Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive fat deposition in the liver, which is often associated with disrupted iron homeostasis. Betaine has been reported to be hepatoprotective, yet whether and how betaine ameliorates high-fat diet-induced disruption of hepatic lipid and iron homeostasis remains elusive. In this study, mice were fed either standard (CON) or high-fat diet (HFD) for 9 weeks to establish a NAFLD model. Mice raised on HF diet were then assigned randomly to HF and HFB groups, HFB group being supplemented with 1% (w/v) of betaine in the drinking water for 13 weeks. Betaine supplementation significantly alleviated excessive hepatic lipid deposition and restored hepatic iron content. Betaine partly yet significantly reversed HFD-induced dysregulation of lipogenic genes such as PRARγ and CD36, as well as the iron-metabolic genes including FPN and HAMP that encodes hepcidin. Similar mitigation effects of betaine were observed for BMP2 and BMP6, the up-stream regulators of hepcidin expression. Betaine significantly rectified disrupted expression of methyl transfer gene, including BHMT, GNMT and DNMT1. Moreover, HFD-modified CpG methylation on the promoter of PRARγ and HAMP genes was significantly reversed by betaine supplementation. These results indicate that betaine alleviates HFD-induced disruption of hepatic lipid and iron metabolism, which is associated with modification of CpG methylation on promoter of lipogenic and iron-metabolic genes.

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Background Glucocorticoid receptor (GR) mediated corticosterone-induced fatty liver syndrome (FLS) in the chicken by transactivation of Fat mass and obesity associated gene (FTO), leading to demethylation of N6-methyladenosine (m6A) and post-transcriptional activation of lipogenic genes. Nutrition is considered the main cause of FLS in the modern poultry industry. Therefore, this study was aimed to investigate whether GR and m6A modification are involved in high-energy and low protein (HELP) diet-induced FLS in laying hens, and if true, what specific m6A sites of lipogenic genes are modified and how GR mediates m6A-dependent lipogenic gene activation in HELP diet-induced FLS in the chicken. Results Laying hens fed HELP diet exhibit excess (P < 0.05) lipid accumulation and lipogenic genes activation in the liver, which is associated with significantly increased (P < 0.05) GR expression that coincided with global m6A demethylation. Concurrently, the m6A demethylase FTO is upregulated (P < 0.05), whereas the m6A reader YTHDF2 is downregulated (P < 0.05) in the liver of FLS chickens. Further analysis identifies site-specific demethylation (P < 0.05) of m6A in the mRNA of lipogenic genes, including FASN, SREBP1 and SCD. Moreover, GR binding to the promoter of FTO gene is highly enriched (P < 0.05), while GR binding to the promoter of YTHDF2 gene is diminished (P < 0.05). Conclusions These results implicate a possible role of GR-mediated transcriptional regulation of m6A metabolic genes on m6A-depenent post-transcriptional activation of lipogenic genes and shed new light in the molecular mechanism of FLS etiology in the chicken.

Scope: Betaine serves as a methyl donor for DNA methylation. Here, the effects of betaine on hippocampal expression of neurogenesis genes and their DNA methylation status across three generations are investigated. Methods and Results: Pregnant rats (F0) are fed control and betaine-supplemented diets throughout gestation and lactation. Female F1 and F2 offspring at weaning, together with the F0 dams, are used in the study. Hippocampal expression of aromatase, estrogen receptor , and estrogen-related receptor is downregulated in F1, together with the estrogen-responsive insulin-like growth factor 2/insulin-like growth factor binding protein 2 (IGF-2/IGFBP2) genes. However, all these genes are upregulated in F2, which follows the same pattern of F0. In agreement with changes in mRNA expression, the imprinting control region (ICR) of IGF-2 gene is hypomethylated in F1 but hypermethylated in F2 and F0. In contrast, the promoter DNA methylation status of all the affected genes is hypermethylated in F1 but hypomethylated in F2 and F0. Methyl transfer enzymes, such as betaine homocysteine methyltransferase and DNA methyltransferase 1, follow the same pattern of transgenerational inheritance. Conclusion: These results indicate that betaine exerts a transgenerational effect on hippocampal expression of estrogen-responsive genes in rat offspring, which is associated with corresponding alterations in DNA methylation on ICR of IGF-2 gene and the promoter of affected genes.

24Betaine as a methyl donor participates in DNA methylation. Here, we sought to investigate 25 the effects of betaine on hippocampal expression of neurogenesis genes and their DNA 26 methylation status across three generations. Pregnant rats (F0) were fed control and 27 betaine-supplemented diets throughout gestation and lactation; and female F1 and F2 28 offspring at weaning, together with the F0 dams, were used in the study. Hippocampal 29 expression of aromatase, estrogen receptor α and estrogen related receptor β was 30 down-regulated in F1, together with the estrogen-responsive IGF-2/IGFBP2 genes. 31 However, all these genes were up-regulated in F2, which follows the same pattern of F0. In 32 agreement with changes in mRNA expression, the imprinting control region (ICR) of 33 IGF-2 gene was hypomethylated in F1 but hypermethylated in F2 and F0. In contrast, the 34 promoter DNA methylation status of all the affected genes was hypermethylated in F1 but 35 hypomethylated in F2 and F0. Betaine homocysteine methyltransferase followed same 36 pattern of transgenerational inheritance. These results indicate that betaine exerts a 37 transgenerational effect on hippocampal expression of estrogen-responsive genes in rat 38 offspring, which is associated with corresponding alterations in DNA methylation on ICR 39 of IGF-2 gene and the promoter of affected genes. 40 42 43 44 3 45 Author Summary 46 Epigenetic transgenerational inheritance is defined as the phenotypic change across 47 generations and germline transmission of epigenetic information without direct genetic 48 manipulation or environmental exposure. Maternal and paternal diet has been shown to 49 have transgenerational behavioral and physiological effects. Maternal intake of methyl 50 donors, including folate, betaine, and choline during different gestation period alters 51 epigenetic modifications of many imprinted genes in the genomic DNA isolated from cord 52blood. Betaine as a methyl donor nutrition plays an important role in brain development. 53However, the effect of maternal betaine supplementation on transgenerational inheritance 54 of imprinted gene IGF-2 in offspring hippocampus is still unknown. Here, we sought to 55 investigate the effects of betaine on rat hippocampal expression of neurogenesis genes and 56 their DNA methylation status across three generations. We found that betaine significantly 57 decreased estrogen-responsive genes IGF-2/IGFBP2 expression in rat F1 offspring, yet 58 increased in F2 offspring, which is associated with corresponding alterations in DNA 59 methylation on ICR of IGF-2 gene and the promoter of affected genes. Our results 60 suggesting that maternal methyl donor nutrition supplementation may be exert an 61 epigenetic transgenerational effect on female offspring. 4 63 Introduction 64Hippocampus is the key structure governing many important functions including learning 65 and memory, as well as navigation and spatial orientation (1). Accumulated studies have 66 shown that estrogen enhances synaptic plasticity, such ...