Qinwei Sun

Maternal dietary protein affects transcriptional regulation of myostatin gene distinctively at weaning and finishing stages in skeletal muscle of Meishan pigs

Myostatin (MSTN) is suggested to mediate the effect of maternal nutrition on offspring phenotype, yet the mechanisms underlying such adaptive gene regulation is elusive. In this study, we determined the effects of maternal dietary protein on transcriptional regulation of MSTN in skeletal muscle of pig offspring. Fourteen Meishan sows were fed either low-protein (LP) or standard-protein (SP) diets throughout gestation and lactation. MSTN expression in the longissimus dorsi muscle was determined both at weaning and finishing stages. Myostatin mRNA abundance was downregulated at weaning, but upregulated at finishing in LP pigs, indicating stage-specific transcriptional regulation. At weaning, CCAAT/enhancer-binding protein beta (C/EBPβ) in muscle nuclear lysate was decreased in LP piglets, associated with diminished binding of C/EBPβ to all the 3 putative binding sites in MSTN promoter. None of the four histone modification marks investigated showed differences between SP and LP piglets. Among 12 microRNAs predicted to target MSTN, none was differently expressed. At finishing stage, C/EBPβ content remained unchanged, but the binding of C/EBPβ to two of the 3 putative binding sites increased in LP pigs. Histone H3 acetylation and histone H3 lysine 27 trimethylation on MSTN promoter were increased, while histone H3 lysine 9 monomethylation was decreased in LP pigs. Moreover, expression of ssc-miR-136 and ssc-miR-500 was significantly reduced. These results indicate that maternal dietary protein affects MSTN expression through distinct regulatory mechanisms at different stages. The immediate effect at weaning is mediated by C/EBPβ binding without epigenetic modifications, whereas the long-term effect at finishing stage involves both C/EBPβ binding and epigenetic regulations, including histone modification and microRNA expression.

Maternal Low-Protein Diet Affects Epigenetic Regulation of Hepatic Mitochondrial DNA Transcription in a Sex-Specific Manner in Newborn Piglets Associated with GR Binding to Its Promoter

Mitochondrial oxidative phosphorylation (OXPHOS) plays an important role in energy homeostasis by controlling electron transfer and ATP generation. Maternal malnutrition during pregnancy affects mitochondrial (mt) DNA-encoded OXPHOS activity in offspring, yet it is unknown whether epigenetic mechanism is involved in the transcriptional regulation of mtDNA-encoded OXPHOS genes. In this study, 14 primiparous purebred Meishan sows were fed either standard- (SP, 12% crude protein) or low-protein (LP; 6% crude protein) diets throughout gestation, and the hepatic expression and transcriptional regulation of mtDNA-encoded OXPHOS genes were analyzed in newborn piglets. Maternal low protein diet decreased hepatic mtDNA copy number in males, but not in females. LP male piglets had significantly higher hepatic AMP concentration and low energy charge, which was accompanied by enhanced mRNA expression of NADH dehydrogenase subunits 6, cytochrome c oxidase subunit 1, 2, 3 and cytochrome b, as well as increased cytochrome c oxidase enzyme activity. In contrast, LP female piglets showed significantly lower hepatic AMP concentrations and higher energy charge with no alterations in OXPHOS gene expression. Moreover, LP males demonstrated higher glucocorticoid receptor (GR) binding to the mtDNA promoter compared with SP males, which was accompanied by lower cytosine methylation and hydroxymethylation on mtDNA promoter. Interestingly, opposite changes were seen in females, which showed diminished GR binding and enriched cytosine methylation and hydroxymethylation on mtDNA promoter. These results suggest that maternal low protein diet during pregnancy causes sex-dependent epigenetic alterations in mtDNA-encoded OXPHOS gene expression, possibly GR is involved in mtDNA transcription regulation.

In Ovo injection of betaine affects hepatic cholesterol metabolism through epigenetic gene regulation in newly hatched chicks.

Betaine is reported to regulate hepatic cholesterol metabolism in mammals. Chicken eggs contain considerable amount of betaine, yet it remains unknown whether and how betaine in the egg affects hepatic cholesterol metabolism in chicks. In this study, eggs were injected with betaine at 2.5 mg/egg and the hepatic cholesterol metabolism was investigated in newly hatched chicks. Betaine did not affect body weight or liver weight, but significantly increased the serum concentration (P < 0.05) and the hepatic content (P < 0.01) of cholesterol. Accordingly, the cholesterol biosynthetic enzyme HMGCR was up-regulated (P < 0.05 for both mRNA and protein), while CYP7A1 which converts cholesterol to bile acids was down-regulated (P < 0.05 for mRNA and P = 0.07 for protein). Moreover, hepatic protein content of the sterol-regulatory element binding protein 1 which regulates cholesterol and lipid biosynthesis, and the mRNA abundance of ATP binding cassette sub-family A member 1 (ABCA1) which mediates cholesterol counter transport were significantly (P < 0.05) increased in betaine-treated chicks. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and adenosylhomocysteinase-like 1 were increased (P < 0.05), which was associated with global genomic DNA hypermethylation (P < 0.05) and diminished gene repression mark histone H3 lysine 27 trimethylation (P < 0.05). Furthermore, CpG methylation level on gene promoters was found to be increased (P < 0.05) for CYP7A1 yet decreased (P < 0.05) for ABCA1. These results indicate that in ovo betaine injection regulates hepatic cholesterol metabolism in chicks through epigenetic mechanisms including DNA and histone methylations.

Coordinated miRNA/mRNA Expression Profiles for Understanding Breed-Specific Metabolic Characters of Liver between Erhualian and Large White Pigs

MicroRNAs (miRNAs) are involved in the regulation of various metabolic processes in the liver, yet little is known on the breed-specific expression profiles of miRNAs in coordination with those of mRNAs. Here we used two breeds of male newborn piglets with distinct metabolic characteristics, Large White (LW) and Erhualian (EHL), to delineate the hepatic expression profiles of mRNA with microarray and miRNAs with both deep sequencing and microarray, and to analyze the functional relevance of integrated miRNA and mRNA expression in relation to the physiological and biochemical parameters. EHL had significantly lower body weight and liver weight at birth, but showed elevated serum levels of total cholesterol (TCH), high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC), as well as higher liver content of cholesterol. Higher serum cortisol and lower serum insulin and leptin were also observed in EHL piglets. Compared to LW, 30 up-regulated and 18 down-regulated miRNAs were identified in the liver of EHL, together with 298 up-regulated and 510 down-regulated mRNAs (FDR<10%). RT-PCR validation of some differentially expressed miRNAs (DEMs) further confirmed the high-throughput data analysis. Using a target prediction algorithm, we found significant correlation between the up-regulated miRNAs and down-regulated mRNAs. Moreover, differentially expressed genes (DEGs), which are involved in proteolysis, were predicted to be mediated by DEMs. These findings provide new information on the miRNA and mRNA profiles in porcine liver, which would shed light on the molecular mechanisms underlying the breed-specific traits in the pig, and may serve as a basis for further investigation into the biological functions of miRNAs in porcine liver.

Sexually dimorphic effects of maternal dietary protein restriction on fetal growth and placental expression of 11β-HSD2 in the pig

Placental 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) inactivates glucocorticoids (GCs) to protect fetuses from over-exposure to maternal GCs, yet how maternal malnutrition affects placental 11β-HSD2 expression is unknown. In this study, Meishan sows were fed standard-protein (SP) or low-protein (LP, 50% of SP) diets and fetuses/newborn piglets were weighed and the corresponding placenta and umbilical cord blood were collected on gestational day 70 and the day of parturition. Significant growth retardation was observed in female, but not male, fetuses (P < 0.05) and the newborns (P < 0.01) of the LP group, which was accompanied by sexually dimorphic expression of 11β-HSD2 in placentas. Female fetuses in LP group showed significant decrease in placental 11β-HSD2 protein content (P < 0.05) and enzyme activity (P < 0.05), whereas male fetuses demonstrated significantly enhanced placental 11β-HSD2 activity (P < 0.05). Serum cortisol levels were significantly higher (P < 0.05) in male piglets compared to females, and the effects of maternal protein restriction on thyroid hormones (T3 and T4) in the umbilical cord blood were also sex dimorphic. Male piglets in LP group had significantly higher T3 (P < 0.01) and lower T4 (P < 0.01), whereas female piglets showed significantly lower T4 (P < 0.01) with no change in T3. As a result, male piglets in LP group exhibited significantly higher T3/T4 ratio compared to female counterparts. These results indicate that the effects of maternal protein restriction on placental 11β-HSD2 expression are gender-dependent in the pig, and thyroid hormones may be involved in such effects.

Maternal dietary protein induces opposite myofiber type transition in Meishan pigs at weaning and finishing stages.

To describe the effects of maternal dietary protein level on offspring skeletal muscle fiber plasticity, 14 Meishan sows were fed on either low-protein (LP) or high-protein (HP) diets throughout gestation and lactation, and the myofiber characteristics of the offspring were observed both at weaning and finishing. Body weight, as well as the longissimus dorsi (LD) and psoas major (PM) muscle weights were significantly higher (P<0.05) in HP piglets at weaning with increased myofiber cross-sectional area and higher expression of myosin heavy chain (MyHC) IIb mRNA and MyHC II protein in LD and IIx mRNA in PM. A conversed transition towards higher proportion of type I fibers in LD, together with decreased MyHC II protein and MyHC IIb mRNA in both LD and PM, was observed at finishing stage in HP group. Although the slaughter weight and meat quality were not affected, the fast-to-slow shift in myofiber types was detected at slaughter.

Maternal betaine supplementation during gestation modifies hippocampal expression of GR and its regulatory miRNAs in neonatal piglets.

Methyl donor nutrients are critical for embryonic development of brain. Hippocampus is the most susceptible brain region to various factors including prenatal supply of methyl donors. Glucocorticoid receptor (GR) expressed in hippocampus is involved in the regulation of energy homeostasis and stress sensitivity. Hippocampal GR expression is highly susceptible to epigenetic regulation, yet the effect of maternal methyl donor supplementation on epigenetic regulation of GR transcription in offspring hippocampus remains unclear. In this study, we fed sows with betaine (3 g/kg) throughout the gestation and analyzed the hippocampal expression of GR mRNA and its variants, as well as the CpG methylation status of the promoter and the microRNAs predicted to target 3’ UTR of porcine GR gene in neonatal piglets. Total GR mRNA (P<0.01) and its variants GR 1-4 (P<0.05) and 1-9,10 (P<0.01), were significantly higher in the hippocampus of betaine-treated piglets, while the content of GR protein was not significantly changed. The CpGs located in the –1650 ~ –1515 segment of GR gene were hypermethylated (P<0.05). The hippocampal expression of miR-130b (P<0.05), miR-181a (P<0.05) and miR-181d (P<0.01) was significantly up-regulated. The targeting efficacy of miR-130b and miR-181d was validated in vitro using dual-luciferase reporter assay system. Our results demonstrate that maternal betaine supplementation during gestation enhances GR mRNA expression in offspring hippocampus, which involves alterations in miRNAs expression.

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.

Breed-specific expression of GR exon 1 mRNA variants and profile of GR promoter CpG methylation in the hippocampus of newborn piglets.

Glucocorticoid receptor (GR) transcription is driven by alternative promoters to produce different exon 1 mRNA variants. CpG methylation on GR promoters profoundly affects GR transcription. GR in hippocampus is critical for energy homeostasis and stress responses, yet it remains unclear whether hippocampal expression of GR exon 1 mRNA variants and the methylation status of GR promoters differ between Large White (LW) and Erhualian (EHL) pigs showing distinct metabolic and stress-coping characteristics. EHL pigs had higher hippocampus weight relative to BW (P<0.01), which was associated with higher serum cortisol level compared with LW pigs. Hippocampal expression of brain-derived neurotrophic factor (P<0.05) was significantly higher, while Bax, a pro-apoptotic gene, was significantly lower in EHL pigs (P<0.05). Hippocampal expression of total GR did not differ between breeds, yet GR exon 1 to 11 mRNA was significantly higher (P<0.01) in EHL pigs, which was associated with a trend of increase (P=0.057) in GR protein content. No significant breed difference was detected for the methylation status across the whole region of the proximal GR promoter, while CpG334 and CpG266.267 were differentially methylated, in a reversed manner, between breeds. The methylation status of CpGs 248, 259, 260, 268 and 271 was negatively correlated (P<0.05) with GR exon 1 to 11 mRNA abundance. Our results provide fundamental information on the breed-specific characteristics of GR and its mRNA variants expression and the status of DNA methylation on the proximal GR promoter in the pig hippocampus.

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.