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 PRAR$\mathsf{\gamma }$ 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 PRAR$\mathsf{\gamma }$ 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.

Our previous study had shown that chronic corticosterone (CORT) exposure causes excessive fat deposition in chicken liver, yet it remains unknown whether it is associated with inflammation and fibrosis. In general, heat shock proteins (HSPs) are activated in response to acute stress to play a cytoprotective role, and this activation is associated with m⁶A-mediated post-transcriptional regulation. However, changes of HSPs and the m⁶A methylation on their mRNAs in response to chronic CORT treatment in chicken liver have not been reported. In this study, chronic CORT exposure induced inflammation and fibrosis in chicken liver, associated with significantly modulated expression of HSPs that was significantly upregulated at mRNA level yet downregulated at protein level. Concurrently, m⁶A methyltransferases METTL3 content was upregulated together with the level of m⁶A methylation on HSPs transcripts. The m⁶A-seq analysis revealed 2-6 significantly (P < 0.05) hypermethylated m⁶A peaks in the mRNA of 4 different species of HSPs in CORT-treated chicken liver. HSP90B1 transcript had 6 differentially methylated m⁶A peaks among which peaks on exon 16 and exon 17 showed 3.14- and 4.72-fold of increase, respectively. Mutation of the 8 predicted m⁶A sites on exon 16 and exon 17 resulted in a significant (P < 0.05) increase in eGFP-fused content of HSP90B1 exon 16 and exon 17 fragment in 293 T cells, indicating a possible role of m⁶A in post-transcriptional regulation of HSPs. In conclusion, chronic CORT exposure induces inflammation and fibrosis in chicken liver along with an increase in the levels and m6A methylation of several HSPs mRNAs; HSPs levels were however reduced under the indicated conditions. Results presented suggest that the reduction in HSPs levels may be associated with m6A methylation in CORT-exposed chickens.

Heat stress (HS) can inhibit the growth performance of broilers and cause substantial economic losses. Alterations in bile acid (BA) pools have been reported to be correlated with chronic HS, yet the specific mechanism and whether it is related to gut microbiota remains unclear. In this study, 40 Rugao Yellow chickens were randomly selected and distributed into two groups (20 broilers in each group) when reaching 56-day age: a chronic heat stress group (HS, 36 ± 1 °C for 8 h per day in the first 7 days and 36 ± 1 °C for 24 h in the last 7 days) and a control group (CN, 24 ± 1 °C for 24 h within 14 days). Compared with the CN group, total BAs’ serum content decreased, while cholic acid (CA), chenodeoxycholic acid (CDCA), and taurolithocholic acid (TLCA) increased significantly in HS broilers. Moreover, 12α-hydroxylase (CYP8B1) and bile salt export protein (BSEP) were upregulated in the liver, and the expression of fibroblast growth factor 19 (FGF19) decreased in the ileum of HS broilers. There were also significant changes in gut microbial composition, and the enrichment of Peptoniphilus was positively correlated with the increased serum level of TLCA. These results indicate that chronic HS disrupts the homeostasis of BA metabolism in broilers, which is associated with alterations in gut microbiota.