Dietary betaine activates hepatic VTGⅡexpression 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 VTGI. Plasma estrogen level did not change,but the hepatic expression of estrogen receptor α(ERα) mR NA 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 mR NA and protein levels. Also, ERα/GR interaction tended to be enhanced in the liver nuclear lysates of betainesupplemented 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.

GR-mediated transcriptional regulation of m^(6)A metabolic genes contributes to dietinduced fatty liver in hens

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(m^(6)A)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 m^(6)A modification are involved in high-energy and low protein(HELP)diet-induced FLS in laying hens,and if true,what specific m^(6)A sites of lipogenic genes are modified and how GR mediates m^(6)A-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 m^(6)A demethylation.Concurrently,the m^(6)A demethylase FTO is upregulated(P<0.05),whereas the m^(6)A reader YTHDF2 is downregulated(P<0.05)in the liver of FLS chickens.Further analysis identifies site-specific demethylation(P<0.05)of m^(6)A 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 m^(6)A metabolic genes on m^(6)A-depenent post-transcriptional activation of lipogenic genes and shed new light in the molecular mechanism of FLS etiology in the chicken.

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 m RNA 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 m RNA 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 c AMP 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.

Chronic corticosterone disrupts the circadian rhythm of CRH expression and m^(6)A RNA methylation in the chicken hypothalamus

Background:Corticotropin-releasing hormone(CRH),the major secretagogue of the hypothalamic-pituitary-adrenal(HPA)axis,is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions.N6-methyladenosine(m^(6)A)RNA methylation is involved in the regulation of circadian rhythm,yet it remains unknown whether CRH expression and m^(6)A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress.Results:Chronic exposure to corticosterone(CORT)eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken.The circadian rhythms of clock genes in hippocampus,hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens.The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened,together with mRNA of other feeding-related neuropeptides.Interestingly,hypothalamic m^(6)A level oscillates in an opposite pattern to CRH mRNA,with lowestm^(6)A level after midnight(ZT18)corresponding to the peak of CRH mRNA before dawn(ZT22).CORT diminished the circadian rhythm of m^(6)A methylation with significantly increased level at night.Further site-specific m^(6)A analysis on 3’UTR of CRH mRNA indicates that higher m^(6)A on 3’UTR of CRH mRNA coincides with lower CRH mRNA at night(ZT18 and ZT22).Conclusions:Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus,leading to dysfunction of HPA axis in the chicken.RNA m^(6)A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.

Constant light in early life induces fear-related behavior in chickens with suppressed melatonin secretion and disrupted hippocampal expression of clock-and BDNF-associated genes

Background: Light management plays an important role in the growth and behavior of broiler chickens. Constant light in early post hatch stage has been a common practice in broiler industry for improving growth performance,while whether and how constant light in early life affects the behavior of broiler chickens is rarely reported.Results: In this study, newly hatched chicks were kept in either constant(24 L:0 D, LL) or(12 L:12 D, LD) photoperiod for 7 d and then maintained in 12 L:12 D thereafter until 21 days of age. Constant light increased the average daily feed intake but not the body weight, which led to higher feed conversion ratio. Chickens in LL group exhibited fear-related behaviors, which was associated with higher corticosterone, lower melatonin and 5-HT levels. Concurrently, constant light exposure increased the mRNA expression of clock-related genes and suppressed the expression of antioxidative genes in the hippocampus. Moreover, brain derived neurotrophic factor/extracel ular signal-regulated kinase(BDNF/ERK) pathway was suppressed in the hippocampus of chickens exposed to constant light in the first week post hatching.Conclusions: These findings indicate that constant light exposure in early life suppress melatonin secretion and disrupts hippocampal expression of genes involved in circadian clock and BDNF/ERK pathway, thereby contributing to fear-related behaviors in the chicken.

Negative effects of long-term feeding of high-grain diets to lactating goats on milk fat production and composition by regulating gene expression and DNA methylation in the mammary gland

Background： It is well known that feeding a high concentrate（HC） diet to lactating ruminants likely induces subacute ruminal acidosis（SARA） and leads to a decrease in milk fat production. However, the effects of feeding a HC diet for long periods on milk fatty acids composition and the mechanism behind the decline of milk fat still remains poorly understood. The aim of this study was to investigate the impact of feeding a HC diet to lactating dairy goats on milk fat yield and fatty acids composition with an emphasis on the mechanisms underlying the milk fat depression. Seventeen mid-lactating dairy goats were randomly allocated to three groups. The control treatment was fed a low-concentrate diet（35% concentrate, n = 5, LC） and there were two high-concentrate treatments（65% concentrate, HC）, one fed a high concentrate diet for a long period（19 wks, n = 7, HL）; one fed a high concentrate diet for a short period of time（4 wk, n = 5, HS）. Milk fat production and fatty acids profiles were measured. In order to investigate the mechanisms underlying the changes in milk fat production and composition,the gene expression involved in lipid metabolism and DNA methylation in the mammary gland were also analyzed.Results： Milk production was increased by feeding the HC diet in the HS and HL groups compared with the LC diet（P 〈 0.01）, while the percentage of milk fat was lower in the HL（P 〈 0.05） but not in the HS group. The total amount of saturated fatty acids（SFA） in the milk was not changed by feeding the HC diet, whereas the levels of unsaturated fatty acids（UFA） and monounsaturated fatty acids（MUFA） were markedly decreased in the HL group compared with the LC group（P 〈 0.05）. Among these fatty acids, the concentrations of C15：0（P 〈 0.01）, C17：0（P 〈 0.01）, C17：1（P 〈 0.01）, C18：1 n-9 c（P 〈 0.05）, C18：3 n-3 r（P 〈 0.01） and C20：0（P 〈 0.01） were markedly lower in the HL group, and the concentrations of C20：0（P 〈 0.05） and C18：3 n-3 r（P 〈 0.01） were lower in the HS group compared with the LC group. However, the concentrations of C18：2 n-6 c（P 〈 0.05） and C20：4 n-6（P 〈 0.05） in the milk fat were higher in the HS group. Real-time PCR results showed that the m RNA expression of the genes involved in milk fat production in the mammary gland was generally decreased in the HL and HS groups compared with the LC group. Among these genes, ACSL1, ACSS1 ＆ 2, ACACA, FAS, SCD, FADS2, and SREBP1 were downregulated in the mammary gland of the HL group（P 〈 0.05）, and the expressions of ACSS2, ACACA, and FADS2 m RNA were markedly decreased in the HS goats compared with the LC group（P 〈 0.05）. In contrast to the gene expression, the level of DNA methylation in the promoter regions of the ACACA and SCD genes was increased in the HL group compared with the LC group（P 〈 0.05）. The levels of ACSL1 protein expression and FAS enzyme activity were also decreased in the mammary gland of the HL compared with the LC group（P 〈 0.05）.Conclusions： Long-term feeding of a HC diet to lactating goats induced milk fat depression and FAs profile shift with lower MUFAs but higher SFAs. A general down-regulation of the gene expression involved in the milk fat production and a higher DNA methylation in the mammary gland may contribute to the decrease in milk fat production in goats fed a HC diet for long time periods.

In ovo leptin administration affects hepatic lipid metabolism and microRNA expression in newly hatched broiler chickens

Background： A leptin-like immunoreactive substance has been found in chicken eggs and has been implicated in serving as a maternal signal to program offspring growth and metabolism. In the present study, we investigated the effects of in ovo leptin administration on hatch weight, serum and hepatic concentrations of metabolites and hormones, as well as on the expression of genes involved in hepatic lipid metabolism and the predicted microRNAs （miRNAs） targeting the affected genes. To this end we injected fertile eggs with either 0.5 μg of recombinant murine leptin or vehicle （PBS） before incubation.Results： Prenatally leptin-exposed chicks showed lower hatch weight, but higher liver weight relative to the body weight, compared to the control group. In ovo leptin treatment increased the hepatic content and serum concentration of leptin in newly hatched chickens. The hepatic contents of triglycerides （TG） and total cholesterol （Tch） were decreased, whereas the serum levels of TG, Tch and apolipoprotein B （ApoB） were increased. The hepatic mRNA expression of sterol regulator element binding protein 1 （SREBP-1c）, SREBP-2, hydroxy-3-methylglutaryl coenzyme A reductase （HMGCR） and cholesterol 7a-hydroxylase 1 （CYP7A1） was significantly up-regulated, as was the protein content of both SREBP-1 c and SREBP-2 in hepatic nuclear extracts of leptin-treated chickens. Moreover, out of 12 miRNAs targeting SREBP-1c and/or HMGCR, five were significantly up-regulated in liver of leptin-treated chicks, including gga-miR-200b and gga-miR-429, which target both SREBP-1c and HMGCR.Conclusions： These results suggest that leptin in ovo decreases hatch weight, and modifies hepatic leptin secretion and lipid metabolism in newly hatched broiler chickens, possibly via microRNA-mediated gene regulation.

Whole transcriptome analysis of RNA expression profiles reveals the potential regulating action of long noncoding RNA in lactating cows fed a high concentrate diet

Subacute ruminal acidosis(SARA)is a common metabolic disease in the dairy farming industry which is usually caused by an excessive amount of high concentrate diet.SARA not only threatens animal welfare but also leads to economic losses in the farming industry.The liver plays an important role in the distribution of nutritional substances and metabolism;however,a high concentrate diet can cause hepatic metabolic disorders and liver injury.Recently,noncoding RNA has been considered as a critical regulator of hepatic disease,however,its role in the bovine liver is limited.In this study,12 mid-lactating dairy cows were randomly assigned to a control(CON)group(40% concentrate of dry matter,n=6)and a SARA group(60% concentrate of dry matter,n=6).After 21 d of treatment,all cows were sacrificed,and liver tissue samples were collected.Three dairy cows were randomly selected from the CON and SARA groups respectively to perform whole transcriptome analysis.More than 20,000 messenger RNA(mRNA),10,000 long noncoding RNA(lncRNA),3,500 circular RNA(circRNA)and 1,000 micro RNA(miRNA)were identified.Furthermore,43 mRNA,121 lncRNA and 3 miRNA were differentially expressed,whereas no obvious differentially expressed circRNA were detected between the 2 groups.Gene Ontology(GO)annotation revealed that the differentially expressed genes were mainly enriched in oxidoreductase activity,stress,metabolism,the immune response,cell apoptosis,and cell proliferation.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the deferentially expressed genes were highly enriched in the phosphatidylinositol 3 kinase(PI3) K-serine/threonine kinase(AKT)signaling pathway(P<0.05).According to KEGG pathway analysis,the differentially expressed lncRNA(DElncRNA)target genes were mainly related to proteasomes,peroxisomes,and the hypoxia-inducible factor-1 signaling pathway(P<0.005).Further bioinformatics and integrative analyses revealed that the lncRNA were strongly correlated with mRNA;therefore,it is reasonable to speculate that lncRNA potentially play important roles in the liver dysfunction induced by SARA.Our study provides a valuable resource for future investigations on the mechanisms of SARA to facilitate an understanding of the importance of lncRNA,and offer functional RNA information.

Maternal butyrate supplementation affects the lipid metabolism and fatty acid composition in the skeletal muscle of offspring piglets

Maternal sodium butyrate(SB)intake has important effects on offspring growth and development.This study aimed to investigate the impacts of maternal SB supplementation during gestation and lactation on fatty acid composition and lipid metabolism in the offspring skeletal muscle of pigs.Twenty sows(Yorkshire,parity 2 to 3)were assigned to the control group(diets without SB,n=10)and SB group(diets with 0.1%SB,n=10).The results showed maternal SB supplementation throughout gestation and lactation increased(P<0.05)body weight of offspring piglets at weaning.The concentrations of triglyceride in plasma and milk were enhanced(P<0.05).Maternal SB induced(P<0.05)lipid accumulation with increased expression of peroxisome proliferator activated receptorγ(PPARγ)by enrichment of the acetylation of H3 acetylation K27(H3 K27)in offspring skeletal muscle.Meanwhile,the concentrations of C18:2 n-6,C18:3 n-3,total polyunsaturated fatty acid(PUFA),n-6 PUFA and n-3 PUFA decreased(P<0.05)in skeletal muscle of weaning piglets derived from SB sows.Together,these results showed that maternal SB supplementation could influence offspring growth performance,lipid metabolism and fatty acid composition of the skeletal muscle.

Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs

Sixteen Large White ?Landrace castrated male pigs were allotted into treatment and control group. The treatment group was injected intramuscularly with recombinant porcine growth hormone (rpGH, 4 mg·d-1) and the control group with vehicle for 28 days. Animals were slaugh-tered 4 h after final injection for liver, longissimus dorsi (LD) muscle and blood sampling. Serum concentration of insulin-like growth factor 1 (IGF-I) and leptin were determined by RIA. The total RNA was extracted from tissues to measure the abundance of growth hormone receptor (GHR), IGF-I mRNA by RT-PCR with 18S rRNA internal standard. Results showed that rpGH enhanced the average daily weight gain by 26.1% (P < 0.05), the serum IGF-I concentration by 70.94% (P < 0.01), decreased serum leptin by 34.8% (P < 0.01). The relative abundance of GHR and IGFmRNA in liver were increased by 24.45% (P < 0.05) and 45.30% (P < 0.01), respectively, but no difference of GHR (P > 0.05) and IGF-I mRNA (P > 0.05) in LD between GH treated and control group was found. These results suggest that rpGH can up-regulate hepatic GHR and IGF-I gene expression and improve animal growth. However the effect of rpGH on GHR and IGF-I gene ex-pression are tissue-specific.