Berberine reduces methylation of the MTTP promoter and alleviates fatty liver induced by a high-fat diet in rats

High-calorie food leads to nonalcoholic fatty liver disease (NAFLD) through dysregulation of genes involved in lipid metabolism, but the precise mechanism remains unclear. DNA methylation represents one of the mechanisms that contributes to dysregulation of gene expression via interaction with environmental factors. Berberine can alleviate fatty liver in db/db and ob/ob mice. Here, we investigated whether DNA methylation is involved in the pathogenesis of NAFLD induced by a high-fat diet (HFD) and whether berberine improves NAFLD through influencing the methylation status of promoters of key genes. HFD markedly decreased the mRNA levels encoding CPT-1α, MTTP, and LDLR in the liver. In parallel, DNA methylation levels in the MTTP promoter of rats with NAFLD were elevated in the liver. Interestingly, berberine reversed the downregulated expression of these genes and selectively inhibited HFD-induced increase in the methylation of MTTP. Consistently, berberine increased hepatic triglyceride (TG) export and ameliorated HFD-induced fatty liver. Furthermore, a close negative correlation was observed between the MTTP expression and its DNA methylation (at sites −113 and −20). These data indicate that DNA methylation of the MTTP promoter likely contributes to its downregulation during HFD-induced NAFLD and, further, that berberine can partially counteract the HFD-elicited dysregulation of MTTP by reversing the methylation state of its promoter, leading to reduced hepatic fat content.     

S-adenosylmethionine upregulates the angiotensin receptor-binding protein ATRAP via the methylation of HuR in NAFLD

Nonalcoholic fatty liver disease (NAFLD) has emerged globally and is associated with inflammatory signaling. The underlying mechanisms remain poorly delineated, although NAFLD has attracted considerable attention and been extensively investigated. Recent publications have determined that angiotensin II (Ang II) plays an important role in stimulating NAFLD progression by causing lipid metabolism disorder and insulin resistance through its main receptor, Ang II type 1 receptor (AT1R). Herein, we explored the effect of supplementary S-adenosylmethionine (SAM), which is the main biological methyl donor in mammalian cells, in regulating AT1R-associated protein (ATRAP), which is the negative regulator of AT1R. We found that SAM was depleted in NAFLD and that SAM supplementation ameliorated steatosis. In addition, in both high-fat diet-fed C57BL/6 rats and L02 cells treated with oleic acid (OA), ATRAP expression was downregulated at lower SAM concentrations. Mechanistically, we found that the subcellular localization of human antigen R (HuR) was determined by the SAM concentration due to protein methylation modification. Moreover, HuR was demonstrated to directly bind ATRAP mRNA and control its nucleocytoplasmic shuttling. Thus, SAM was suggested to upregulate ATRAP protein expression by maintaining the export of its mRNA from the nucleus. Taken together, our findings suggest that SAM can positively regulate ATRAP in NAFLD and may have various potential benefits for the treatment of NAFLD.Subject terms: Lipidomics, Metabolomics     

Folic acid supplementation alters the DNA methylation profile and improves insulin resistance in high-fat-diet-fed mice

Folic acid (FA) supplementation may protect from obesity and insulin resistance, the effects and mechanism of FA on chronic high-fat-diet-induced obesity-related metabolic disorders are not well elucidated. We adopted a genome-wide approach to directly examine whether FA supplementation affects the DNA methylation profile of mouse adipose tissue and identify the functional consequences of these changes. Mice were fed a high-fat diet (HFD), normal diet (ND) or an HFD supplemented with folic acid (20 μg/ml in drinking water) for 10 weeks, epididymal fat was harvested, and genome-wide DNA methylation analyses were performed using methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mice exposed to the HFD expanded their adipose mass, which was accompanied by a significant increase in circulating glucose and insulin levels. FA supplementation reduced the fat mass and serum glucose levels and improved insulin resistance in HFD-fed mice. MeDIP-seq revealed distribution of differentially methylated regions (DMRs) throughout the adipocyte genome, with more hypermethylated regions in HFD mice. Methylome profiling identified DMRs associated with 3787 annotated genes from HFD mice in response to FA supplementation. Pathway analyses showed novel DNA methylation changes in adipose genes associated with insulin secretion, pancreatic secretion and type 2 diabetes. The differential DNA methylation corresponded to changes in the adipose tissue gene expression of Adcy3 and Rapgef4 in mice exposed to a diet containing FA. FA supplementation improved insulin resistance, decreased the fat mass, and induced DNA methylation and gene expression changes in genes associated with obesity and insulin secretion in obese mice fed a HFD.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Maternal betaine supplementation attenuates glucocorticoid-induced hepatic lipid accumulation through epigenetic modification in adult offspring rats

There are lots of reports about alleviation of NAFLD by dietary supplements of betaine. However, it remains unclear whether maternal betaine supplementation can also ameliorate NAFLD in offspring. Hence, twenty pregnant rats were fed with a basal diet with or without betaine (1%), and then the female offspring rats were raised at 3 months of age followed by 3 weeks of physiological saline or dexamethasone in a dose of 0.1 mg/kg body mass every day via intraperitoneal injection. In this study, maternal betaine supplementation significantly (P<.05) reduced the increase of hepatic triglycerides concentration in dexamethasone-induced rats, which is associated with the expression of hepatic lipogenic genes (ACC1, FASN and SCD1). Moreover, the hypomethylation of lipogenic genes in dexamethasone-induced rats were reserved by prenatal betaine exposure. Furthermore, the increase of hepatic GR or SP1 content in dexamethasone-injected rats were significantly decreased (P<.05), which were in line with the binding of GR or SP1 to lipogenic genes, in betaine -exposed rats. Together, these results suggest that maternal betaine supplementation attenuates dexamethason-induced fatty liver in the female adult offspring rats, which may be attributed to DNA methylation and GR or SP1-mediated the regulation of lipogenic genes.     

In Ovo Injection of Betaine Affects Hepatic Cholesterol Metabolism through Epigenetic Gene Regulation in Newly Hatched Chicks

Betaine is reported to regulate hepatic cholesterol metabolism in mammals. Chicken eggs contain considerable amount of betaine, yet it remains unknown whether and how betaine in the egg affects hepatic cholesterol metabolism in chicks. In this study, eggs were injected with betaine at 2.5 mg/egg and the hepatic cholesterol metabolism was investigated in newly hatched chicks. Betaine did not affect body weight or liver weight, but significantly increased the serum concentration (P < 0.05) and the hepatic content (P < 0.01) of cholesterol. Accordingly, the cholesterol biosynthetic enzyme HMGCR was up-regulated (P < 0.05 for both mRNA and protein), while CYP7A1 which converts cholesterol to bile acids was down-regulated (P < 0.05 for mRNA and P = 0.07 for protein). Moreover, hepatic protein content of the sterol-regulatory element binding protein 1 which regulates cholesterol and lipid biosynthesis, and the mRNA abundance of ATP binding cassette sub-family A member 1 (ABCA1) which mediates cholesterol counter transport were significantly (P < 0.05) increased in betaine-treated chicks. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and adenosylhomocysteinase-like 1 were increased (P < 0.05), which was associated with global genomic DNA hypermethylation (P < 0.05) and diminished gene repression mark histone H3 lysine 27 trimethylation (P < 0.05). Furthermore, CpG methylation level on gene promoters was found to be increased (P < 0.05) for CYP7A1 yet decreased (P < 0.05) for ABCA1. These results indicate that in ovo betaine injection regulates hepatic cholesterol metabolism in chicks through epigenetic mechanisms including DNA and histone methylations.     

Dietary and supplemental maternal methyl-group donor intake and cord blood DNA methylation

Maternal nutrition is critically involved in the development and health of the fetus. We evaluated maternal methyl-group donor intake through diet (methionine, betaine, choline, folate) and supplementation (folic acid) before and during pregnancy in relation to global DNA methylation and hydroxymethylation and gene specific (IGF2 DMR, DNMT1, LEP, RXRA) cord blood methylation. A total of 115 mother-infant pairs were enrolled in the MAternal Nutrition and Offspring's Epigenome (MANOE) study. The intake of methyl-group donors was assessed using a food-frequency questionnaire. LC-MS/MS and pyrosequencing were used to measure global and gene specific methylation, respectively. Dietary intake of methyl-groups before and during pregnancy was associated with changes in LEP, DNMT1, and RXRA cord blood methylation. Statistically significant higher cord blood LEP methylation was observed when mothers started folic acid supplementation more than 6 months before conception compared with 3–6 months before conception (34.6 ± 6.3% vs. 30.1 ± 3.6%, P = 0.011, LEP CpG1) or no folic acid used before conception (16.2 ± 4.4% vs. 13.9 ± 3%, P = 0.036 for LEP CpG3 and 24.5 ± 3.5% vs. 22.2 ± 3.5%, P = 0.045 for LEP mean CpG). Taking folic acid supplements during the entire pregnancy resulted in statistically significantly higher cord blood RXRA methylation as compared with stopping supplementation in the second trimester (12.3 ± 1.9% vs. 11.1 ± 2%, P = 0.008 for RXRA mean CpG). To conclude, long-term folic acid use before and during pregnancy was associated with higher LEP and RXRA cord blood methylation, respectively. To date, pregnant women are advised to take a folic acid supplement of 400 µg/day from 4 weeks before until 12 weeks of pregnancy. Our results suggest significant epigenetic modifications when taking a folic acid supplement beyond the current advice.     

Betaine supplementation prevents fatty liver induced by a high-fat diet: effects on one-carbon metabolism

The purpose of this study was to examine the effects of betaine supplementation on the regulation of one-carbon metabolism and liver lipid accumulation induced by a high-fat diet in rats. Rats were fed one of three different liquid diets: control diet, high-fat diet and high-fat diet supplemented with betaine. The control and high-fat liquid diets contained, respectively, 35 and 71 % of energy derived from fat. Betaine supplementation involved the addition of 1 % (g/L) to the diet. After three weeks on the high-fat diet the rats had increased total liver fat concentration, liver triglycerides, liver TBARS and plasma TNF-α. The high-fat diet decreased the hepatic S-adenosylmethionine concentration and the S-adenosylmethionine/S-adenosylhomocysteine ratio compared to the control as well as altering the expression of genes involved in one-carbon metabolism. Betaine supplementation substantially increased the hepatic S-adenosylmethionine concentration (~fourfold) and prevented fatty liver and hepatic injury induced by the high-fat diet. It was accompanied by the normalization of the gene expression of BHMT, GNMT and MGAT, which code for key enzymes of one-carbon metabolism related to liver fat accumulation. In conclusion, the regulation of the expression of MGAT by betaine supplementation provides an additional and novel mechanism by which betaine supplementation regulates lipid metabolism and prevents accumulation of fat in the liver.     

Sex-specific epigenetic alterations of the hypothalamic Agrp-Pomc system do not explain ‘diabesity’ in the offspring of high-fat diet (HFD) overfed maternal rats

Maternal high-fat diet (HFD) overfeeding pre- and during pregnancy and lactation may ‘program’ a ‘diabesity’ predisposition in the offspring, for inconclusive reasons. Acquired alterations of the hypothalamic promoter methylation and mRNA expression of the satiety neurohormone Pomc are possibly of critical importance here. We investigated within one developmental approach, including male and female rats, the sex-specific DNA methylation pattern and corresponding mRNA expression of both Pomc and its endogenous functional antagonist Agrp in the hypothalamus of adult HFD offspring. Obesity and diabetic disturbances occurred in both male and female HFD offspring, accompanied by altered Pomc promoter methylation pattern. However, this was not related to significant Pomc mRNA expression alterations. In contrast, male-specific alterations of Agrp promoter methylation were found, even associated with reduced mRNA expression of this orexigenic/anabolic Pomc antagonist. In conclusion, acquired epigenetic alterations of the hypothalamic Agrp-Pomc system hardly explain the ‘diabesity’ phenotype in HFD offspring, while distinct vulnerability and functionality of Agrp promoter and related genomic regions methylation should be further investigated.     

Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites

Whereas in Escherichia coli DNA mismatch repair is directed to the newly synthesized strand due to its transient lack of adenine methylation, the molecular determinants of strand discrimination in eukaryotes are presently unknown. In mammalian cells, cytosine methylation within CpG sites may represent an analogous and mechanistically plausible means of targeting mismatch correction. Using HeLa nuclear extracts, we conducted a systematic analysis in vitro to determine whether cytosine methylation participates in human DNA mismatch repair. We prepared a set of A·C heteroduplex molecules that were either unmethylated, hemimethylated or fully methylated at CpG sequences and found that the methylation status persisted under the assay conditions. However, no effect on either the time course or the magnitude of mismatch repair events was evident; only strand discontinuities contributed to strand bias. By western analysis we demonstrated that the HeLa extract contained MED1 protein, which interacts with MLH1 and binds to CpG-methylated DNA; supplementation with purified MED1 protein was without effect. In summary, human DNA mismatch repair operates independently of CpG methylation status, and we found no evidence supporting a role for CpG hemimethylation as a strand discrimination signal.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Cell specific patterns of methylation in the human placenta

Epigenetic processes, such as DNA methylation, are known to regulate tissue specific gene expression. We explored this concept in the placenta to define whether DNA methylation is cell-type specific. Cytotrophoblasts and fibroblasts were isolated from normal midtrimester placentas. Using immunocytochemistry, we demonstrated 95% purity for cytotrophoblasts and 60–70% for fibroblasts. We compared DNA methylation profiles from cytotrophoblasts, fibroblasts and whole placental villi using bisulfite modified genomic DNA hybridized to the Illumina Methylation27 array. Euclidean cluster analysis of the DNA methylation profiles showed two main clusters, one containing cytotrophoblasts and placenta, the other fibroblasts. Differential methylation analysis identified 442 autosomal CpG sites that differed between cytotrophoblasts and fibroblasts, 315 between placenta and fibroblasts and 61 between placenta and cytotrophoblasts. Three candidate methylation differences were validated by targeted pyrosequencing assays. Pyrosequencing assays were developed for CpG sites less methylated in cytotrophoblasts than fibroblasts mapping to the promoter region of the beta subunit of human chorionic gonadotropin 5 (CGB5), as well as two CpG sites mapping to each of two tumor suppressor genes. Our data suggest that epigenetic regulation of gene expression is likely to be a key factor in the functional specificity of cytotrophoblasts. These data are proof of principle for cell-type specific epigenetic regulation in placenta and demonstrate that the methylation profile of placenta is mainly driven by cytotrophoblasts.Key words: cytotrophoblast purification, placental fibroblast purification, DNA methylation, epigenetics, placenta, cell type-specific methylation