| Brain contains more than 20%of the total cholesterol in the body.Cholesterol in the brain is essential for neuronal development and brain functions,especially during early embryonic and early postnatal stages.Hypothalamus is a regulatory center for autonomic functions including growth and behavior,yet studies on cholesterol homeostasis and its functional relevance in the chicken are lacking.Betaine is a feed additive used to promote growth performance in livestock animals.Betaine acts as a methyl donor to participate in methylation processes that are critical for epigenetic gene regulation.Previous studies have shown that prenatal betaine exposure plays an important role in programming postnatal hepatic cholesterol metabolism in mammals and avian species,yet it is unknown whether betaine may exert an intergenerational programming effect on hypothalamic cholesterol homeostasis in the chicken.In this study,3 experiments were carried out to investigate the effects of,(1)prenatal betaine exposure by in ovo injection on cholesterol content and the expression of cholesterol metabolic genes in the hypothalamus of cockerels;(2)feeding hens with betaine-supplemented diet on hypothalamic expression of cholesterol metabolic genes in F1 cockerels;and(3)prenatal betaine exposure in hens on hypothalamic expression of cholesterol metabolic genes in F2 cockerels.We elucidated the betaine effects on cholesterol homeostasis in association with aggressive behaviors and growth performance in cockerels.Hypothalamic expression of genes involved in betaine/methionine cycle and methyl transfer were determined together with the modifications of DNA methylation on the promoter of affected genes.1.Prenatal betaine exposure modulates hypothalamic expression of cholesterol metabolic genes in cockerels through modifications of DNA methylationPreviously we reported that in ovo administration of betaine modulates hepatic cholesterol metabolism in the chicken,yet it remains unknown whether prenatal betaine affects the cholesterol content and the expression of cholesterol metabolic genes in the 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-days of age.Maternal betaine significantly(P = 0.05)increased the body weight and suppressed aggressive behaviour 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 methyltransferases1(P<0.05),which was associated with alterations of CpG methylation on the promoter of modified cholesterol metabolic genes.These results indicate that prenatal betaine modulates hypothalamic cholesterol metabolism in cockerels through changing DNA methylation on the promoter of cholesterol metabolic genes.2.Dietary betaine supplementation in hens modulates hypothalamic expression of cholesterol metabolic genes in F1 cockerels through modification of DNA methylationPrenatal betaine exposure is reported to modulate hypothalamic cholesterol metabolism in chickens,yet it remains unknown whether feeding hens with betaine may affect hypothalamic cholesterol metabolism in F1 offspring.In this study,hens were fed with basal or betaine-supplemented(0.5%)for 30 days,and the eggs were collected for incubation.The hatchlings were raised under the same condition up to 56 days of age.Betaine-treated group showed significantly(P<0.05)increased hypothalamic content and blood concentration of total cholesterol and cholesterol ester.Concordantly,hypothalamic expression of SREBP2,HMGCR,and LDLR was significantly up-regulated at both mRNA and protein levels(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 mRNA expression of regulatory neuropeptides including BDNF and CRH,and a significant up-regulation of NPY mRNA expression.Moreover,genes involved in methyl transfer cycle were also modulated.DNA methyltransferase 1 and betaine-homocysteine methyltransferase 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 with modification of promoter DNA methylation in F1 cockerels.3.Prenatal betaine exposure in hens modulates hypothalamic expression of cholesterol metabolic genes in F2 cockerels with modification of promoter DNA methylationIn ovo administration of betaine was reported to modulate cholesterol metabolism in chickens post-hatching,yet it remains unknown whether such effects can be transmitted to next generation.In this 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.Hens in control and betaine groups were then artificially inseminated with sperms collected from cocks of the respective group.Eggs were incubated,and the hatchlings of the second generation(F2)were raised up to 64 days 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,such as SREBP2 and HMGCR,were significantly down-regulated(P<0.05),together with CYP46A1 and ABCA1 that are responsible for cholesterol conversion and excretion.These changes coincided with a significant down-regulation of mRNA expression of regulatory neuropeptides including BDNF,NPY,and CRH.Moreover,genes involved in methyl transfer cycle were also modified.Betaine-homocysteine methyltransferase(P<0.05)was down regulated,yet DNA methyltransferase1 tended to be up regulated(P = 0.06).SAM/SAH ratio was higher in the hypothalamus of betaine-treated F2 cockerels,which was associated with significantly modified CpG methylation on the promoter of affected genes.These results indicate that betaine modulates hypothalamic expression of genes involved in cholesterol metabolism and brain functions with modification of promoter DNA methylation in F2 cockerels. |
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