Betaine alleviates high-fat diet-induced fatty liver and prenatal betaine programs offspring hepatic lipid metabolism. Excessive corticosterone (CORT) exposure causes fatty liver in chickens, yet it remains unknown whether and how prenatal betaine modulates the susceptibility of CORT-induced fatty liver later in life. In this study, fertilized eggs were injected with saline or betaine before incubation, and the hatchlings were raised at 8 weeks of age followed by 7 days of subcutaneous CORT injection. CORT-induced fatty liver was less severe in betaine-treated chickens, with significantly reduced oil-red staining and hepatic triglyceride content (P < 0.05). The protective effect of prenatal betaine was associated with significantly up-regulated expression of PPARα and CPT1α, as well as mitochondrial DNA (mtDNA)-encoded genes (P < 0.05). Moreover, betaine rescued CORT-induced alterations in methionine cycle genes, which coincided with modifications of CpG methylation on CPT1α gene promoter and mtDNA D-loop regions. Furthermore, the elevation of hepatic GR protein content after CORT treatment was significantly reduced (P < 0.05), while the reduction of GR binding to the control region of affected genes was significantly increased (P < 0.05), in betaine-treated chickens. These results indicate that in ovo betaine injection protects the juvenile chickens from CORT-induced fatty liver.

In avian species, liver lipid metabolism plays an important role in egg laying performance. Previous studies indicate that betaine supplementation in laying hens improves egg production. However, it remains unclear if betaine improves laying performance by affecting hepatic lipid metabolism and what mechanisms are involved. We fed laying hens a 0.5% betaine-supplemented diet for 4 wks to investigate its effect on hepatic lipids metabolism in vivo and confirmed its mechanism via in vitro experiments using embryonic chicken hepatocytes. Results showed that betaine supplemented diet enhanced laying production by 4.3% compared with normal diet, accompanied with increased liver and plasma triacylglycerol concentrations ( P < 0.05) in hens. Simultaneously, key genes involved in hepatic lipid synthesis, such as sterol regulatory element binding protein 1 (SREBP-1), fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase 1 (SCD1) were markedly upregulated at the mRNA level ( P < 0.05). Western blot results showed that SREBP-1 and SCD1 protein levels were also increased ( P < 0.05). Moreover, mRNA expression of main apolipoprotein components of yolk-targeted lipoproteins, apolipoprotein B (ApoB) and apolipoprotein-V1 (ApoV1), in addition to microsomal triglyceride transfer proteins, which is closely related to the synthesis and release of very-low density lipoprotein, were also markedly elevated ( P < 0.05). Methylated DNA immunoprecipitation combined with PCR detects reduction of methylation levels in certain regions of the above gene promoters. Chromatin immunoprecipitation PCR assays showed increased binding of glucocorticoid receptor (GR) to SREBP1 and ApoB gene promoters. Similar results of ApoV1 gene expression were obtained from cultured hepatocytes treated with betaine. Additionally, betaine increased the expression of GR and some genes involved in methionine cycle in vitro. These results suggest that betaine supplementation could alter the expression of liver lipid synthesis and transport-related genes by modifying the methylation status and GR binding on their promoter and hence promote the synthesis and release of yolk precursor substances in the liver.

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Cholesterol is essential for neuronal development and brain function. Previously we reported that in ovo administration of betaine modulates hepatic cholesterol metabolism in the chicken, yet it remains unknown whether maternal betaine affects the cholesterol content and the expression of cholesterol metabolic genes in chicken hypothalamus. In this study, eggs were injected with saline or betaine at 2.5 mg/egg, and the hatchlings were raised under the same condition until 64 d of age. Maternal betaine significantly (P = 0.05) increased the body weight and suppressed aggressive behavior of 64-day-old cockerels, in association with significantly (P < 0.05) up-regulated expression of 5-HTR1A receptor in the hypothalamus. Concurrently, betaine in ovo significantly increased (P < 0.05) the hypothalamic content of total cholesterol and cholesterol ester, which coincided with significantly up-regulated (P < 0.05) hypothalamic expression of cholesterol biosynthetic genes, such as sterol-regulatory element binding protein 2 and 3-hydroxy-3-methyl-glutaryl-CoA reductase as well as acetyl-CoA cholesterol acyltransferase 1, which converts free cholesterol to cholesterol ester for storage. In contrast, low density lipoprotein receptor, which mediates the cholesterol uptake, was significantly down-regulated (P < 0.05). In ovo betaine administration significantly enhanced the expression of betaine-homocysteine methyltransferase and DNA methyltransferase 1 (P < 0.05), which was associated with alterations of CpG methylation on the promoter of modified cholesterol metabolic genes. These results indicate that maternal betaine modulates hypothalamic cholesterol metabolism in cockerels through modifying DNA methylation on the promoter of cholesterol metabolic genes.