Porcine Mammary Epithelial Cells Research Articles

Maternal sodium acetate supplementation promotes lactation performance of sows and their offspring growth performance.

Milk yield and composition are critical determining factors for the early growth and development of neonates. The objective of this experiment was to comprehensively evaluate the effects of dietary sodium acetate (SA) supplementation on the milk yield and composition of sows and the growth performance of their offspring. A total of 80 sows (Landrace×Yorkshire, 3 to 6 parity) were randomly assigned to 2 groups (with or without 0.1% SA) from d 85 of gestation to d 21 of lactation. The result shows that maternal 0.1% SA supplementation significantly increased sows milk yield, milk fat, immunoglobulin A (IgA) and IgG content in milk (P<0.05), with the up-regulation of short-chain fatty acids receptors (GPR41 and GPR43) expression and the activation of mammalian target of rapamycin complex C1 (mTORC1) signaling pathway. Consistently, in our invitro experiment, SA also activated mTORC1 signaling in porcine mammary epithelial cells (P<0.05). Furthermore, the improvement of milk quality and quantity caused by maternal SA supplementation led to the increase in body weight (BW) and average daily weight gain (ADG) of weaning piglets, with the improvement of gut health and colonization of the beneficial bacteria (P<0.05). In conclusion, maternal supplementation of 0.1% SA improved the lactation performance (milk yield and milk fat) of sows, possibly with the activation of GPR41/GPR43-mTORC1 signaling. Furthermore, enhanced milk quality improved growth performance, gut health and the colonization of beneficial microbial flora of their piglets.

Leucine and arginine enhance milk fat and milk protein synthesis via the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways.

Amino acids (AAs) not only constitute milk protein but also stimulate milk synthesis through the activation of mTORC1 signaling, but which amino acids that have the greatest impact on milk fat and protein synthesis is still very limited. In this study, we aimed to identify the most critical AAs involved in the regulation of milk synthesis and clarify how these AAs regulate milk synthesis through the G-protein-coupled receptors (GPCRs) signaling pathway. In this study, a mouse mammary epithelial cell line (HC11) and porcine mammary epithelial cells (PMECs) were selected as study subjects. After treatment with different AAs, the amount of milk protein and milk fat synthesis were detected. Activation of mTORC1 and GPCRs signaling induced by AAs was also investigated. In this study, we demonstrate that essential amino acids (EAAs) are crucial to promote lactation by increasing the expression of genes and proteins related to milk synthesis, such as ACACA, FABP4, DGAT1, SREBP1, α-casein, β-casein, and WAP in HC11 cells and PMECs. In addition to activating mTORC1, EAAs uniquely regulate the expression of calcium-sensing receptor (CaSR) among all amino-acid-responsive GPCRs, which indicates a potential link between CaSR and the mTORC1 pathway in mammary gland epithelial cells. Compared with other EAAs, leucine and arginine had the greatest capacity to trigger GPCRs (p-ERK) and mTORC1 (p-S6K1) signaling in HC11 cells. In addition, CaSR and its downstream G proteins Gi, Gq, and Gβγ are involved in the regulation of leucine- and arginine-induced milk synthesis and mTORC1 activation. Taken together, our data suggest that leucine and arginine can efficiently trigger milk synthesis through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways. We found that the G-protein-coupled receptor CaSR is an important amino acid sensor in mammary epithelial cells. Leucine and arginine promote milk synthesis partially through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 signaling systems in mammary gland epithelial cells. Although this mechanism needs further verification, it is foreseeable that this mechanism may provide new insights into the regulation of milk synthesis.

Silibinin Alleviates Lipopolysaccharide Induced Inflammation in Porcine Mammary Epithelial Cells via mTOR/NF-κB Signaling Pathway.

Inflammatory responses reduce milk production in lactating sow. Silymarin (Silibinin is main component) reduces the inflammatory reaction and increases milk yield in lactating sow in the previous study. In the present study, silibinin may be a previously unrecognized nutrients in inflammatory resolution in porcine mammary epithelial cells (PMECs) is hypothesized. PMECs are treated with or without lipopolysaccharide (LPS) in the absence or presence of silibinin to test cell viability, cell cycle, cell apoptosis, cellular inflammatory factors, and signaling protein phosphorylation and expression. Silibinin promotes the proliferation of PMEC independent of the estrogen pathway. In LPS-induced damage of PMECs, silibinin protects cell proliferation, as well as reduced cell apoptosis. Silibinin reverses the LPS-induced increase in tumor necrosis factor-alpha (TNF-α) expression compared with control. In addition, silibinin accentuates the LPS-induced decrease in the key proteins phosphorylated-ribosomal protein S6 (p-S6) and phosphorylated-mammalian target of rapamycin (p-mTOR) of the mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, silibinin reverses the increase in phosphorylated-nuclear factor-kappa B p65 (p-NF-κB p65), phosphorylated-Ikappab-alpha (p-IκB-α), and phosphorylated-Mitogen-activated protein kinase p38 (p-MAPK p38) expression in LPS-induced damage in PMECs. This study highlights silibinin-mTOR/NF-κB axis plays an important role in the control of inflammation in PMECs, and suggests that silibinin may be an effective dietary strategy to alleviate the inflammatory response in lactating sow.

Effects of Boron on Fat Synthesis in Porcine Mammary Epithelial Cells.

This study aimed to investigate the effect of boron on porcine mammary epithelial cells (PMECs) survival, cell cycle, and milk fat synthesis. PMECs from boron-treated groups were exposed to 0-80mmol/L boric acid concentrations. Cell counting kit-8 and flow cytometry assays were performed to assess cell survival and the cell cycle, respectively. Triacylglycerol (TAG) levels in PMECs and culture medium were determined by a triacylglycerol kit while PMECs lipid droplet aggregation was investigated via oil red staining. Milk fat synthesis-associated mRNA levels were determined by quantitative real-time polymerase chain reaction (qPCR) while its protein expressions were determined by Western blot. Low (0.2, 0.3, 0.4mmol/L) and high (> 10mmol/L) boron concentrations significantly promoted and inhibited cell viabilities, respectively. Boron (0.3mmol/L) markedly elevated the abundance of G2/M phase cells. Ten mmol/L boron significantly increased the abundances of G0/G1 and S phase cells, but markedly suppressed G2/M phase cell abundance. At 0.3mmol/L, boron significantly enhanced ERK phosphorylation while at 0.4, 0.8, 1, and 10mmol/L, it markedly decreased lipid droplet diameters. Boron (10mmol/L) significantly suppressed ACACA and SREBP1 protein expressions. The FASN protein levels were markedly suppressed by 0.4, 0.8, 1, and 10mmol/L boron. Both 1 and 10mmol/L markedly decreased FASN and SREBP1 mRNA expressions. Ten mmol/L boron significantly decreased PPARα mRNA levels. Low concentrations of boron promoted cell viability, while high concentrations inhibited PMECS viabilities and reduced lipid droplet diameters, which shows the implications of boron in pregnancy and lactation.

Taurine alleviates oxidative stress in porcine mammary epithelial cells by stimulating the Nrf2-MAPK signaling pathway.

The high incidence of oxidative stress in sows during late gestation and lactation affects mammary gland health, milk yield, and milk quality. Recently, we found that supplementing maternal diets with 1% taurine improved antioxidant capability and enhanced growth performance in offspring; however, the mechanisms underlying these are unknown. This study aimed to investigate the cytoprotective effects and the mechanism of taurine in mitigating oxidative stress in porcine mammary epithelial cells (PMECs). PMECs were pretreated with 0-2.0 mM taurine for 12 h and then subjected to oxidative injury with 500 μM hydrogen peroxide (H2O2). Pretreatment with taurine attenuated decreased cell viability, enhanced superoxide dismutase, and reduced the intracellular reactive oxygen species accumulation after H2O2 exposure. Taurine also prevented H2O2-induced endoplasmic reticulum stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) was essential to the cytoprotective effects of taurine on PMECs, as Nrf2 knockdown significantly inhibited taurine-induced cytoprotection against oxidative stress. Moreover, we confirmed that Nrf2 induction by taurine was mediated through the inactivation of the p38/MAPK pathway. Overall, taurine supplementation has beneficial effects on redox balance regulation and may protect against oxidative stress in lactating animals.

Niacin/β-hydroxybutyrate regulates milk fat and milk protein synthesis via the GPR109A/Gi/mTORC1 pathway.

As a crucial receptor of BHBA and niacin, GPR109A is largely expressed in the mammary gland. However, the role of GPR109A in milk synthesis and its underlying mechanism is still largely unknown. In this study, we first investigated the effect of GPR109A agonists (niacin/BHBA) on milk fat and milk protein synthesis in a mouse mammary epithelial cell line (HC11) and PMECs (porcine mammary epithelial cells). The results showed that both niacin and BHBA promote milk fat and milk protein synthesis with the activation of mTORC1 signaling. Importantly, knockdown GPR109A attenuated the niacin-induced increase of milk fat and protein synthesis and the niacin-induced activation of mTORC1 signaling. Furthermore, we found that GPR109A downstream G protein-Gαi and -Gβγ participated in the regulation of milk synthesis and the activation of mTORC1 signaling. Consistent with the finding in vitro, dietary supplementation with niacin increases milk fat and protein synthesis in mice with the activation of GPR109A-mTORC1 signaling. Collectively, GPR109A agonists promote the synthesis of milk fat and milk protein through the GPR109A/Gi/mTORC1 signaling pathway.

Z. morio Hemolymph Relieves E. coli-Induced Mastitis by Inhibiting Inflammatory Response and Repairing the Blood-Milk Barrier.

Escherichia coli (E. coli) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio (Z. morio) hemolymph against mastitis pathogens. In this study, we established E. coli-induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli-induced mastitis in vivo and in vitro. In mice with E. coli, Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4+ T/CD8+ T, and increased the production of IL-2 triggered by E. coli. Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli-induced mastitis. In E. coli-stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli-induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli-induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli-induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.

Branched-chain amino acids regulate intracellular protein turnover in porcine mammary epithelial cells.

Dietary supplementation with branched-chain amino acids (BCAAs) to lactating sows has been reported to enhance their milk production, but the underlying mechanisms remain largely unknown. This study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that individual BCAAs or their mixture stimulates protein synthesis and inhibit proteolysis in PMECs. Cells were cultured at 37°C in customizedDulbecco's modified Eagle medium containing 5mmol/L D-glucose, 1mmol/L L-phenylalanine, L-[ring-2,4-3H]phenylalanine, 0.1 (control), 0.25, 0.5, 1, or 2mmol/L L-leucine, L-isoleucine or L-valine or an equimolar mixture of the three BCAAs. The culture medium also contained physiological concentrations of other amino acids found in the plasma of lactating sows. Proliferation, protein synthesis, proteolysis, β-casein production, the mechanistic target of rapamycin (mTOR) signaling, and the ubiquitin-proteasome pathway were determined for PMECs. Cell proliferation and abundances of phosphorylated mTOR, eukaryotic translation initiation factor 4E-binding protein 1, and ribosomal protein S6 kinase β-1 proteins increased (P < 0.05), but abundances of ubiquitinated protein and 20S proteasome decreased (P < 0.05) when extracellular concentrations of L-leucine, L-isoleucine, L-valine, or an equimolar mixture of BCAAs were increased from 0.1 to 2mmol/L. Compared with the control, 0.25, 0.5, 1 or 2mmol/L BCAAs enhanced (P < 0.01) protein (including β-casein) synthesis, while decreasing (P < 0.05) proteolysis in PMECs in a dose-dependent manner. Collectively, our results indicate that physiological concentrations of BCAAs regulate protein turnover in mammary epithelial cells to favor net protein synthesis through stimulating the mTOR signaling pathway and inhibiting the ubiquitin-proteasome pathway.

L-Leucine stimulates proliferation and casein secretion in porcine mammary epithelial cells through mTOR-p70S6K-4EBP1 signalling pathway

Milk production determines health status and optimal growth of suckling neonates. Recent studies revealed that the secretion of leucine into sow milk from the mammary gland is significantly lower than the amount taken up by the mammary gland from the blood, suggesting the potential and fundamental role in udder metabolism. However, little is known about the underlying mechanism. In this study, porcine mammary epithelial cells (PMECs) were employed to investigate the effects of leucine on protein synthesis and mammalian target of rapamycin (mTOR) signalling. Results showed that leucine supplementation significantly increased the proliferation of PMECs in a dose-dependent manner from 0 to 1 mmol/l. Secretions of four casein subunits were significantly increased by addition of leucine. The phosphorylation of proteins related to mTOR signalling, including mTOR, eukaryotic translation initiation factor 4E-binding protein, and p70 ribosomal protein S6 kinase, were accordingly upregulated by leucine supplementation. Casein secretion was not affected by leucine treatment when mTOR signalling was inhibited by rapamycin. This suggests that leucine stimulates protein synthesis through the mTOR signalling pathway. These findings facilitate the utilization of dietary leucine supplementation to improve milk production in lactating sows.

The Selenium Yeast vs Selenium Methionine on Cell Viability, Selenoprotein Profile and Redox Status via JNK/ P38 Pathway in Porcine Mammary Epithelial Cells.

Comprehensive studies have been conducted to compare the effect of organic and inorganic selenium previously, but there is still limited knowledge about the difference between organic selenium (Se) from varied sources despite the widely use of organic Se in both animal and human being nutrient additives. In the present study, we systemically compared the effect of two different types of organic Se including selenium yeast (SeY) and selenium methionine (Sel-Met) on cell viability, selenoprotein transcriptome, and antioxidant status in porcine mammary epithelial cells (PMECs) and the results indicated that appropriate addition of SeY and Sel-Met both significantly promoted cell viability and up-regulated the mRNA expression of most selenopreoteins including DIOs, GPXs, and TrxRs family et al. (P < 0.05). Besides, two different sources of Se supplementation both greatly improved redox status with higher levels of T-AOC, SOD, and CAT (P < 0.05), while less content of MDA (P < 0.05), and reduced protein expression of cleaved-caspase-3 (P < 0.05) to mitigate cell apoptosis. Furthermore, the key proteins related to p38/JNK pathway including p38, p-p38, JNK, and p-JNK were apparently reduced in the groups with both of SeY and Sel-Met (P < 0.05). Interestingly we found that the changes induced by SeY supplementation in cell viability, selenoprotein transcriptome, antioxidative capacity, and anti-apoptosis were comprehensively greater compared with same levels addition of Sel-Met in PEMCs (P < 0.05). In conclusion, both SeY and Sel-Met promoted cell viability and attenuated cell apoptosis by regulating the selenoprotein expression and antioxidative capacity via p38/JNK signaling pathway in PMEC, but SeY has more efficient benefits than that of Sel-Met.

Effects of Selenomethionine on Cell Viability, Selenoprotein Expression and Antioxidant Function in Porcine Mammary Epithelial Cells.

This study investigated the effects of selenomethionine (Se-Met) on the cell viability, selenoprotein expression, and antioxidant function of porcine mammary epithelial cells (pMECs) to reveal the underlying molecular mechanism of Se-Met on the lactation performance and antioxidant capacity of sows in vitro. The pMECs were used as an in vitro model and were treated with various concentrations of Se-Met (0, 0.5, 1, 2, and 4 μM). Cells were analyzed for cell viability, selenoprotein transcriptome, selenoprotein expression, and antioxidant enzyme activities. The results showed that, with increasing Se-Met concentrations, cell viability first increased and then decreased at 24, 48, or 72 h posttreatment with maximum values at 0.5-μM Se-Met. As the Se-Met concentrations increased, the mRNA expression of 17 selenoproteins first upregulated and then downregulated, with maximum values at 0.5-μM Se-Met. The 17 selenoproteins included SEPHS2, SELENOP, GPX1, GPX2, GPX3, GPX6, TXNRD1, SELENOK, SELENOW, DIO1, DIO2, DIO3, SELENOF, SELENOS, SELENOH, SELENOI, and SELENOT. Additionally, the protein expression levels of SEPHS2, SELENOP, GPX1, and TXNRD1 and the activities of glutathione peroxidase and thioredoxin were highest at 0.5-μM Se-Met. In conclusion, 0.5-μM Se-Met promotes cell viability partially by improving selenoprotein expression and antioxidant function in pMECs, which provides evidence for the potential ability of Se-Met to improve mammary gland health in sows.

Development of a Pig Mammary Epithelial Cell Culture Model as a Non-Clinical Tool for Studying Epithelial Barrier-A Contribution from the IMI-ConcePTION Project.

Simple SummaryThe information about the risks related to the use of medication during breastfeeding is lacking for most commonly used drugs. The ConcePTION project aims to fill this gap using multiple approaches. Within the project, the pig has been selected as the most appropriate in vivo animal model. In agreement with the application of the “3Rs” principle (Replacement, Reduction and Refinement) and international legislations, the present paper reports the establishment of cellular lines of porcine mammary epithelial cells as a valid tool to study the mammary epithelial barrier function in vitro.The ConcePTION project aims at generating further knowledge about the risks related to the use of medication during breastfeeding, as this information is lacking for most commonly used drugs. Taking into consideration multiple aspects, the pig model has been considered by the consortium as the most appropriate choice. The present research was planned to develop an efficient method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study the mammary epithelial barrier in vitro. Mammary gland tissues were collected at a local slaughterhouse, dissociated and the selected cellular population was cultured, expanded and characterized by morphology, cell cycle analysis and immunophenotyping. Their ability to create a barrier was tested by TEER measurement and sodium fluorescein transport activity. Expression of 84 genes related to drug transporters was evaluated by a PCR array. Our results show that primary cells express epithelial cell markers: CKs, CK18, E-Cad and tight junctions molecules ZO-1 and OCL. All the three pMEC cellular lines were able to create a tight barrier, although with different strengths and kinetics, and express the main ABC and SLC drug transporters. In conclusion, in the present paper we have reported an efficient method to obtain primary pMEC lines to study epithelial barrier function in the pig model.

Artemisinin Protects Porcine Mammary Epithelial Cells against Lipopolysaccharide-Induced Inflammatory Injury by Regulating the NF-κB and MAPK Signaling Pathways.

Simple SummarySow mastitis is a serious breast disease that can cause severe inflammation, agalaxia and even lead to death of piglets. Porcine mammary epithelial cells (pMECs) are the main cell types that affect sow milk secretion, therefore, when swine mastitis occurs, the inflammatory response of pMECs directly affects the mammary gland health and sow’s lactation ability. Promoting the health of mammary gland epithelial cells is an important method for treating mastitis. Thus, in the current study, we investigated the effects of artemisinin on the inflammatory response of pMECs induced by lipopolysaccharide (LPS), and proposed a potential anti-inflammatory mechanism. We confirmed that artemisinin can reduce the inflammatory damage of pMECs induced by LPS by inhibiting MAPK and NF-κB signaling pathways. Pretreatment of pMECs with artemisinin showed enhanced anti-inflammatory activity against LPS-induced inflammation. Artemisinin could be a useful, safe and natural anti-inflammatory feed additive to prevent sow mastitis.Artemisinin performs a variety of biological functions, such as anti-cancer, anti-inflammatory, anti-viral, and anti-oxidant effects. However, the effects of artemisinin on sow mastitis have not been studied. The results of the current study showed that mRNA expression abundance and content of the inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) were significantly increased when using 50 μg/mL LPS to stimulate pMECs for 24 h (p < 0.05). Pretreatment with 20 μM artemisinin weakened LPS-induced inflammatory damage in pMECs and decreased mRNA expression abundance and the content of inflammatory factors (IL-1β, IL-6, and TNF-α) in pMECs (p < 0.05). Mechanistically, artemisinin inhibited LPS-induced activation of the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. In summary, the pretreatment of pMECs with artemisinin showed enhanced anti-inflammatory activity against LPS-induced inflammation.

Metabolic Transition of Milk Triacylglycerol Synthesis in Response to Varying Levels of Three 18-Carbon Fatty Acids in Porcine Mammary Epithelial Cells.

This study aimed to examine the effects of increasing levels of three 18-carbon fatty acids (stearate, oleate and linoleate) on mammary lipogenesis, and to evaluate their effects on the milk lipogenic pathway in porcine mammary epithelial cells (pMECs). We found that increasing the three of 18-carbon fatty acids enhanced the cellular lipid synthesis in a dose-dependent manner, as reflected by the increased (triacylglycerol) TAG content and cytosolic lipid droplets in pMECs. The increased lipid synthesis by the three 18-carbon fatty acids was probably caused by the up-regulated expression of major genes associated with milk fat biosynthesis, including CD36 (long chain fatty acid uptake); GPAM, AGPAT6, DGAT1 (TAG synthesis); PLIN2 (lipid droplet formation); and PPARγ (regulation of transcription). Western blot analysis of CD36, DGAT1 and PPARγ proteins confirmed this increase with the increasing incubation of 18-carbon fatty acids. Interestingly, the mRNA expressions of ACSL3 and FABP3 (fatty acids intracellular activation and transport) were differentially affected by the three 18-carbon fatty acids. The cellular mRNA expressions of ACSL3 and FABP3 were increased by stearate, but were decreased by oleate or linoleate. However, the genes involved in fatty acid de novo synthesis (ACACA and FASN) and the regulation of transcription (SREBP1) were decreased by incubation with increasing concentrations of 18-carbon fatty acids. In conclusion, our findings provided evidence that 18-carbon fatty acids (stearate, oleate and linoleate) significantly increased cytosolic TAG accumulation in a dose-dependent manner, probably by promoting lipogenic genes and proteins that regulate the channeling of fatty acids towards milk TAG synthesis in pMECs.

Lactobacillus johnsonii L531 Ameliorates Escherichia coli-Induced Cell Damage via Inhibiting NLRP3 Inflammasome Activity and Promoting ATG5/ATG16L1-Mediated Autophagy in Porcine Mammary Epithelial Cells

Escherichia coli (E. coli), a main mastitis-causing pathogen in sows, leads to mammary tissue damage. Here, we explored the effects of Lactobacillus johnsonii L531 on attenuating E. coli-induced inflammatory damage in porcine mammary epithelial cells (PMECs). L. johnsonii L531 pretreatment reduced E. coli adhesion to PMECs by competitive exclusion and the production of inhibitory factors and decreased E. coli-induced destruction of cellular morphology and ultrastructure. E. coli induced activation of NLRP3 inflammasome associated with increased expression of NLRP3, ASC, and cleaved caspase-1, however, L. johnsonii L531 inhibited E. coli-induced activation of NLRP3 inflammasome. Up-regulation of interleukin (Il)-1β, Il-6, Il-8, Il-18, tumor necrosis factor alpha, and chemokine Cxcl2 expression after E. coli infection was attenuated by L. johnsonii L531. E. coli infection inhibited autophagy, whereas L. johnsonii L531 reversed the inhibitory effect of E. coli on autophagy by decreasing the expression of autophagic receptor SQSTM1/p62 and increasing the expression of autophagy-related proteins ATG5, ATG16L1, and light chain 3 protein by Western blotting analysis. Our findings suggest that L. johnsonii L531 pretreatment restricts NLRP3 inflammasome activity and induces autophagy through promoting ATG5/ATG16L1-mediated autophagy, thereby protecting against E. coli-induced inflammation and cell damage in PMECs.

Valine supplementation during late pregnancy in gilts increases colostral protein synthesis through stimulating mTOR signaling pathway in mammary cells

Mammary gland development during late pregnancy in sows is a major factor affecting the composition of colostrum and milk and the pre-weaning growth of piglets, while valine is essential for protein and nitrogen metabolism in mammary gland of sow. However, the effects of valine and its underlying mechanism on mammary gland development during late pregnancy are still unclear. Here, we hypothesized that dosage of dietary valine during late pregnancy will affect protein synthesis of colostrum in gilts. The results showed that supplementation of valine during late pregnancy significantly increased content of protein (P < 0.01), fat (P = 0.02) and solids-non-fat (P = 0.04) in colostrum. Our in vitro study also confirmed that valine supplementation increased protein synthesis and cell proliferation in porcine mammary epithelial cells (PMEC). Furthermore, these changes were associated with elevated phosphorylation levels of mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase (S6) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1) in valine-supplemented cells, which could be effectively blocked by the antagonists of mTOR. These findings indicated that valine enhanced mammary gland development and protein synthesis in colostrum via the mTOR signaling pathway. These results, using an in vivo and in vitro model, helped to understand the beneficial effects of dietary valine supplementation on gilts.

DNA methylation analysis of porcine mammary epithelial cells reveals differentially methylated loci associated with immune response against Escherichia coli challenge

BackgroundEpigenetic changes such as cytosine (CpG) DNA methylations regulate gene expression patterns in response to environmental cues including infections. Microbial infections induce DNA methylations that play a potential role in modulating host-immune response. In the present study, we sought to determine DNA methylation changes induced by the mastitis causing Escherichia coli (E. coli) in porcine mammary epithelial cells (PMEC). Two time points (3 h and 24 h) were selected based on specific transcriptomic changes during the early and late immune responses, respectively.ResultsDNA methylation analysis revealed 561 and 898 significant (P < 0.01) differentially methylated CpG sites at 3 h and 24 h after E. coli challenge in PMEC respectively. These CpG sites mapped to genes that have functional roles in innate and adaptive immune responses. Significantly, hypomethylated CpG sites were found in the promoter regions of immune response genes such as SDF4, SRXN1, CSF1 and CXCL14. The quantitative transcript estimation indicated higher expression associated with the DNA CpG methylation observed in these immune response genes. Further, E. coli challenge significantly reduced the expression levels of DNMT3a, a subtype of de novo DNA methylation enzyme, in PMEC indicating the probable reason for the hypomethylation observed in the immune response genes.ConclusionsOur study revealed E. coli infection induced DNA methylation loci in the porcine genome. The differentially methylated CpGs were identified in the regulatory regions of genes that play important role in immune response. These results will help to understand epigenetic mechanisms for immune regulation during coliform mastitis in pigs.

Tryptophan Supplementation Increases Reproduction Performance, Milk Yield, and Milk Composition in Lactating Sows and Growth Performance of Their Piglets.

Tryptophan (Trp) can produce bioactive compounds for appetite regulation, calcium mobilization, and mammary gland homeostasis via a serotonin pathway. This study evaluated the effects of Trp supplementation on the reproduction performance, milk yield, and composition of lactating sows, growth performance of their piglets, and the secretion function of porcine mammary epithelial cells (PMECs). The infrared emulsion analyzer and ELISA analyses revealed that feeding sows with a 0.12% Trp addition increased ( P < 0.05) sow average daily feed intake, milk yield, milk calcium concentration, average daily gain of piglets, fatty acid synthase (FAS) and lactose synthase (LS), β-casein secretion, intracellular Ca2+ level, the expression of calcium binding protein CaM, and the activity of CaMKII. In a cellular experiment of PMECs treated with Trp, ELISA and flow cytometry analyses revealed that the pretreatment of a Trp hydroxylase inhibitor reduced ( P < 0.05) FAS and LS synthesis, the intracellular Ca2+ level, and the activity of CaMKII. In conclusion, Trp supplementation at 0.12% increased sows' reproductive performance, milk yield, and calcium concentration and piglets' growth performance. Milk yield increased by Trp was linked to 5-hydroxytryptamine-mediated synthesis of FAS, LS, and β-casein in PMECs, while the increase in calcium concentration was attributed to increasing CaM expression and CAMKII activity.

Valine increases milk fat synthesis in mammary gland of gilts through stimulating AKT/MTOR/SREBP1 pathway†.

Lactating mammary glands are among the most active lipogenic organs and provide a large percentage of bioactive lipids and calories for infant growth. The branched-chain amino acid (BCAA) valine is known to modulate fatty acids synthesis in adipose tissue; however, its effects on fat metabolism and the underlying mechanisms in mammary glands remain to be determined. Valine supplementation during late pregnancy significantly increased the contents of total milk fat, triglyceride, sphingomyelin, and polyunsaturated fatty acids in the colostrum of gilts. Further study in porcine mammary epithelial cells (PMECs) confirmed that valine upregulated the phosphorylation levels of AKT-activated MTOR and subsequently induced the nuclear accumulation of sterol regulatory element binding protein 1 (SREBP1), thus increasing the expression of proteins related to fatty acids synthesis and intracellular triacylglycerol content. Inhibition of AKT/MTOR signaling or silencing of SREBP1 in PMECs downregulates the expression of proteins related to fatty acids synthesis and intracellular triacylglycerol content. Our findings indicated that valine enhanced milk fat synthesis of colostrum in porcine mammary glands via the AKT/MTOR/SREBP1 signaling pathway.

Regulation of protein synthesis in porcine mammary epithelial cells by L-valine.

This study was conducted to determine the catabolism of L-valine in porcine mammary epithelial cells (PMECs) and its role in stimulating protein synthesis in these cells. PMECs were incubated with 0.05-, 0.10-, 0.25-, 0.5-, and 1.0-mM L-valine at 37oC for 2h. Cell viability and expressions of α-lactalbumin and β-casein were measured after culture with L-valine for 3days. L-[1-14C]valine was used to study valine catabolism, whereas [3H]phenylalanine was employed as a tracer to determine protein synthesis and degradation in PMECs. The abundances of proteins involved in the mTOR signaling pathway and the mRNA levels for the relatedkey genes were determined using the western blot and RT-PCR techniques, respectively. Cell numbers and the synthesis of proteins (including α-lactalbumin and β-casein) were greater (P<0.05) in the presence of 0.5-mM L-valine, compared with 0.05- or 0.1-mM L-valine.L-Valine at 0.5 mM also enhanced (P < 0.05) the production of α-lactalbumin by PMECs, in comparison with 0.25 mM L-valine. Increasing the extracellular concentration of L-valine from 0.05 to 0.5 mM stimulated protein synthesis in a concentration-dependent mannerwithout affecting proteolysis. Although L-valine was actively transaminated in PMECs, its α-ketoacid product (α-ketoisovalerate) at 0.05-0.2mM did not affect protein synthesis or degradation in the cells. Thus, the effect of L-valine on protein synthesis was independent of its metabolism to yield α-ketoisovalerate. At the molecular level, 0.5-mM L-valine increased (P<0.05) the mRNA levels for Ras, ERK1/2, and p70S6K, and the abundances of mTOR, p-4EBP1, total 4EBP1, p-ERK1/2, and total ERK1/2 proteins. These findings establish the critical role of L-valine in enhancing PMEC growth and milk protein synthesis possibly by regulating the mTOR and Ras/ERK signaling pathways. Further studies are warranted to understand how L-valine regulates gene expression and mTOR activation in PMECs.