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      Regulation of amino acid transporters in the mammary gland from late pregnancy to peak lactation in the sow

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          Abstract

          Background

          Milk protein is crucial for milk quality in sows and health of newborn piglets. Plasma amino acids (AA) in sows are important precursors for milk protein synthesis in the mammary gland. In order to study the regulation of AA transported in sow mammary glands and possible underlying mechanisms, we measured the expression of genes coding for milk proteins, AA transporter expressions, and plasma AA concentrations in sows at three different physiological stages (D-17, D1 and D17 of lactation), and then further investigated the regulation of AA transport across the cell membrane by adaptive mechanisms using pig mammary epithelial cells (PMEC) as an in vitro model. PMEC were cultured in DMEM:F12 with 4 amino acid concentrations (0 × AA complex, 1 × AA complex, 5 × AA complex, and 25 × AA complex). Classes of AA complexes evaluated in this study included neutral AAs ( L-Ala + L-Ser +  L-Cys), acidic AAs ( L-Asp, L-Glu) and neutral + basic AAs ( L-Ala + L-Ser +  L-Cys +  L-Lys).

          Results

          Our results indicated that mRNA expression of genes coding for milk protein (αs1-casein, αs2-casein, β-casein and κ-casein) increased significantly with the advance of physiological stage ( P < 0.05), and plasma concentrations of most AAs including threonine, serine, glutamate, alanine, valine, cysteine, methionine, isoleucine and tyrosine were greater at D1 of lactation compared with D-17 and D17 of lactation ( P < 0.05). Additionally, protein and gene expressions of AA transporters including excitatory AA transporter 3 (EAAT3), alanine/serine/cysteine/threonine transporter (ASCT1) and sodium-coupled neutral AA transporter 1 (SNAT2) were greater in lactating sow mammary glands compared with sow mammary glands in late pregnancy ( P < 0.05). The mRNA expressions of SLC38A2, SLC1A1, SLC6A14 increased significantly in the cell mediums supplemented with 5 × and 25 × of AA complexes compared with those cells cultured in DMEM/F12 cell medium ( P < 0.05). The mRNA expressions of SLC38A, SLC1A4, and SLC6A14 also increased in EBSS cell medium compared to DMEM/F12. However, only mRNA expression of SLC38A decreased when AA complex was added into EBSS ( P < 0.05).

          Conclusion

          AA transportation was positively regulated in sow mammary glands with the advance of physiological stage from late pregnancy to peak of lactation and AA transporters in PMECs were adaptively regulated by changed AA concentrations.

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          Most cited references 36

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          A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics.

          Real-time reverse transcription (RT) PCR is currently the most sensitive method for the detection of low-abundance mRNAs. Two relative quantitative methods have been adopted: the standard curve method and the comparative C(T) method. The latter is used when the amplification efficiency of a reference gene is equal to that of the target gene; otherwise the standard curve method is applied. Based on the simulation of kinetic process of real-time PCR, we have developed a new method for quantitation and normalization of gene transcripts. In our method, the amplification efficiency for each individual reaction is calculated from the kinetic curve, and the initial amount of gene transcript is derived and normalized. Simulation demonstrated that our method is more accurate than the comparative C(T) method and would save more time than the relative standard curve method. We have used the new method to quantify gene expression levels of nine two-pore potassium channels. The relative levels of gene expression revealed by our quantitative method were broadly consistent with those estimated by routine RT-PCR, but the results also showed that amplification efficiencies varied from gene to gene and from sample to sample. Our method provides a simple and accurate approach to quantifying gene expression level with the advantages that neither construction of standard curve nor validation experiments are needed.
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            Key stages in mammary gland development. Secretory activation in the mammary gland: it's not just about milk protein synthesis!

            The transition from pregnancy to lactation is a critical event in the survival of the newborn since all the nutrient requirements of the infant are provided by milk. While milk contains numerous components, including proteins, that aid in maintaining the health of the infant, lactose and milk fat represent the critical energy providing elements of milk. Much of the research to date on mammary epithelial differentiation has focused upon expression of milk protein genes, providing a somewhat distorted view of alveolar differentiation and secretory activation. While expression of milk protein genes increases during pregnancy and at secretory activation, the genes whose expression is more tightly regulated at this transition are those that regulate lipid biosynthesis. The sterol regulatory element binding protein (SREBP) family of transcription factors is recognized as regulating fatty acid and cholesterol biosynthesis. We propose that SREBP1 is a critical regulator of secretory activation with regard to lipid biosynthesis, in a manner that responds to diet, and that the serine/threonine protein kinase Akt influences this process, resulting in a highly efficient lipid synthetic organ that is able to support the nutritional needs of the newborn.
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              Amino acid transporters: roles in amino acid sensing and signalling in animal cells.

              Amino acid availability regulates cellular physiology by modulating gene expression and signal transduction pathways. However, although the signalling intermediates between nutrient availability and altered gene expression have become increasingly well documented, how eukaryotic cells sense the presence of either a nutritionally rich or deprived medium is still uncertain. From recent studies it appears that the intracellular amino acid pool size is particularly important in regulating translational effectors, thus, regulated transport of amino acids across the plasma membrane represents a means by which the cellular response to amino acids could be controlled. Furthermore, evidence from studies with transportable amino acid analogues has demonstrated that flux through amino acid transporters may act as an initiator of nutritional signalling. This evidence, coupled with the substrate selectivity and sensitivity to nutrient availability classically associated with amino acid transporters, plus the recent discovery of transporter-associated signalling proteins, demonstrates a potential role for nutrient transporters as initiators of cellular nutrient signalling. Here, we review the evidence supporting the idea that distinct amino acid "receptors" function to detect and transmit certain nutrient stimuli in higher eukaryotes. In particular, we focus on the role that amino acid transporters may play in the sensing of amino acid levels, both directly as initiators of nutrient signalling and indirectly as regulators of external amino acid access to intracellular receptor/signalling mechanisms.
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                Author and article information

                Contributors
                +86-13650886262 , wtguan@scau.edu.cn
                Journal
                J Anim Sci Biotechnol
                J Anim Sci Biotechnol
                Journal of Animal Science and Biotechnology
                BioMed Central (London )
                1674-9782
                2049-1891
                8 April 2018
                8 April 2018
                2018
                : 9
                Affiliations
                [1 ]ISNI 0000 0000 9546 5767, GRID grid.20561.30, Department of Animal Science, , South China Agricultural University, ; Guangzhou, 510642 China
                [2 ]National Engineering Research Center for Breeding Swine Industry, Guangzhou, 510642 China
                [3 ]ISNI 0000 0001 2173 6074, GRID grid.40803.3f, Department of Animal Science, , North Carolina State University, ; Raleigh, NC 27695 USA
                Article
                250
                10.1186/s40104-018-0250-4
                5891945
                5d07cec7-5483-4962-a933-ddc70bc5950b
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31402082
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                Animal science & Zoology

                amino acid transporter, lactation, mammary epithelial cell, milk protein, sow

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