Lactoferrin Attenuates Intestinal Barrier Dysfunction and Inflammation by Modulating the MAPK Pathway and Gut Microbes in Mice.

BACKGROUND: Deoxynivalenol (DON) is a major mycotoxin present in staple foods (particularly in cereal products) that induces intestinal inflammation and disrupts intestinal integrity. Lactoferrin (LF) is a multifunctional protein that contributes to maintaining intestinal homeostasis and improving host health. However, the protective effects of LF on DON-induced intestinal dysfunction remain unclear. OBJECTIVES: This study aimed to investigate the effects of LF on DON-induced intestinal dysfunction in mice, and its underlying protective mechanism. METHODS: Male BALB/c mice (5 wk old) with similar body weights were divided into 4 groups (n = 6/group) and treated as follows for 5 wk: Veh [peroral vehicle daily, commercial (C) diet]; LF (peroral 10 mg LF/d, C diet); DON (Veh, C diet containing 12 mg DON/kg); and LF + DON (peroral 10 mg LF/d, DON diet). Intestinal morphology, tight junction proteins, cytokines, and microbial community were determined. Data were analyzed by 2-factor ANOVA or Kruskal-Wallis test. RESULTS: The DON group exhibited lower final body weight (-12%), jejunal villus height (VH; -41%), and jejunal occludin expression (-36%), and higher plasma IL-1ß concentration (+85%) and jejunal Il1b mRNA expression (+98%) compared with the Veh group (P < 0.05). In contrast, final body weight (+19%), jejunal VH (+49%), jejunal occludin (+53%), and intelectin 1 protein expression (+159%) were greater in LF + DON compared with DON (P < 0.05). Additionally, jejunal Il1b mRNA expression (-31%) and phosphorylation of p38 and extracellular signal regulated kinase 1/2 (-40% and - 38%) were lower in LF + DON compared with DON (P < 0.05). Furthermore, the relative abundance of Clostridium XIVa (+181%) and colonic butyrate concentration (+53%) were greater in LF + DON compared with DON (P < 0.05). CONCLUSIONS: Our study highlights a promising antimycotoxin approach using LF to alleviate DON-induced intestinal dysfunction by modulating the mitogen-activated protein kinase pathway and gut microbial community in mice.

Targeted inhibition of PPARα ameliorates CLA-induced hypercholesterolemia via hepatic cholesterol biosynthesis reprogramming.

BACKGROUND & AIMS: Disruption of lipid metabolism is largely linked to metabolic disorders, such as hypercholesterolemia (HCL) and liver steatosis. While cholesterol metabolic re-programmers can serve as targets for relevant interventions. Here we explored the dietary conjugated linoleic acids (CLA)-induced HCL in mice and the molecular regulation behind it. METHODS: A high dose of CLA supplementation in the diet was used to induce HCL in mice and was found to cause a hyper-activated cholesterol biosynthesis programme in the liver, leading to cholesterol metabolism dysregulation. The effects of a small-molecule drug targeting PPARα, i.e., GW6471 were studied in vivo in mice fed diets with CLA supplementation for 28 days, and in primary hepatocytes derived from HCL-mice in vitro. RESULTS: We demonstrate that CLA induced HCL and liver steatosis through multiple pathways. Among which was the PPARα-mediated cholesterogenesis. It was found to cooperate with SREBP2 via binding to Hmgcr and Dhcr7 (genes encoding key enzymes of the cholesterol biosynthetic pathway) and recruits the histone marks H3K27ac and H3K4me1 and cofactors. PPARα inhibition disrupts its physical association with SREBP2 by blocking cobinding of PPARα and SREBP2 to the genomic DNA response element. We showed that NR RORγ functions as an essential mediator that facilitates the interaction of PPARα and SREBP2 to modulate the cholesterol biosynthesis genes expression. CONCLUSIONS: Our study unravels that the small-molecule compound GW6471 exerts an attractive therapeutic effect for CLA-induced HCL, involving multiple pathways with the "PPARα-RORγ-SREBP2" being a potential complex player in this hepatic cholesterol biosynthesis programming.

A Recombinant Genotype I Japanese Encephalitis Virus Expressing a Gaussia Luciferase Gene for Antiviral Drug Screening Assay and Neutralizing Antibodies Detection.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in humans throughout Asia. In the past twenty years, the emergence of the genotype I (GI) JEV as the dominant genotype in Asian countries has raised a significant threat to public health security. However, no clinically approved drug is available for the specific treatment of JEV infection, and the commercial vaccines derived from the genotype III JEV strains merely provided partial protection against the GI JEV. Thus, an easy-to-perform platform in high-throughput is urgently needed for the antiviral drug screening and assessment of neutralizing antibodies specific against the GI JEV. In this study, we established a reverse genetics system for the GI JEV strain (YZ-1) using a homologous recombination strategy. Using this reverse genetic system, a gaussia luciferase (Gluc) expression cassette was inserted into the JEV genome to generate a reporter virus (rGI-Gluc). The reporter virus exhibited similar growth kinetics to the parental virus and remained genetically stable for at least ten passages in vitro. Of note, the bioluminescence signal strength of Gluc in the culture supernatants was well correlated with the viral progenies determined by viral titration. Taking advantage of this reporter virus, we established Gluc readout-based assays for antiviral drug screening and neutralizing antibody detection against the GI JEV. These Gluc readout-based assays exhibited comparable performance to the assays using an actual virus and are less time consuming and are applicable for a high-throughput format. Taken together, we generated a GI JEV reporter virus expressing a Gluc gene that could be a valuable tool for an antiviral drug screening assay and neutralization assay.

Co-option of PPARα in the regulation of lipogenesis and fatty acid oxidation in CLA-induced hepatic steatosis.

Nonalcoholic-fatty-liver-disease (NAFLD) is the result of imbalances in hepatic lipid partitioning and is linked to dietary factors. We demonstrate that conjugated linoleic acid (CLA) when given to mice as a dietary supplement, induced an enlarged liver, hepatic steatosis, and increased plasma levels of fatty acid (FA), alanine transaminase, and triglycerides. The progression of NAFLD and insulin resistance was reversed by GW6471 a small-molecule antagonist of peroxisome proliferator-activated receptor α (PPARα). Transcriptional profiling of livers revealed that the genes involved in FA oxidation and lipogenesis as two core gene programs controlled by PPARα in response to CLA and GW6471 including Acaca and Acads. Bioinformatic analysis of PPARα ChIP-seq data set and ChIP-qPCR showed that GW6471 blocks PPARα binding to Acaca and Acads and abolishes the PPARα-mediated local histone modifications of H3K27ac and H3K4me1 in CLA-treated hepatocytes. Thus, our findings reveal a dual role of PPARα in the regulation of lipid homeostasis and highlight its druggable nature in NAFLD.

The Circadian Physiology: Implications in Livestock Health.

Circadian rhythms exist in almost all types of cells in mammals. Thousands of genes exhibit approximately 24 h oscillations in their expression levels, making the circadian clock a crucial regulator of their normal functioning. In this regard, environmental factors to which internal physiological processes are synchronized (e.g., nutrition, feeding/eating patterns, timing and light exposure), become critical to optimize animal physiology, both by managing energy use and by realigning the incompatible processes. Once the circadian clock is disrupted, animals will face the increased risks of diseases, especially metabolic phenotypes. However, little is known about the molecular components of these clocks in domestic species and by which they respond to external stimuli. Here we review evidence for rhythmic control of livestock production and summarize the associated physiological functions, and the molecular mechanisms of the circadian regulation in pig, sheep and cattle. Identification of environmental and physiological inputs that affect circadian gene expressions will help development of novel targets and the corresponding approaches to optimize production efficiency in farm animals.

GR-mediated FTO transactivation induces lipid accumulation in hepatocytes via demethylation of m6A on lipogenic mRNAs.

Chronic stress or excessive exposure to glucocorticoids (GC) contributes to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Glucocorticoid receptor (GR) mediates the action of GC, but its downstream signalling is not fully understood. Fat mass and obesity associated (FTO) protein and its demethylation substrate N6-methyladenosine (m6A) are both reported to participate in the regulation of lipid metabolism, yet it remains unknown whether they are involved in GC-induced hepatic lipid accumulation as new components of GR signalling. In this study, we use both in vivo and in vitro models of GC-induced hepatic lipid accumulation and demonstrate that the activation of lipogenic genes and accumulation of lipid in liver cells are mediated by GR-dependent FTO transactivation and m6A demethylation on mRNA of lipogenic genes. Targeted mutation of m6A methylation sites and FTO knockdown further validated the role of m6A on 3'UTR of sterol regulatory element-binding transcription factor 1 and stearoyl-CoA desaturase mRNAs. Finally, FTO knockdown significantly alleviated dexamethasone-induced fatty liver in mice. These results demonstrate a role of GR-mediated FTO transactivation and m6A demethylation in the pathogenesis of NAFLD and provide new insight into GR signalling in the regulation of fat metablism in the liver.

Maternal high-protein diet modulates hepatic growth axis in weaning piglets by reprogramming the IGFBP-3 gene.

PURPOSE: The aim of this study was to investigate the effects of maternal high dietary protein intake on the hepatic growth axis in offspring. METHODS: Fourteen primiparous purebred Meishan sows were fed either a standard-protein (SP, n = 7) diet or a high-protein (HP, 150% of SP, n = 7) diet during pregnancy. Offspring (one male and one female per group, n = 14) on day 70 of the embryonic stage and on days 1, 35 and 180 after birth were selected, weighed and killed. Serum samples were analyzed for Tch, insulin and insulin-like growth factor-binding protein 3 (IGFBP-3) levels. Liver samples were analyzed for IGFBP-3 and IGF-I mRNA expression by qRT-PCR and for IGFBP-3, IGF1R and growth hormone receptor (GHR) protein expression by Western blotting. The underlying mechanism of IGFBP-3 regulation was determined by methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation (ChIP). RESULTS: High-protein exposure resulted in significantly higher body and liver weights of piglets, and it increased their serum T3 and T4 levels at birth and/or at weaning. Furthermore, the IGFBP-3 protein content in the liver and serum was significantly reduced in the HP-exposed weaning piglets, whereas at the transcriptional level IGFBP-3 mRNA expression was downregulated in the livers of HP group piglets. Finally, DNA hypermethylation and higher enrichment of the histone repressive marks H3K27me3 and H3K9me3 were observed. CONCLUSIONS: Taken together, these results suggest that a maternal high-protein diet during gestation epigenetically reprograms IGFBP-3 gene expression to modulate the hepatic growth axis in weaning piglets.

Amino acid starvation-induced autophagy is involved in reduced subcutaneous fat deposition in weaning piglets derived from sows fed low-protein diet during gestation and lactation : Autophagy is involved in reduced fat deposition in maternal low-protein piglets.

PURPOSE: The study aimed to determine the effects of maternal low-protein (LP) diet on subcutaneous fat deposition of weaning piglets and the potential mechanism. METHODS: Sows were fed either a standard protein (SP, 15 and 18% crude protein) or a LP diet (50% protein levels of SP) throughout pregnancy and lactation. Subcutaneous fat and blood were sampled from male piglets at 28 days of age. Serum biochemical metabolites and hormone concentrations were detected with the enzymatic colorimetric methods. Serum-free amino acid (FAA) levels were measured by amino acid auto-analyzer. The mRNA and protein were measured by qRT-PCR and Western blot. RESULTS: Body weight, back fat thickness, triglycerides concentrations in subcutaneous fat tissue, and serum, as well as FFA concentrations were significantly reduced in LP piglets when compared with SP piglets. Further studies showed that mRNA and protein expression of acetyl-CoA carboxylase and fatty acid synthetase, two key enzymes of de novo lipogenesis, were significantly reduced in LP piglets, while mRNA expression and the lipolytic enzymes activities of lipolysis genes, adipose triglyceride lipase and hormone-sensitive lipase, were significantly increased. Furthermore, expression of autophagy-related gene 7 and autophagy maker gene microtubule-associated protein 1A/1B-light chain 3 (LC 3) as well as the conversion of LC3I to LC3II were significantly elevated, along with the expression of activating transcription factor-4 and eukaryotic translation initiation factor-2a. CONCLUSION: These results indicate that amino acid starvation-induced autophagy is involved in reduced subcutaneous fat deposition in maternal LP weaning piglets, demonstrating links between maternal protein restriction and offspring fat deposition.

Gestational dietary betaine supplementation suppresses hepatic expression of lipogenic genes in neonatal piglets through epigenetic and glucocorticoid receptor-dependent mechanisms.

Methyl donors play critical roles in nutritional programming through epigenetic regulation of gene expression. Here we fed gestational sows with control or betaine-supplemented diets (3g/kg) throughout the pregnancy to explore the effects of maternal methyl-donor nutrient on neonatal expression of hepatic lipogenic genes. Betaine-exposed piglets demonstrated significantly lower liver triglyceride content associated with down-regulated hepatic expression of lipogenic genes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD) and sterol regulatory element-binding protein-1c. Moreover, s-adenosyl methionine to s-adenosyl homocysteine ratio was elevated in the liver of betaine-exposed piglets, which was accompanied by DNA hypermethylation on FAS and SCD gene promoters and more enriched repression histone mark H3K27me3 on SCD gene promoter. Furthermore, glucocorticoid receptor (GR) binding to SCD gene promoter was diminished along with reduced serum cortisol and liver GR protein content in betaine-exposed piglets. GR-mediated SCD gene regulation was confirmed in HepG2 cells in vitro. Dexamethasone (Dex) drastically increased the luciferase activity of porcine SCD promoter, while the deletion of GR response element on SCD promoter significantly attenuated Dex-mediated SCD transactivation. In addition, miR-let-7e, miR-1285 and miR-124a, which respectively target porcine SCD, ACC and GR, were significantly up-regulated in the liver of betaine-exposed piglets, being in accordance with decreased protein content of these three genes. Taken together, our results suggest that maternal dietary betaine supplementation during gestation attenuates hepatic lipogenesis in neonatal piglets via epigenetic and GR-mediated mechanisms.

Butyrate stimulates adipose lipolysis and mitochondrial oxidative phosphorylation through histone hyperacetylation-associated ß3 -adrenergic receptor activation in high-fat diet-induced obese mice.

NEW FINDINGS: What is the central question of this study? Butyrate can prevent diet-induced obesity through increasing energy expenditure. However, it is unclear whether ß3 -adrenergic receptors (ARß3) mediate butyrate-induced adipose lipolysis. What is the main finding and its importance? Short-term oral administration of sodium butyrate is effective in alleviating diet-induced obesity through activation of ARß3-mediated lipolysis in white adipose tissue. Butyrate can prevent diet-induced obesity through increasing energy expenditure. However, it is unclear whether ARß3 mediates butyrate-induced adipose lipolysis. In this study, weaned mice were were fed control (Con) or high-fat (HF) diet for 8 weeks to establish obesity. High-fat diet-induced obese mice maintained on the HF diet were divided into two subgroups; the HFB group was gavaged with 80 mg sodium butyrate (SB) per mouse every other day for 10 days, whereas the HF group received vehicle. Chromatin immunoprecipitation assay was performed to determine the status of histone H3 lysine 9 acetylation (H3K9Ac) on the promoter of the ß3 -adrenergic receptor (ARß3) gene in epididymal white adipose tissue. It was shown that five gavage doses of SB significantly alleviated HF diet-induced obesity and restored plasma leptin concentration to the control level. Protein contents of ARß3 and PKA, as well as ATGL and p-HSL (Ser563), were significantly upregulated in the HFB group compared with the HF group. Mitochondrial oxidative phosphorylation was enhanced by SB treatment. Sodium butyrate significantly increased the expression of four out of 13 mitochondrial DNA-encoded genes and significantly upregulated the protein contents of peroxisome proliferator-activated receptor-γ coactivator 1α and COX4. Moreover, SB administration enhanced the expression of ARß3 and its downstream signalling. The G protein-coupled receptor 43 and p-CREB (Ser133) were significantly stimulated by SB. In addition, an active transcription marker, H3K9Ac, was significantly enriched on the promoter of the ARß3 gene. Our results indicate that short-term oral administration of SB is effective in alleviating diet-induced obesity through activation of the ARß3-mediated lipolysis in the epididymal white adipose tissue.

Maternal protein restriction depresses the duodenal expression of iron transporters and serum iron level in male weaning piglets.

To investigate the effects of maternal dietary protein restriction on offspring Fe metabolism, twenty-four second-parity Landrace×Yorkshire sows were randomly allocated to standard-protein (SP) and low-protein (LP) groups. The SP sows were fed diets containing 15 and 18 % crude protein throughout pregnancy and lactation, respectively, whereas the LP sows were subjected to 50 % dietary protein restriction. Offspring birth weight was not affected, but the body weight at weaning (P=0·06) and average daily gain (P=0·01) of the female piglets were significantly decreased. Serum Fe level in the LP piglets was markedly decreased at weaning, especially in males (P=0·03). Serum ferritin level (P=0·08) tended to be lower, yet serum transferrin was greatly higher (P=0·01) in male weaning piglets of the LP group. Duodenal expression of the divalent metal transporter 1 (DMT1) and ferroportin (FPN) was surprisingly reduced (P<0·05) at the level of protein, but not at the mRNA level, in male weaning piglets of the LP group. Male weaning piglets born to the LP sows exhibited higher hepatic hepcidin levels (P=0·09), lower hepatic expression of transferrin (P<0·01) and transferrin receptor 1 (P<0·05) at the level of mRNA. However, no significant differences were observed for hepatic Fe storage, ferritin, transferrin and transferrin receptor 1 protein expression in male weaning piglets of the two groups. These results indicate that maternal protein restriction during pregnancy and lactation influences growth of female offspring at weaning, reduces duodenal expression of Fe transporters (DMT1 and FPN) and decreases serum Fe level in male weaning piglets.

Epigenetic and SP1-mediated regulation is involved in the repression of galactokinase 1 gene in the liver of neonatal piglets born to betaine-supplemented sows.

PURPOSE: In this study, we sought to investigate the effects of maternal betaine supplementation on the expression and regulation of GALK1 gene in the liver of neonatal piglets. METHODS: Sixteen sows of two groups were fed control or betaine-supplemented diets (3 g/kg), respectively, throughout the pregnancy. Newborn piglets were individually weighed immediately after birth, and one male piglet close to mean body weight from the same litter was selected and killed before suckling. Serum samples of newborn piglets were analyzed for biochemical indexes, hormone and amino acid levels. Liver samples were analyzed for GALK1 expression by real-time PCR and western blotting, while GALK1 regulational mechanism was analyzed by methylated DNA immunoprecipitation, chromatin immunoprecipitation and microRNAs expression. RESULTS: Betaine-exposed neonatal piglets had lower serum concentration of galactose, which was associated with significantly down-regulated hepatic GALK1 expression. The repression of GALK1 mRNA expression was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3 on its promoter. Binding sites of SP1, GR and STAT3 were predicted on GALK1 promoter, and decreased SP1 protein content and lower SP1 binding to GALK1 promoter were detected in the liver of betaine-exposed piglets. Furthermore, the expression of miRNA-149 targeting GALK1 was up-regulated in the liver of betaine-exposed piglets, along with elevated miRNAs-processing enzymes Dicer and Ago2. CONCLUSIONS: Our results suggest that maternal dietary betaine supplementation during gestation suppresses GALK1 expression in the liver of neonatal piglets, which involves complex gene regulation mechanisms including DNA methylation, histone modification, miRNAs expression and SP1-mediated transcriptional modulation.

Low-protein diet fed to crossbred sows during pregnancy and lactation enhances myostatin gene expression through epigenetic regulation in skeletal muscle of weaning piglets.

PURPOSE: This study was aimed to investigate the effects of a maternal low-protein diet on transcriptional regulation of the myostatin (MSTN) gene in skeletal muscle of weaning piglets. METHODS: Sows were fed either a standard-protein (SP, 15 and 18 % crude protein) or a low-protein (LP, 50 % protein level of SP) diet throughout pregnancy and lactation. Longissimus dorsi muscle was sampled from male piglets at 28 days of age. The mRNA was determined by RT-PCR, and protein was measured by Western blot. Chromatin immunoprecipitation assay was used to determine the binding of transcription factors and histone H3 modifications on the MSTN gene promoter. RESULTS: The maternal LP diet significantly decreased body weight and average daily gain (P < 0.05), which was associated with significantly lower plasma concentration of urea nitrogen and total protein (P < 0.05), as well as decreased muscle RNA content (P < 0.05). MSTN mRNA (P < 0.05) was significantly increased, together with enhanced (P < 0.05) mRNA and protein expression of forkhead box class O family member protein 3 (FoxO3), and a tendency of an increase (P = 0.10) in glucocorticoid receptor (GR) mRNA in the muscle of LP piglets. Furthermore, the binding of both FoxO3 and GR to the MSTN gene promoter was significantly higher (P < 0.05) in muscle of LP piglets, together with significantly enriched (P < 0.05) gene activation markers, H3K9Ac and H3K4me3. CONCLUSION: These results indicate that MSTN mediates maternal LP diet-induced growth retardation, through epigenetic regulation involving FoxO3 and GR binding to its promoter.

Intravenous injection of microvesicle-delivery miR-130b alleviates high-fat diet-induced obesity in C57BL/6 mice through translational repression of PPAR-γ.

BACKGROUND: We have shown previously that microvesicle (MV)-delivered miR-130b (miR-130b-MV) is able to target PPAR-γ and subsequently reduce the lipid accumulation in vitro. However, the in vivo effect of miR-130b on fat deposition and glucose homeostasis remains unknown. RESULTS: Three-week-old C57BL/6 mice were fed a high-fat diet for 8 weeks and then intravenously injected with MV-packaged scrambled control microRNA (miRNA) or miR-130b every other day for 10 days. Glucose tolerance test was performed and body weight, epididymal fat weight, as well as the expression of lipid metabolic genes were determined. We showed that mice fed on high-fat diet for 8 weeks demonstrated significantly higher body weight, elevated blood glucose and impaired glucose tolerance. miR-130b-MV injection significantly reduced body weight and epididymal fat weight and partly restored glucose tolerance. miR-130b expression was significantly increased in the epididymal fat after miR-130b-MV injection while the protein content of its target gene PPAR-γ was significantly suppressed, together with a significant up-regulation of the lipolysis genes, hormone sensitive lipase, monoglyceride lipase and leptin. Moreover, miR-130b-MV injection increased the expression of miR-378a and miR-378-3p that are reported to participate in the regulation of fat deposition. CONCLUSION: Our results indicate that miR-130b-MV is able to reduce the epididymal fat deposition and partly restore glucose tolerance, through translational repression of PPAR-γ in a high-fat diet-induced obese mouse model.

Dietary betaine supplementation to gestational sows enhances hippocampal IGF2 expression in newborn piglets with modified DNA methylation of the differentially methylated regions.

PURPOSE: The adequate supply of methyl donors is critical for the normal development of brain. The purpose of the present study was to investigate the effects of maternal betaine supplementation on hippocampal gene expression in neonatal piglets and to explore the possible mechanisms. METHODS: Gestational sows were fed control or betaine-supplemented (3 g/kg) diets throughout the pregnancy. Immediately after birth, male piglets were killed, and the hippocampus was dissected for analyses. The mRNA abundance was determined by reverse transcription real-time polymerase chain reaction. Protein content was measured by Western blot, and DNA methylation was detected by methylated DNA immunoprecipitation assay. RESULTS: Prenatal betaine supplementation did not alter the body weight or the hippocampus weight, but increased the hippocampal DNA content as well as the mRNA expression of proliferation-related genes. Prenatal betaine supplementation increased serum level of methionine (P < 0.05) and up-regulated (P < 0.05) the mRNA and protein expression of betaine-homocysteine methyltransferase, glycine N-methyltransferase and DNA methyltransferase 1 in the neonatal hippocampus. Hippocampal expression of insulin growth factor II (IGF2) and its receptors IGF1R and IGF2R were all significantly up-regulated (P < 0.05) in betaine-treated group, together with a significant activation (P < 0.01) of the downstream extracellular signal-regulated kinase 1/2. Moreover, the differentially methylated region (DMR) 1 and 2 on IGF2 locus was found to be hypermethylated (P < 0.05) in the hippocampus of betaine-treated piglets. CONCLUSIONS: These results indicate that maternal betaine supplementation enhances betaine/methionine metabolism and DNA methyltransferase expression, causes hypermethylation of DMR on IGF2 gene, which was associated with augmented expression of IGF2 and cell proliferation/anti-apoptotic markers in the hippocampus of neonatal piglets.

Maternal dietary betaine supplementation modifies hepatic expression of cholesterol metabolic genes via epigenetic mechanisms in newborn piglets.

To elucidate the effects of maternal dietary betaine supplementation on hepatic expression of cholesterol metabolic genes in newborn piglets and the involved epigenetic mechanisms, we fed gestational sows with control or betaine-supplemented diets (3 g/kg) throughout pregnancy. Neonatal piglets born to betaine-supplemented sows had higher serum methionine concentration and hepatic content of betaine, which was associated with significantly up-regulated hepatic expression of glycine N-methyltransferase. Prenatal betaine exposure increased hepatic cholesterol content and modified the hepatic expression of cholesterol metabolic genes in neonatal piglets. Sterol regulatory element-binding protein 2 was down-regulated at both mRNA and protein levels, while 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) was down-regulated at the mRNA level, but up-regulated at the protein level, in betaine-exposed piglets. The transcriptional repression of HMGCR was associated with CpG island hypermethylation and higher repressive histone mark H3K27me3 (histone H3 lysine 27 trimethylation) on the promoter, whereas increased HMGCR protein content was associated with significantly decreased expression of miR-497. Furthermore, LDL receptor was significantly down-regulated at both mRNA and protein levels in the liver of betaine-exposed piglets, which was associated with promoter CpG hypermethylation. In addition, the expression of cholesterol-27α-hydroxylase (CYP27α1) was up-regulated at both mRNA and protein levels, while the expression of cholesterol-7α-hydroxylase (CYP7α1) was increased at the mRNA level, but unchanged at the protein level associated with increased expression of miR-181. These results indicate that maternal betaine supplementation increases hepatic cholesterol content in neonatal piglets through epigenetic regulations of cholesterol metabolic genes, which involve alterations in DNA and histone methylation and in the expression of microRNA targeting these genes.

Maternal Low-protein Diet Alters Ovarian Expression of Folliculogenic and Steroidogenic Genes and Their Regulatory MicroRNAs in Neonatal Piglets.

Maternal malnutrition during pregnancy may give rise to female offspring with disrupted ovary functions in adult age. Neonatal ovary development predisposes adult ovary function, yet the effect of maternal nutrition on the neonatal ovary has not been described. Therefore, here we show the impact of maternal protein restriction on the expression of folliculogenic and steroidogenic genes, their regulatory microRNAs and promoter DNA methylation in the ovary of neonatal piglets. Sows were fed either standard-protein (SP, 15% crude protein) or low-protein (LP, 7.5% crude protein) diets throughout gestation. Female piglets born to LP sows showed significantly decreased ovary weight relative to body weight (p<0.05) at birth, which was accompanied with an increased serum estradiol level (p<0.05). The LP piglets demonstrated higher ratio of bcl-2 associated X protein/B cell lymphoma/leukemia-2 mRNA (p<0.01), which was associated with up-regulated mRNA expression of bone morphogenic protein 4 (BMP4) (p<0.05) and proliferating cell nuclear antigen (PCNA) (p<0.05). The steroidogenic gene, cytochrome P450 aromatase (CYP19A1) was significantly down-regulated (p<0.05) in LP piglets. The alterations in ovarian gene expression were associated with a significant down-regulation of follicle-stimulating hormone receptor mRNA expression (p<0.05) in LP piglets. Moreover, three microRNAs, including miR-423-5p targeting both CYP19A1 and PCNA, miR-378 targeting CYP19A1 and miR-210 targeting BMP4, were significantly down-regulated (p<0.05) in the ovary of LP piglets. These results suggest that microRNAs are involved in mediating the effect of maternal protein restriction on ovarian function through regulating the expression of folliculogenic and steroidogenic genes in newborn piglets.

The Metabolic Pathway of Bile Secretion Is Vulnerable to Salmonella enterica Exposure in Porcine Intestinal Epithelial Cells.

Pigs can be colonized with Salmonella enterica and become established carriers. However, the mechanisms of the host's response to Salmonella enterica infection are largely unclear. This study was constructed with the Salmonella enterica infection model in vitro using porcine intestinal epithelial cells (IPEC-J2). Transcriptome profiling of IPEC-J2 cells was carried out to characterize the effect of Salmonella enterica infection and lipopolysaccharide (LPS) treatment, in which LPS-induced inflammation was a positive control. At first, Salmonella enterica infection increased the cell apoptosis rate and induced an inflammation response in IPEC-J2. Then, the up-regulated genes were enriched in metabolic pathways, such as those for bile secretion and mineral absorption, while down-regulated genes were enriched in immune-related pathways, such as the Toll-like receptor signaling and p53 signaling pathways. Moreover, we found 368 up-regulated genes and 101 down-regulated genes in common. Then, an integrative analysis of the transcriptomic profile under Salmonella enterica infection and LPS treatment was conducted, and eight up-regulated genes and one down-regulated gene were detected. Among them, AQP8 is one critical gene of the bile secretion pathway, and its mRNA and protein expression were increased significantly under Salmonella enterica infection and LPS treatment. Thus, the AQP8 gene and bile secretion pathway may be important in IPEC-J2 cells under Salmonella enterica infection or LPS treatment.

Alternatives to antibiotics in pig production: looking through the lens of immunophysiology.

In the livestock production system, the evolution of porcine gut microecology is consistent with the idea of "The Hygiene Hypothesis" in humans. I.e., improved hygiene conditions, reduced exposure to environmental microorganisms in early life, and frequent use of antimicrobial drugs drive immune dysregulation. Meanwhile, the overuse of antibiotics as feed additives for infectious disease prevention and animal growth induces antimicrobial resistance genes in pathogens and spreads related environmental pollutants. It justifies our attempt to review alternatives to antibiotics that can support optimal growth and improve the immunophysiological state of pigs. In the current review, we first described porcine mucosal immunity, followed by discussions of gut microbiota dynamics during the critical weaning period and the impacts brought by antibiotics usage. Evidence of in-feed additives with immuno-modulatory properties highlighting probiotics, prebiotics, and phytobiotics and their cellular and molecular networking are summarized and reviewed. It may provide insights into the immune regulatory mechanisms of antibiotic alternatives and open new avenues for health management in pig production.

Lithocholic Acid Alleviates Deoxynivalenol-Induced Inflammation and Oxidative Stress via PPARγ-Mediated Epigenetically Transcriptional Reprogramming in Porcine Intestinal Epithelial Cells.

Deoxynivalenol (DON) is a common mycotoxin that induces intestinal inflammation and oxidative damage in humans and animals. Given that lithocholic acid (LCA) has been suggested to inhibit intestinal inflammation, we aimed to investigate the protective effects of LCA on DON-exposed porcine intestinal epithelial IPI-2I cells and the underlying mechanisms. Indeed, LCA rescued DON-induced cell death in IPI-2I cells and reduced DON-stimulated inflammatory cytokine levels and oxidative stress. Importantly, the nuclear receptor PPARγ was identified as a key transcriptional factor involved in the DON-induced inflammation and oxidative stress processes in IPI-2I cells. The PPARγ function was found compromised, likely due to the hyperphosphorylation of the p38 and ERK signaling pathways. In contrast, the DON-induced inflammatory responses and oxidative stress were restrained by LCA via PPARγ-mediated reprogramming of the core inflammatory and antioxidant genes. Notably, the PPARγ-modulated transcriptional regulations could be attributed to the altered recruitments of coactivator SRC-1/3 and corepressor NCOR1/2, along with the modified histone marks H3K27ac and H3K18la. This study emphasizes the protective actions of LCA on DON-induced inflammatory damage and oxidative stress in intestinal epithelial cells via PPARγ-mediated epigenetically transcriptional reprogramming, including histone acetylation and lactylation.