both LXRα and LXRß with dissociation constants of 2.2 and 73.2µM, respectively. Cell-free FRET analysis demonstrated that cyanidin induces the recruitment of co-activator peptide for LXRα and LXRß with EC50 of 3.5µM and 125.2µM, respectively. In addition, intracellular cholesterol and triglyceride (TG) concentrations were reduced in macrophages following cyanidin stimulation. In cultured hepatocytes, cyanidin mildly induced SREBP1c gene expression but marginally affected cellular TG concentrations as well as reduced cellular cholesterol accumulations which activated the expression of genes for reverse cholesterol transport. Two cyanidin metabolites, procatechic acid and phloroglucinaldehyde, did not directly bind or activate LXRs. These results demonstrate that cyanidin is a direct ligand for both LXRα and LXRß, suggesting that cyanidin may operate, at least in part, through modulation of cellular LXR activity.

Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-α.

Peroxisome proliferator-activated receptor-alpha (PPARα) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPARα agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPARα agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPARα. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P<0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPARα via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

Melissa officinalis essential oil reduces plasma triglycerides in human apolipoprotein E2 transgenic mice by inhibiting sterol regulatory element-binding protein-1c-dependent fatty acid synthesis.

We investigated the hypolipidemic effects of Melissa officinalis essential oil (MOEO) in human APOE2 transgenic mice and lipid-loaded HepG2 cells. Plasma TG concentrations were significantly less in APOE2 mice orally administered MOEO (12.5 µg/d) for 2 wk than in the vehicle-treated group. Cellular TG and cholesterol concentrations were also significantly decreased in a dose- (400 and 800 mg/L) and time- (12 and 24 h) dependent manner in HepG2 cells stimulated with MOEO compared with controls. Mouse hepatic transcriptome analysis suggested MOEO feeding altered several lipid metabolic pathways, including bile acid and cholesterol synthesis and fatty acid metabolism. In HepG2 cells, the rate of fatty acid oxidation, as assessed using [1-(14)C]palmitate, was unaltered; however, the rate of fatty acid synthesis quantified with [1-(14)C]acetate was significantly reduced by treatment with 400 and 800 mg/L MOEO compared with untreated controls. This reduction was due to the decreased expression of SREBP-1c and its responsive genes in fatty acid synthesis, including FAS, SCD1, and ACC1. Subsequent chromatin immunoprecipitation analysis further demonstrated that the binding of p300/CBP-associated factor, a coactivator of SREBP-1c, and histone H3 lysine 14 acetylation at the FAS, SCD1, and ACC1 promoters were significantly reduced in the livers of APOE2 mice and HepG2 cells treated with MOEO compared with their controls. Additionally, MOEO stimulation in HepG2 cells induced bile acid synthesis and reduced the nuclear form of SREBP-2, a key transcription factor in hepatic cholesterol synthesis. These findings suggest that the intake of phytochemicals with pleasant scent could have beneficial metabolic effects.

Ethyl 2,4,6-trihydroxybenzoate is an agonistic ligand for liver X receptor that induces cholesterol efflux from macrophages without affecting lipid accumulation in HepG2 cells.

The present study reports a novel liver X receptor (LXR) activator, ethyl 2,4,6-trihydroxybenzoate (ETB), isolated from Celtis biondii. Using a reporter gene assay, time-resolved fluorescence resonance energy transfer (TR-FRET), and surface plasmon resonance (SPR) analysis, we showed that ETB directly bound to and stimulated the transcriptional activity of LXR-α and LXR-ß. In macrophages, hepatocytes, and intestinal cells, ETB suppressed cellular cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-ß-responsive genes. Notably, ETB did not induce lipogenic gene expression or cellular triglyceride accumulation in hepatocytes. These results suggest that ETB is a dual-LXR modulator that regulates the expression of key genes in cholesterol homeostasis in multiple cells without inducing lipid accumulation in HepG2 cells.

Iristectorigenin B isolated from Belamcanda chinensis is a liver X receptor modulator that increases ABCA1 and ABCG1 expression in macrophage RAW 264.7 cells.

A novel liver X receptor (LXR) modulator, iristectorigenin B isolated from Belamcanda chinensis, stimulated the transcriptional activity of both LXR-α and LXR-ß. In macrophages, iristectorigenin B suppressed cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-ß-responsive genes, ATP-binding cassette transporters A1 and G1. It did not induce hepatic lipid accumulation nor the expression of the lipogenesis genes sterol regulatory element-binding protein-1c, fatty acid synthase, and stearoyl-CoA desaturase-1. Iristectorigenin B thus is a dual-LXR agonist that regulates the expression of key genes in cholesterol homeostasis in macrophage cells without inducing hepatic lipid accumulation.

Acute effect of high-dose isoflavones from Pueraria lobata (Willd.) Ohwi on lipid and bone metabolism in ovariectomized mice.

We investigated the acute metabolic effects of isoflavones from Pueraria lobata (Willd.) Ohwi (IPL) in ovariectomized (OVX) mice. After 4 weeks of IPL feeding at 500 mg/day/kg body weight (OVX500), plasma 17ß-estradiol concentrations were significantly higher (+25%, p < 0.05), whereas plasma triglyceride levels were significantly lower in OVX mice (-15%, p < 0.05) compared with controls. Abdominal adipose tissue weight was marginally reduced in IPL-fed groups compared with OVX controls and the plasma levels of liver enzymes were unchanged. In addition, IPL significantly inhibited the reduction of bone mineral density in the femurs of OVX mice (OVX200, +22%; OVX500, +26%; p < 0.05) compared with controls after 4 weeks of IPL feeding. In quantitative polymerase chain reaction analysis the expression of aromatase was significantly suppressed and SULT1E1 was increased by IPL feeding, showing that IPL feeding may not alter the risk for breast cancer in mice. Our results suggest that IPL could ameliorate menopausal symptoms in mice. Further studies will confirm the effects of IPL in humans.

Restoration of autophagy by puerarin in ethanol-treated hepatocytes via the activation of AMP-activated protein kinase.

We investigated the effects of puerarin, the major isoflavone in Kudzu roots, on the regulation of autophagy in ethanol-treated hepatocytes. Incubation in ethanol (100 mM) for 24 h reduced cell viability by 20% and increased the cellular concentrations of cholesterol and triglycerides by 40% and 20%, respectively. Puerarin stimulation significantly recovered cell viability and reduced cellular lipid accumulation to a level comparable to that in untreated control cells. Ethanol incubation reduced autophagy significantly as assessed by microtubule-associated protein1 light chain 3 (LC3) expression using immunohistochemistry and immunoblot analysis. The reduced expression of LC3 was restored by puerarin in a dose-dependent manner in ethanol-treated cells. The effect of puerarin on mammalian targets of rapamycin (mTOR), a key regulator of autophagy, was examined in ethanol-treated hepatocytes. Immunoblotting revealed that puerarin significantly induced the phosphorylation of 5'AMP-activated protein kinase (AMPK), thereby suppressing the mTOR target proteins S6 ribosomal protein and 4E-binding protein 1. These data suggest that puerarin restored the viability of cells and reduced lipid accumulation in ethanol-treated hepatocytes by activating autophagy via AMPK/mTOR-mediated signaling.

Ursolic acid is a PPAR-α agonist that regulates hepatic lipid metabolism.

In this study, we confirmed that ursolic acid, a plant triterpenoid, activates peroxisome proliferator-activated receptor (PPAR)-α in vitro. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses do not show direct binding of ursolic acid to the ligand-binding domain of PPAR-α; however, ursolic acid enhances the binding of PPAR-α to the peroxisome proliferator response element in PPAR-α-responsive genes, alters the expression of key genes in lipid metabolism, significantly reducing intracellular triglyceride and cholesterol concentrations in hepatocytes. Thus, ursolic acid is a PPAR-α agonist that regulates the expression of lipid metabolism genes, but it is not a direct ligand of PPAR-α.

The efficacy and safety of epidermal growth factor in treatment of diabetic foot ulcers: the preliminary results.

OBJECTIVE: To evaluate the efficacy and safety of recombinant human epidermal growth factor (rh-EGF) in healing foot ulcers in diabetic patients. METHODS: A total of 28 subjects with foot ulcers were recruited into the pilot study. Patients who had obvious peripheral arterial disease, trans-tibial amputation, plastic surgery or skin flap, and skin graft were excluded. The properly debrided wounds and the non closure wounds after toe amputation were included. When the wounds became clean or uninfected, they received twice-a-day treatment with 0.005% Easyef and hydrocolloid dressing. The size and severity of the wounds were evaluated. Others such as blood sugar, renal and hepatic function, serum albumin, vascular condition, foot infection or osteomyelitis were assessed. RESULTS: All of 28 patients had positive response of granulation (100%). Complete healing was noted in 13 out of 23 subjects and finished 8-week follow-up (56.5%). The rates of wound closure were 43.3%, 59.9%, 68.7%, and 84.8% in week 2, 4, 6 and 8, respectively, regardless of the severity. Being dropped out, three patients needed further interventions. No skin allergic reaction. Over-granulation was observed in one female patient (3.7%), but as minor. CONCLUSIONS: Easyef has positive effects on healing of moderate-to-severe foot ulcers and demonstrated being safe to diabetic patients. The drug had high tolerability and compliance.

Kaempferol reduces hepatic triglyceride accumulation by inhibiting Akt.

In this paper, we studied the mechanism of the triglyceride (TG)-lowering effect of kaempferol in vitro and in vivo. Kaempferol showed LXR agonistic activities without inducing TGs or the expression of several lipogenic genes in cultured cells. A luciferase and qPCR analysis showed that kaempferol increased the transactivation of PPARα and PPARδ and stimulated gene expression associated with fatty acid oxidation and uptake in hepatocytes. More importantly, kaempferol inhibited protein kinase B (Akt) activity and suppressed SREBP-1 activation via multiple mechanisms, including through increasing Insig-2a expression, reducing SREBP-1 phosphorylation, and increasing GSK-3 phosphorylation. Collectively, these actions inhibited the SREBP-1 activation process. Furthermore, as an Akt/mTOR pathway inhibitor, kaempferol led to the induction of hepatic autophagy and resulted in a decrease in lipid droplet formation in the mouse liver. These findings demonstrate that kaempferol exerts its TG-lowering effect via Akt inhibition and activation of PPARα and PPARδ. PRACTICAL APPLICATIONS: Kaempferol is a major dietary flavonoid in various plant-based foods, and it is used as a valuable ingredient in functional foods, with numerous beneficial properties such as anticancer, antioxidant, and anti-atherosclerotic activities. Kaempferol exerts its TG-lowering effect via Akt inhibition and activation of PPARα and PPARδ. Currently, the number of people with hyperlipidemia is rapidly growing in both developed and developing societies; thus, we propose that kaempferol could be used for therapeutic interventions aimed at the treatment of these individuals.

Kaempferol ameliorates symptoms of metabolic syndrome by regulating activities of liver X receptor-ß.

Kaempferol is a dietary flavonol previously shown to regulate cellular lipid and glucose metabolism. However, its molecular mechanisms of action and target proteins have remained elusive, probably due to the involvement of multiple proteins. This study investigated the molecular targets of kaempferol. Ligand binding of kaempferol to liver X receptors (LXRs) was quantified by time-resolved fluorescence resonance energy transfer and surface plasmon resonance analyses. Kaempferol directly binds to and induces the transactivation of LXRs, with stronger specificity for the ß-subtype (EC50 = 0.33 µM). The oral administration of kaempferol in apolipoprotein-E-deficient mice (150 mg/day/kg body weight) significantly reduced plasma glucose and increased high-density lipoprotein cholesterol levels and insulin sensitivity compared with the vehicle-fed control. Kaempferol also reduced plasma triglyceride concentrations and did not cause liver steatosis, a common side effect of potent LXR activation. In immunoblotting analysis, kaempferol reduced the nuclear accumulation of sterol regulatory element-binding protein-1 (SREBP-1). Our results show that the suppression of SREBP-1 activity and the selectivity for LXR-ß over LXR-α by kaempferol contribute to the reductions of plasma and hepatic triglyceride concentrations in mice fed kaempferol. They also suggest that kaempferol activates LXR-ß and suppresses SREBP-1 to enhance symptoms in metabolic syndrome.

Extraction of squalene as value-added product from the residual biomass of Schizochytrium mangrovei PQ6 during biodiesel producing process.

Today microalgae represent a viable alternative source of squalene for commercial application. The species Schizochytrium mangrovei, a heterotrophic microalga, has been widely studied and provides a high amount of squalene, polyunsaturated fatty acids and has good profiles for biodiesel production. Our work was aimed at examining the squalene contents in Vietnam's heterotrophic marine microalga S. mangrovei PQ6 biomass and residues of the biodiesel process from this strain. Thin-layer chromatography and high-performance liquid chromatography (HPLC) methods were successfully applied to the determination of squalene in S. mangrovei PQ6. The squalene content and production of S. mangrovei PQ6 reached 33.00 ± 0.02 and 33.04 ± 0.03 mg g(-1) of dry cell weight; and 0.992 g L(-1) and 1.019 g L(-1) in 30 and 150 L bioreactors, respectively after 96 h of fermentation. In addition, squalene was also detected in spent biomass (approximately 80.10 ± 0.03 mg g(-1) of spent biomass) from the S. mangrovei PQ6 biodiesel production process. The structure of squalene in residues of the biodiesel process was confirmed from its nuclear magnetic resonance spectra. The results obtained from our work suggest that there is tremendous potential in the exploitation of squalene as a value-added by-product besides biodiesel from S. mangrovei PQ6 to reduce biodiesel price.

Hypolipidemic effect of Goami-3 rice (Oryza sativa L. cv. Goami-3) on C57BL/6J mice is mediated by the regulation of peroxisome proliferator-activated receptor-α and -γ.

We investigated the hypolipidemic effects of Goami-3 rice (GR; Oryza sativa L. cv. Goami-3), a newly developed strain with high levels of amylose and fibers. Diet-induced obese mice were fed three types of isocaloric diets for 8 weeks: a high-fat diet, a high-fat diet with GR or control rice (CR; O. sativa L. cv. Ilpumbyeo). Mice fed GR exhibited a significant reduction in body fat (-23%), total cholesterol (-20%) and triglyceride concentrations (-30%) compared to mice fed CR. The mice fed GR showed induction of peroxisome proliferator-activated receptor (PPAR)-α and inhibition of γ expressions in the liver and adipose tissue. The reduced adiposity of mice fed GC was supported by changes in the expression of genes related to lipid accumulation and hydrolysis in adipose tissues and the plasma concentrations of insulin, adiponectin and leptin. Principal components analysis with gas chromatography-time-of-flight mass spectrometry-based metabolomic data revealed that the average level of specific plasma metabolites in the GR group was statistically different from that in the other groups after 4weeks. These metabolites included propionic acid, valine, leucine and proline. Based on partial least-squares analysis, the plasma concentrations of valine were inversely correlated with the high-density lipoprotein (HDL) to non-HDL and HDL to total cholesterol ratios. In conclusion, GR feeding for 8 weeks significantly improved dyslipidemia and adiposity in diet-induced obese mice by regulating gene expression of PPARs and its target genes. Key plasma metabolites (including valine) were significantly altered by the hypolipidemic effects of GR.

Correction: Hepatic Lipid Accumulation Alters Global Histone H3 Lysine 9 and 4 Trimethylation in the Peroxisome Proliferator-Activated Receptor Alpha Network.

[This corrects the article DOI: 10.1371/journal.pone.0044345.].

Hepatic lipid accumulation alters global histone h3 lysine 9 and 4 trimethylation in the peroxisome proliferator-activated receptor alpha network.

Recent data suggest that the etiology of several metabolic diseases is closely associated with transcriptome alteration by aberrant histone methylation. We performed DNA microarray and ChIP-on-chip analyses to examine transcriptome profiling and trimethylation alterations to identify the genomic signature of nonalcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease. Transcriptome analysis showed that steatotic livers in high-fat diet-fed apolipoprotein E2 mice significantly altered the expression of approximately 70% of total genes compared with normal diet-fed control livers, suggesting that hepatic lipid accumulation induces dramatic alterations in gene expression in vivo. Also, pathway analysis suggested that genes encoding chromatin-remodeling enzymes, such as jumonji C-domain-containing histone demethylases that regulate histone H3K9 and H3K4 trimethylation (H3K9me3, H3K4me3), were significantly altered in steatotic livers. Thus, we further investigated the global H3K9me3 and H3K4me3 status in lipid-accumulated mouse primary hepatocytes by ChIP-on-chip analysis. Results showed that hepatic lipid accumulation induced aberrant H3K9me3 and H3K4me3 status in peroxisome proliferator-activated receptor alpha and hepatic lipid catabolism network genes, reducing their mRNA expression compared with non-treated control hepatocytes. This study provides the first evidence that epigenetic regulation by H3K9me3 and H3K4me3 in hepatocytes may be involved in hepatic steatosis and the pathogenesis of NAFLD. Thus, control of H3K9me3 and H3K4me3 represents a potential novel NAFLD prevention and treatment strategy.