Glucose-6-phosphatase (G6PC) plays an important role in glucose homeostasis because it catalyzes the final steps of gluconeogenesis and glycogenolysis. Maternal malnutrition during pregnancy affects G6PC activity, yet it is unknown whether epigenetic regulations of the G6PC gene are also affected. In this study, we fed primiparous, purebred Meishan sows either standard-protein (SP; 12% crude protein) or low-protein (LP; 6% crude protein) diets throughout gestation and analyzed hepatic G6PC expression in both male and female newborn piglets. The epigenetic regulation of G6PC, including DNA methylation, histone modifications, and micro RNA (miRNA), was determined to reveal potential mechanisms. Male, but not female, LP piglets had a significantly lower serum glucose concentration and greater hepatic G6PC mRNA expression and enzyme activity. Also, in LP males, glucocorticoid receptor binding to the G6PC promoter was lower compared with SP males, which was accompanied by hypomethylation of the G6PC promoter. Modifications in histones also were gender dependent; LP males had less histone H3 and histone H3 lysine 9 trimethylation and more histone H3 acetylation and histone H3 lysine 4 trimethylation on the G6PC promoter compared with the SP males, whereas LP females had more H3 and greater H3 methylation compared with their SP counterparts. Moreover, two miRNA, ssc-miR-339-5p and ssc-miR-532-3p, targeting the G6PC 3' untranslated region were significantly upregulated by the LP diet only in females. These results suggest that a maternal LP diet during pregnancy causes hepatic activation of G6PC gene expression in male piglets, which possibly contributes to adult-onset hyperglycemia.

There is limited knowledge about the impact of long-term feeding a high-concentrate (HC) diet on rumen microbiota, metabolome, and host cell functions. In this study, a combination of mass spectrometry-based metabolomics techniques, 454 pyrosequencing of 16S rDNA genes, and RT-PCR was applied to evaluate the changes of ruminal microbiota composition, ruminal metabolites, and related genes expression in rumen epithelial cells of lactating goats received either a 35% concentrate diet or a 65% concentrate diet for 4 or 19 weeks, respectively. Results show that feeding a HC diet reduced the microbiota diversity and led to the disorders of metabolism in the rumen. The concentrations of lactate, phosphorus, NH3-N and endotoxin Lipopolysaccharide in ruminal fluids, and plasma histamine, lactate and urine N (UN) were increased significantly in goats fed with a HC diet. A significant increase of genes expression related to volatile fatty acids transport, cell apoptosis, and inflammatory responses were also observed in goats fed with a HC diet. Correlation analysis revealed some potential relationships between bacteria abundance and metabolites concentrations. Our findings indicate that a HC diet can induce ruminal microbiota dysbiosis and metabolic disorders, thus increasing risks to host health and potential harm to the environment.

Studies on the effect of a high-concentrate (HC) diet on the hindgut microbiota and metabolome of ruminants are rarely reported. We used 454 pyrosequencing of 16S rDNA genes and gas chromatography-mass spectrometry to evaluate the effects of long-term feeding (HL) or short-term (HS) feeding of an HC diet on changes in bacterial microbiota and their metabolites in the hindgut, with Guanzhong goat as a ruminant model. Results indicated that an HC diet decreased bacterial diversity and induced metabolic disorder in the hindgut. The levels of lactate, endotoxin (lipopolysaccharide, LPS), and volatile fatty acid concentrations were higher in the intestinal digesta of the HC goats than in those of the LC goats (P < 0.05). The level of beta-alanine decreased, whereas the levels of stigmasterol and quinic acid decreased in the cecal and colonic digesta of the HC goats. At the genus level, the abundance of Clostridium and Turicibacter was significantly increased in both the colonic and cecal digesta of the HC goats. Several potential relationships between metabolites and several microbial species were revealed in this study. The mRNA expression of the genes functionally associated with nutrients transport, including NHE2, NHE3, MCT1, and MCT4 were significantly downregulated in the colonic mucosa by the HC diet (P < 0.05). The expression levels of the genes related to the inflammatory response, including TLR4, MYD88, TNF-α, and IL-1β were markedly upregulated in the cecal mucosa by the HC diet (P < 0.05). Our results indicate that an HC diet induces microbiota dysbiosis, metabolic disorders, and mucosal damage in the hindgut of goats.

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.

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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.

Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5′-untranslated exons 1–4, 1–5, 1–6, 1–7, 1–8, 1–9,10 and 1–11. Among all these mRNA variants, exons 1–4 and 1–5, as well as the total GR were expressed significantly (P<0.05) higher in the liver of newborn piglets of Large White (LW) compared with Erhualian, a Chinese indigenous breed. Overall level of CpG methylation in the region flanking exons 1–4 and 1–5 did not show breed difference. However, nuclear content of Sp1, p-CREB and GR in the liver was significantly (P<0.05) higher in LW piglets, associated with enhanced binding of p-CREB, and higher level of histone H3 acetylation in 1–4 and 1–5 promoters. In contrast, GR binding to promoters of exons 1–4 and 1–5 was significantly diminished in LW piglets, implicating the presence of negative GREs. These results indicate that the difference in the hepatic expression of GR transcript variants between two breeds of pigs is determined, at least partly, by the disparity in the binding of transcription factors and the enrichment of histone H3 acetylation to the promoters.

BackgroundTo understand the impact of feeding a high-concentrate diet to mid-lactating goats for a long time on liver metabolism and inflammatory response, two dimensional polyacrylamide gel electrophoresis (2-DE) and real-time PCR method were employed to detect proteins differentially expressed in liver and their mRNAs expression in goats fed high concentrate diet (HC) or low concentrate diet (LC). Twelve lactating dairy goats were randomly assigned to either a HC diet group (65 % concentrate of dry matter; n = 6) or a LC diet group (35 % concentrate of dry matter; n = 6) for 10 wk.ResultsTwenty differentially expressed spots (≥2.0-fold changes) in the hepatic tissues were excised and successfully identified using MALDI TOF/TOF. Of these, 8 proteins were up-regulated, while the rest 12 proteins were down-regulated in HC goats compared to LC. Differential expressed proteins including alpha enolase 1 (ENO1), glutamate dehydrogenase 1 (GLUD1), glutathione S-transferase A1 (GSTA1), ATP synthase subunit 5β (ATP5β), superoxide dismutase [Cu-Zn] (SOD1), cytochrom c oxidase subunit Via (COX6A1) and heat shock protein 60 (HSP60) were further verified by real-time PCR and/or western blot at mRNA or protein expression level. Consistent with the 2-DE results, a significant decrease of β-actin protein expression and SOD enzyme activity was observed in liver of HC goats (P < 0.05), while ENO1 protein expression was significantly up-regulated in HC compared to LC goats (P < 0.05) . However, western blot analysis did not show a significant difference of hepatic HSP60 protein between HC and LC group, which did not match the decrease of HSP60 content detected by 2-DE analysis. Real-time PCR showed that glutathione S-transferase P1 (GSTP1) and SOD1 mRNA expression was significantly decreased in liver of HC goats, while cytochrom c oxidase (COX3) and ATPase 8 (ATP8) mRNAs expression were markedly increased compared to LC (P < 0.05). Gene Ontology (GO) analysis revealed that HC diet resulted in altered expression of proteins related to catalytic and mitochondrial metabolism in the liver, and may increase the stress response with up-regulating the expression of differentiation 14 (CD14) cluster and serum amyloid A (SAA) as well as C-reactive protein (CRP) in the liver.ConclusionsThese results suggest that feeding high concentrate diet to lactating goats for 10 wk leads to the activation of the inflammatory response, and decreases the anti-oxidant capacity, and subsequently impairs the mitochondrial function in the liver.Electronic supplementary materialThe online version of this article (doi:10.1186/s40104-016-0065-0) contains supplementary material, which is available to authorized users.