In ovo injection of betaine promotes adrenal steroidogenesis in pre-hatched chicken fetuses.

Corticosterone is critical for the maturation and survival of chicken fetus around hatching. Betaine is used as a feed additive in poultry industry to promote growth and mitigate stress. However, it remains unknown whether betaine could affect adrenal corticosterone synthesis in pre-hatching chicken fetuses. In this study, betaine (2.5 mg/egg) was injected into developing chicken fetuses at d 11 of incubation (E11) and its impact on adrenal steroidogenesis was investigated at day 19 (E19). Plasma corticosterone concentration was significantly (P < 0.05) elevated in betaine-treated fetuses, together with increased adrenal expression of melanocortin 2 receptor and steroidogenic acute regulatory protein. Accordingly, the corticosterone biosynthetic enzymes, such as cytochrome P450 family 11 subfamily A member 1, 3ß-hydroxysteroid dehydrogenase and cytochrome P450 family 21 subfamily A member 2, as well as cholesterol biosynthesis or regulation-related genes, such as sterol regulatory element-binding protein 1, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and low-density lipoprotein receptor, were all significantly (P < 0.05) upregulated in betaine group. Meanwhile, steroidogenic factor-1 and glucocorticoid receptor were significantly (P < 0.05) enhanced, whereas expression of dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene, a nuclear receptor known as a repressor of adrenal steroidogenesis, was significantly (P < 0.05) downregulated. Betaine significantly (P < 0.05) increased adrenal expression of genes involved in one-carbon metabolism and DNA methylation, such as S-adenosyl homocysteine hydrolase, betaine-homocysteine-methyltransferase, methionine adenosyl transferase and DNA methyltransferases, yet the promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated. These results indicate that in ovo injection of betaine promotes adrenal glucocorticoid synthesis in chicken fetuses before hatching, which involves alterations in DNA methylation.

Dietary betaine improves egg-laying rate in hens through hypomethylation and glucocorticoid receptor-mediated activation of hepatic lipogenesis-related genes.

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.

Fetal betaine exposure modulates hypothalamic expression of cholesterol metabolic genes in offspring cockerels with modification of promoter DNA methylation.

In documents, maternal betaine modulates hypothalamic cholesterol metabolism in chicken posthatchings, but it remains unclear whether this effect can be passed on by generations. In present study, eggs were injected with saline or betaine at 2.5 mg/egg, and the hatchlings (F1) were raised under the same condition until sexual maturation. Both the control group and the betaine group used artificial insemination to collect sperm from their cockerels. Fertilized eggs were incubated, and the hatchlings of the following generation (F2) were raised up to 64 D of age. F2 cockerels in betaine group showed significantly (P < 0.05) lower body weight, which was associated with significantly decreased (P < 0.05) hypothalamic content of total cholesterol and cholesterol ester. Concordantly, hypothalamic expression of cholesterol biosynthetic genes, SREBP2 and HMGCR, were significantly downregulated (P < 0.05), together with cholesterol conversion-related and excretion-related genes, CYP46A1 and ABCA1. These changes coincided with a significant downregulation in mRNA expression of regulatory neuropeptides including brain-derived neurotrophic factor, neuropeptide Y, and corticotropin-releasing hormone. Moreover, genes involved in methyl transfer cycle were also modified. Betaine homocysteine methyltransferase (P < 0.05) was downregulated, yet DNA methyltransferase1 tended to be upregulated (P = 0.06). S-adenosyl methionine/S-adenosylhomocysteine ratio was higher in the hypothalamus of betaine-treated F2 cockerels, which was associated with significantly modified CpG methylation on the promoter of those affected genes. These results suggested that betaine might regulate central cholesterol metabolism and hypothalamic expression of genes related to brain function by altering promoter DNA methylation in F2 cockerels.

Maternal betaine suppresses adrenal expression of cholesterol trafficking genes and decreases plasma corticosterone concentration in offspring pullets.

BACKGROUND: Laying hens supplemented with betaine demonstrate activated adrenal steroidogenesis and deposit higher corticosterone (CORT) in the egg yolk. Here we further investigate the effect of maternal betaine on the plasma CORT concentration and adrenal expression of steroidogenic genes in offspring pullets. RESULTS: Maternal betaine significantly reduced (P < 0.05) plasma CORT concentration and the adrenal expression of vimentin that is involved in trafficking cholesterol to the mitochondria for utilization in offspring pullets. Concurrently, voltage-dependent anion channel 1 and steroidogenic acute regulatory protein, the two mitochondrial proteins involved in cholesterol influx, were both down-regulated at mRNA and protein levels. However, enzymes responsible for steroid syntheses, such as cytochrome P450 family 11 subfamily A member 1 and cytochrome P450 family 21 subfamily A member 2, were significantly (P < 0.05) up-regulated at mRNA or protein levels in the adrenal gland of pullets derived from betaine-supplemented hens. Furthermore, expression of transcription factors, such as steroidogenic factor-1, sterol regulatory element-binding protein 1 and cAMP response element-binding protein, was significantly (P < 0.05) enhanced, together with their downstream target genes, such as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, LDL receptor and sterol regulatory element-binding protein cleavage-activating protein. The promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated, although methyl transfer enzymes, such as AHCYL, GNMT1 and BHMT were up-regulated. CONCLUSIONS: These results indicate that the reduced plasma CORT in betaine-supplemented offspring pullets is linked to suppressed cholesterol trafficking into the mitochondria, despite the activation of cholesterol and corticosteroid synthetic genes associated with promoter hypomethylation.

Dietary betaine activates hepatic VTGII expression in laying hens associated with hypomethylation of GR gene promoter and enhanced GR expression.

BACKGROUND: Vitellogenin (VTG) is a precursor of egg yolk proteins synthesized within the liver of oviparous vertebrates. Betaine is an important methyl donor that is reported to improve egg production of laying hens with an unclear mechanism. In the present study, we fed betaine-supplemented diet (0.5%) to laying hens for 4 wk and investigated its effect on VTGII expression in the liver. RESULTS: Betaine did not affect chicken weight, but significantly (P < 0.05) increased egg laying rate accompanied with a significant (P < 0.05) increase in hepatic concentration and plasma level of VTGII. Plasma estrogen level did not change, but the hepatic expression of estrogen receptor α (ERα) mRNA was significantly (P < 0.05) up-regulated. Betaine did not affect the protein content of ERα, but significantly (P < 0.05) increased hepatic expression of glucocorticoid receptor (GR) at both mRNA and protein levels. Also, ERα/GR interaction tended to be enhanced in the liver nuclear lysates of betaine-supplemented hens as determined by co-immunoprecipitation. Furthermore, dietary betaine supplementation significantly increased (P < 0.05) the hepatic expression of methyl-transfer enzymes, such as BHMT, GNMT, and DNMT1, which was associated with higher SAM/SAH ratio and hypomethylation of GR promoter regions. CONCLUSIONS: Betaine activates hepatic VTGII expression in association with modified DNA methylation of GR gene promoter, GR expression and ERα/GR interaction. Activation of hepatic VTGII expression may contribute, at least partly, to improved egg production in betaine-supplemented hens.

Dietary betaine supplementation in hens modulates hypothalamic expression of cholesterol metabolic genes in F1 cockerels through modification of DNA methylation.

Betaine is widely used in animal nutrition to promote growth, development and methyl donor during methionine metabolism through nutritional reprogramming via regulation of gene expression. Prenatal betaine exposure is reported to modulate hypothalamic cholesterol metabolism in chickens, yet it remains unknown whether feeding hens with betaine-supplemented diet may affect hypothalamic cholesterol metabolism in F1 offspring. In this study, hens were fed with basal or betaine-supplemented (0.5%) for 30days, and the eggs were collected for incubation. The hatchlings were raised under the same condition up to 56days of age. Betaine-treated group showed significantly (P<0.05) higher plasma concentration of total cholesterol and HDL-cholesterol, together with increased hypothalamic content of total cholesterol and cholesterol ester. Concordantly, hypothalamic gene expression of SREBP2, HMGCR, and LDLR was significantly up regulated (P<0.05). Also, mRNA abundances of SREBP1, ACAT1 and APO-A1 were up-regulated, while that of CYP46A1 was significantly down-regulated (P<0.05). These changes coincided with a significant down-regulation of BDNF and CRH, and a significant up-regulation of NPY mRNA expression. Moreover, genes involved in methyl transfer cycle were also modulated. DNMT1 and BHMT were up-regulated (P<0.05) at both mRNA and protein levels, which was associated with significant modifications of CpG methylation on the promoter of SREBP-1, SREBP-2 and APO-A1 genes as detected by bisulfate sequencing. These results indicate that feeding betaine to hens modulates hypothalamic expression of genes involved in cholesterol metabolism and brain functions in F1 cockerels with modification of promoter DNA methylation.

Dietary betaine supplementation increases adrenal expression of steroidogenic acute regulatory protein and yolk deposition of corticosterone in laying hens.

Betaine, an important methyl donor, is known to execute epigenetic regulation of gene expression via nutritional reprogramming. Herein, we explore whether feeding a betaine-supplemented diet to laying hens would affect corticosteroid biosynthesis in the adrenal gland and corticosterone deposition in eggs, in correlation with the expression of methyl transfer enzymes and the promoter DNA methylation status of affected genes. Rugao yellow-feathered laying hens at 38 weeks of age were assigned to Control and Betaine groups, fed basal and betaine-supplemented diets, respectively, for four weeks. Betaine supplementation significantly increased (P < 0.05) the average laying rate, while the body weight and egg quality remained unchanged. Plasma concentrations of cholesterol and low-density lipoprotein-cholesterol were also higher (P < 0.05) in the Betaine group. Moreover, eggs in the Betaine group contained higher corticosterone in the yolk, which was associated with up-regulation of steroidogenesis genes in adrenal glands. Steroidogenic acute regulatory protein (StAR), the rate-limiting protein responsible for transporting cholesterol to the inner mitochondrial membrane, was significantly activated (P < 0.05), together with its transcription factors steroidogenic factor-1 (SF-1) and glucocorticoid receptor. Also, betaine supplementation significantly up-regulated (P < 0.05) the adrenal mRNA expression of adenosyl homocysteinase-like 1 and DNA methyltransferases1 and 3a. Bisulfite sequencing analysis revealed significant hypomethylation in several CpG sites within the promoter region of SF-1 gene in the adrenal gland. These results indicate that dietary supplementation of betaine in hens activates adrenal expression of StAR, possibly through epigenetic regulation of SF-1 gene.

Prenatal betaine exposure alleviates corticosterone-induced inhibition of CYP27A1 expression in the liver of juvenile chickens associated with its promoter DNA methylation.

Sterol 27-hydroxylase (CYP27A1) plays an important role in cholesterol homeostasis by degrading cholesterol to bile acids. Betaine can alleviate high-fat diet-induced hepatic cholesterol accumulation and maternal betaine treatment programs the hepatic expression of CYP27A1 in offspring. Excessive corticosterone (CORT) exposure causes hepatic cholesterol deposition in chickens, yet it remains unknown whether prenatal betaine modulates CORT-induced cholesterol accumulation in chicken liver later in life and whether it involves epigenetic gene regulation of CYP27A1. In this study, fertilized eggs were injected with saline or betaine at 2.5mg/egg before incubation, and the hatchlings were raised under the same condition till 56days of age followed by 7days of subcutaneous CORT injection. Plasma concentrations of total cholesterol (Tch), HDL- and LDL-cholesterol were significantly increased (P<0.05), after CORT challenge, in both control and betaine groups. However, prenatal betaine exposure prevented CORT-induced increase (P<0.05) in hepatic Tch content. Hepatic expression of cholesterol biosynthesis genes and ACAT1 protein that esterifies cholesterol for storage, were activated in both control and betaine groups upon CORT challenge. However, betaine-treated chickens were protected from CORT-induced repression (P<0.05) in LXR and CYP27A1 expression in the liver. CORT-induced down-regulation of LXR and CYP27A1 coincided with significantly increased (P<0.05) CpG methylation on their promoters, which was significantly ameliorated in betaine-treated chickens. These results suggest that in ovo betaine injection alleviates CORT-induced hepatic cholesterol deposition most probably through epigenetic regulation of CYP27A1 and LXR genes in juvenile chickens.

In ovo injection of betaine alleviates corticosterone-induced fatty liver in chickens through epigenetic modifications.

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.

Prenatal betaine exposure modulates hypothalamic expression of cholesterol metabolic genes in cockerels through modifications of DNA methylation.

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.