Β-casein Synthesis Research Articles

Examination of methionine stimulation of gene expression in dairy cow mammary epithelial cells using RNA-sequencing.

In this research communication, a cell model with elevated β-CASEIN synthesis was established by stimulating bovine mammary epithelial cells with 0.6 mM methionine, and the genome-wide gene expression profiles of methionine-stimulated cells and untreated cells were investigated by RNA sequencing. A total of 458 differentially expressed genes (DEGs; 219 upregulated and 239 downregulated) were identified between the two groups. Gene Ontology (GO) analysis showed that the two highest-ranked GO terms in 'molecular function' category were 'binding' and 'catalytic activity', suggesting that milk protein synthesis in methionine-stimulated cells requires induction of gene expression to increase metabolic activity. Kyoto Encyclopedia of Genes and Genomes analysis revealed that within the 'environmental information processing' category, the subcategory that is most highly enriched for DEGs was 'signal transduction'. cGMP-PKG, Rap1, calcium, cAMP, PI3K-AKT, MAPK, and JAK-STAT are the pathways with the highest number of DEGs, suggesting that these signaling pathways have potential roles in mediating methionine-induced milk protein synthesis in bovine mammary epithelial cells. This study provides valuable insights into the physiological and metabolic adaptations in cells stimulated with methionine. Understanding the regulation of this transition is essential for effective intervention in the lactation process.

High levels of fatty acids inhibit β-casein synthesis through suppression of the JAK2/STAT5 and mTOR signaling pathways in mammary epithelial cells of cows with clinical ketosis.

Ketosis is a metabolic disease of dairy cows often characterized by high concentrations of ketone bodies and fatty acids, but low milk protein and milk production. The Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) and the mechanistic target of rapamycin (mTOR) signaling pathways are central for the regulation of milk protein synthesis. The effect of high levels of fatty acids on these pathways and β-casein synthesis are unknown in dairy cows with clinical ketosis. Mammary gland tissue and blood samples were collected from healthy (n = 15) and clinically-ketotic (n = 15) cows. In addition, bovine mammary epithelial cells (BMEC) were treated with fatty acids, methionine (Met) or prolactin (PRL), respectively. In vivo, the serum concentration of fatty acids was greater (P > 0.05) and the percentage of milk protein (P > 0.05) was lower in cows with clinical ketosis. The JAK2-STAT5 and mTOR signaling pathways were inhibited and the abundance of β-casein was lower in mammary tissue of cows with clinical ketosis (P > 0.05). In vitro, high levels of fatty acids inhibited the JAK2-STAT5 and mTOR signaling pathways (P > 0.05) and further decreased the β-casein synthesis (P > 0.05) in BMEC. Methionine or PRL treatment, as positive regulators, activated the JAK2-STAT5 and mTOR signaling pathways to increase the β-casein synthesis. Importantly, the high concentration of fatty acids attenuated the positive effect of Met or PRL on mTOR, JAK2-STAT5 pathways and the abundance of β-casein (P > 0.05). Overall, these data indicate that the high concentrations of fatty acids that reach the mammary cells during clinical ketosis inhibit mTOR and JAK2-STAT5 signaling pathways, and further suppress β-casein synthesis.

OGR1 negatively regulates β-casein and triglyceride synthesis and cell proliferation via the PI3K/AKT/mTOR signaling pathway in goat mammary epithelial cells

Goat milk in some cases is less allergenic than cow milk, therefore, more people drink goat milk in the world, so it is necessary for us to improve the yield and quality of goat milk. Previous studies have shown that some genes are closely related to lactation. Ovarian cancer G protein-coupled 1 (OGR1) is a G protein-coupled receptor discovered recently. OGR1 is widely found in various tissues of organisms and is involved in cell skeleton reorganization, carcinogenesis, cell proliferation, and apoptosis by regulating multiple signaling pathways in cells. However, the modulating effect of OGR1 in lactation is still unknown. Therefore, the objective of this study is to investigate the function of OGR1 in goat mammary epithelial cells (GMECs). Flow cytometry, CCK8, EDU, enzyme-linked immunosorbent assay, and triglyceride test kit assays were performed and we found that OGR1 regulated Bcl-2/Bax ratio, Fas protein expression as well as the phosphorylation of AKT and mammalian target of rapamycin (mTOR). si-OGR1 could enhance the proliferation of GMECs by promoting G1/S phase progression and the synthesis of β-casein and triglyceride. By contrast, OGR1 repressed GMECs proliferation and down-regulated the synthesis of β-casein and triglyceride by blocking the PI3K/AKT/mTOR signaling pathway in GMECs.

Laminin-dependent integrin β1 signaling regulates milk protein synthesis via prolactin/STAT5 pathway in bovine mammary epithelial cells

Successful induction of milk protein synthesis relies on prolactin/STAT5. In mice, both laminin and β1 integrin were necessary for STAT5 activity induced by prolactin treatment, resulting in transcriptional activation of β-casein. However, the mechanism by which β1 integrin increases the bovine milk protein synthesis is not well known. In order to display the crosstalk between integrin signaling and lactogenic signaling, we investigated the mechanism by which laminin mediated lactogenic effects via interaction with β1 integrin on bovine mammary epithelial cells (BMECs). Therefore, localization of β1 integrin was examined by immunofluorescence. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting. The results showed that β1 integrin were detected in basal mammary cells and basal membrane surface of adherent BMECs. However, basal distribution of β1 integrin was not sufficient to increase β-casein synthesis in the absence of integrin activation by laminin. A lactogenic hormone cocktail of insulin, hydrocortisone, and prolactin stimulated overall lactogenic effects, including upregulated expression of β1 integrin, activation of prolactin/STAT5 signaling, and consequent increase of β-casein synthesis. In response to a 24 h prolactin treatment, the abundance of STAT5, β1 integrin, and β-casein in BMECs with laminin was higher compared to that with a control substratum. Meanwhile, laminin-dependent lactogenic effects were inhibited by blocking β1 integrin function, resulting in attenuated STAT5 activity and decreased β-casein synthesis. These results indicated that β1 integrin was a key mediator of the laminin-dependent prolactin/STAT5 signaling, which regulated the sustained STAT5 activity necessary for β-casein expression in BMECs.

Seryl-tRNA synthetase is involved in methionine stimulation of β-casein synthesis in bovine mammary epithelial cells.

Despite the well-characterised mechanisms of amino acids (AA) regulation of milk protein synthesis in mammary glands (MG), the underlying specific AA regulatory machinery in bovine MG remains further elucidated. As methionine (Met) is one of the most important essential and limiting AA for dairy cows, it is crucial to expand how Met exerts its regulatory effects on dairy milk protein synthesis. Our previous work detected the potential regulatory role of seryl-tRNA synthetase (SARS) in essential AA (EAA)-stimulated bovine casein synthesis. Here, we investigated whether and how SARS participates in Met stimulation of casein production in bovine mammary epithelial cells (BMEC). With or without RNA interference against SARS, BMEC were treated with the medium in the absence (containing all other EAA and devoid of Met alone)/presence (containing 0·6 mm of Met in the medium devoid of Met alone) of Met. The protein abundance of β-casein and members of the mammalian target of rapamycin (mTOR) and general control nonderepressible 2 (GCN2) pathways was determined by immunoblot assay after 6 h treatment, the cell viability and cell cycle progression were determined by cell counting and propidium iodide-staining assay after 24 h treatment, and protein turnover was determined by l-[ring-3H5]phenylalanine isotope tracing assay after 48 h treatment. In the absence of Met, there was a general reduction in cell viability, total protein synthesis and β-casein production; in contrast, total protein degradation was enhanced. SARS knockdown strengthened these changes. Finally, SARS may work to promote Met-stimulated β-casein synthesis via affecting mTOR and GCN2 routes in BMEC.

ASCT2 Is Involved in SARS-Mediated β-Casein Synthesis of Bovine Mammary Epithelial Cells with Methionine Supply.

The methionine (Met) uptake into mammary cells depends upon the corresponding amino acid (AA) transporters, which play a regulatory role in the mammary protein production beyond transport. Our previous studies have identified that seryl-tRNA synthetase (SARS) could be a novel mediator to regulate essential AA-stimulated casein synthesis in primary bovine mammary epithelial cells (BMECs). However, the regulatory mechanisms of Met in milk protein production in dairy cows remain further clarified. Here, we aimed to investigate the effects of Met on milk protein synthesis in BMECs and explore the underlying mechanism. The effects of Met on the AA transporter, casein synthesis, and the related signaling pathway were evaluated in the BMECs treated with 0.6 mM Met for 6 h combined with or without the inhibition of AA transporter (ASCT2, a neutral AA transporter) activity by the corresponding inhibitor (GPNA). Besides, the effects of SARS on the cells were mainly evaluated in the BMECs treated with 0.6 mM Met for 6 h together with or without SARS knockdown by RNAi interference. The gene expression of AA transporters and pathway-related genes were analyzed by the real-time quantitative polymerase chain reaction method, and the protein expression of related proteins were determined by the western blot assay. Results showed that 0.6 mM Met remarkably enhanced cell growth and β-casein synthesis compared to the supply of other Met concentrations. Among 13 amino acid transporters, 0.6 mM Met highly increased ASCT2 expression. This Met-stimulated ASCT2 expression and the enhanced mammary intracellular Met uptake were both decreased by the addition of 500 μM GPNA, an inhibitor of ASCT2. In the presence of 0.6 mM Met, the inhibition of ASCT2 activity (by GPNA) and SARS expression (by RNAi) both reduced β-casein synthesis. Additionally, 0.6 mM Met increased the gene expression of mTOR, S6K1, 4EBP1, and Akt; in contrast, the inhibition of ASCT2 by GPNA lowered the gene expression of these four genes. Collectively, this work suggests that ASCT2 is involved in the SARS-mediated Met stimulation of β-casein synthesis through enhancing mammary Met uptake and activating the mTOR signaling pathway in BMECs.

CRTC2 Is a Key Mediator of Amino Acid-Induced Milk Fat Synthesis in Mammary Epithelial Cells.

Amino acids can stimulate milk fat synthesis, but the underlying molecular mechanism is still largely unknown. In this study, we studied the regulatory role and corresponding molecular mechanism of cAMP response element-binding protein-regulated transcription coactivator 2 (CRTC2) in amino acid-induced milk fat synthesis in mammary epithelial cells. We showed that leucine and methionine stimulated CRTC2 but not p-CRTC2(Ser171) expression and nuclear localization in cow mammary epithelial cells. Knockdown of CRTC2 decreased milk fat synthesis and sterol regulatory element binding protein 1c (SREBP-1c) expression and activation, whereas its overexpression had the opposite effects. Neither knockdown nor overexpression of CRTC2 affected β-casein synthesis and phosphorylation of the machanistic target of rapamycin (mTOR), suggesting that CRTC2 only regulates milk fat synthesis. CRTC2 knockdown abolished the stimulation of leucine and methionine on SREBP-1c expression and activation. Knockdown or overexpression of CRTC2 did not affect the protein level of cAMP-response element-binding protein (CREB) and its phosphorylation but decreased or increased the binding of p-CREB to the promoter of SREBP-1c gene and its mRNA expression, respectively. Mutation of Ser171 of CRTC2 did not alter the stimulation of CRTC2 on SREBP-1c expression and activation, further suggesting that CRTC2 functions in the nucleus. mTOR inhibition by rapamycin totally blocked the stimulation of leucine and methionine on CRTC2 expression. The expression of CRTC2 was dramatically higher in the mouse mammary gland of lactation period, compared with that of the dry and puberty periods, whereas p-CRTC2(Ser171) was not changed, further supporting that CRTC2 is a key transcription coactivator for milk fat synthesis. These results uncover that CRTC2 is a key transcription coactivator of amino acid-stimulated mTOR-mediated milk fat synthesis in mammary epithelial cells.

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.

Sestrin2 Negatively Regulates Casein Synthesis through the SH3BP4-mTORC1 Pathway in Response to AA Depletion or Supplementation in Cow Mammary Epithelial Cells.

Sestrin2 (SESN2) negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) pathway and casein synthesis in response to amino acid (AA) depletion in cow mammary epithelial cells (CMECs); however, the underlying mechanism is unclear. In the current study, the regulation of SESN2 on AA-mediated β-casein (CSN2) synthesis in CMECs and its mechanism were investigated. Overexpression and silencing of SESN2 demonstrated that SESN2 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway. Co-immunoprecipitation analysis showed that SESN2 interacted with SH3 domain-binding protein 4 (SH3BP4). Overexpression and silencing of SH3BP4 demonstrated that SH3BP4 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway and that SESN2 negatively regulated expression of CSN2 and mTORC1 pathway through the SH3BP4 in the presence and absence of AA. The absence or presence of AA demonstrated that AA negatively regulated expression and nuclear localization of activating transcription factor 4 (ATF4). Overexpression and silencing of ATF4 demonstrated that AA negatively regulated SESN2 expression through ATF4. Together, these results indicate that SESN2 negatively regulates the mTORC1 pathway and subsequent CSN2 synthesis through the SH3BP4 in response to AA absence or presence in CMECs.

U2AF65 enhances milk synthesis and growth of bovine mammary epithelial cells by positively regulating the mTOR-SREBP-1c signalling pathway.

U2 snRNP auxiliary factor 65kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The function of U2AF65 in alternative splicing has been identified; however, the essential physiological role of U2AF65 remains poorly understood. In this study, we investigated the regulatory role of U2AF65 in milk synthesis and growth of bovine mammary epithelial cells (BMECs). Our results showed that U2AF65 localizes in the nucleus. Treatment with amino acids (Met and Leu) and hormones (prolactin and β-estradiol) upregulated the expression of U2AF65 in these cells. U2AF65 overexpression increased the synthesis of β-casein, triglycerides, and lactose; increased cell viability; and promoted proliferation of BMECs. Furthermore, our results showed that U2AF65 positively regulated mTOR phosphorylation and expression of mature mRNA of mTOR and SREBP-1c. Collectively, our findings demonstrate that U2AF65 regulates the mRNA expression of signalling molecules (mTOR and SREBP-1c) involved in milk synthesis and growth of BMECs, possibly via controlling the splicing and maturation of these mRNAs. U2 snRNP auxiliary factor 65kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The essential physiological role of U2AF65 remains poorly understood. In the present study, we confirmed that U2AF65 functions as a positive regulator of milk synthesis in and proliferation of bovine mammary epithelial cells via the mTOR-SREBP-1c signalling pathway. Therefore, our study uncovers the regulatory role of U2AF65 in milk synthesis and cell proliferation.

Seryl-tRNA synthetase-mediated essential amino acids regulate β-casein synthesis via cell proliferation and mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells

Essential amino acids (EAA) play an important role in promoting milk protein synthesis in primary bovine mammary epithelial cells (BMEC). However, the regulatory mechanisms involved in the relationship between EAA and milk protein synthesis have not been fully explored. This study examined the effects of seryl-tRNA synthetase (SARS) on EAA-stimulated β-casein synthesis, cell proliferation, and the mammalian target of rapamycin (mTOR) system in BMEC. First, BMEC were cultured in medium either lacking all EAA (-EAA) or that included all EAA (+EAA) for 12 h. The BMEC were then supplemented with the opposing treatments (-EAA supplemented with +EAA and vice versa) for 0 h, 10 min, 0.5 h, 1 h, 6 h, or 12 h, respectively. After the treatment-specific time allotment, proteins were collected for Western blotting. Subsequently, a 2 × 2 factorial design was used to evaluate the interactive of SARS inhibition (control or SARS inhibited) and EAA supply (+EAA or -EAA) on gene and protein abundance, cell viability, and cell cycle in BMEC. Based on the data obtained in the first experiment, the changes in protein abundance of β-casein and SARS depended on EAA treatment time in similar patterns. The protein abundance of β-casein, SARS, and mammalian target of rapamycin (mTOR)-related proteins, cell viability, cell cycle progression, and the mRNA abundance of cyclin D1 (CCND1, cell cycle progression marker) and marker of proliferation Ki-67 (MKI67, cell proliferation marker) were stimulated by the presence of EAA. Correspondingly, when cells were deprived of EAA, cell proliferation and abundance of these proteins and genes were reduced overall. Moreover, the decreases in these aspects were further exacerbated by inhibiting SARS, suggesting that an interaction between EAA and SARS is important for regulating protein synthesis. The results indicated that SARS stimulated the mTOR signaling pathway when EAA were present, enhanced EAA-stimulated cell proliferation, and contributed to increased β-casein production in BMEC.

Dipeptide (Methionyl-Methionine) Transport and Its Effect on β-Casein Synthesis in Bovine Mammary Epithelial Cells

Background/Aims: The aim of this study was to investigate the transport properties and utilization of methionyl-methionine dipeptide (Met-Met) in β-casein (β-CN) synthesis in bovine mammary epithelial cells (BMECs). Methods: The transport properties were studied for the effects of time, pH, concentration, temperature and inhibitors using Met-Met-FITC in BMECs. BMECs were treated with different concentrations of Met-Met (0, 20, 40, 80, 120 and 160 µg/ml). In several experiments, the cells were treated with Janus kinase 2 (JAK2) inhibitor (tyrphostin AG-490, 50 µM) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin, 100 ng/ml). Results: The uptake of Met-Met-FITC by BMECs was rapid during the first fifteen minutes and became saturated after 15 minutes. The transport of Met-Met-FITC in BMECs exhibited a Michaelis constant of 52.4 µM and maximum transport velocity of 14.8 pmol/min/mg protein. The uptake of Met-Met-FITC in BMECs was pH-dependent, peaked at pH 6.5 and was significantly inhibited by other peptides, including Met-Lys, Lys-Lys, Gly-Met, Gly-Leu and Met-Leu. Knocking down the peptide transporter 2 (PepT2) with small interference RNA markedly decreased Met-Met-FITC uptake. Met-Met concentration-dependently increased the PepT2 expression and β-CN synthesis in BMECs with an optimal concentration of 80 µg/ml. At 80 µg/ml, Met-Met also enhanced the cell viability and cyclin D1 expression and promoted cell cycle transition from G1 phase to S phase. In addition, 80 µg/ml Met-Met increased the mRNA abundance of JAK2 and signal transducer and activator of transcription 5 (STAT5) and enhanced the phosphorylation of JAK2, STAT5, mTOR, p70 ribosomal S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1. The inhibition of JAK2 and mTOR significantly decreased Met-Met-induced increase in cell viability and β-CN synthesis in BMECs. Conclusion: Our data elucidated the properties of peptide transporter and its effect on β-CN synthesis in BMECs. Met-Met, taken up by PepT2, enhances cell proliferation and promotes β-CN synthesis by activating JAK2-STAT5 and mTOR signaling pathways in BMECs.

Kisspeptin-10 Induces β-Casein Synthesis via GPR54 and Its Downstream Signaling Pathways in Bovine Mammary Epithelial Cells.

Kisspeptins (Kps) play a key role in the regulation of GnRH axis and as an anti-metastasis agent by binding with GPR54. Recently, we observed that the expression of GPR54 was higher in the lactating mammary tissues of dairy cows with high-quality milk (0.81 ± 0.13 kg/day of milk protein yield; 1.07 ± 0.18 kg/day of milk fat yield) than in those with low-quality milk (0.51 ± 0.14 kg/day of milk protein yield; 0.67 ± 0.22 kg/day of milk fat yield). We hypothesized that Kp-10 might regulate the milk protein, β-casein (CSN2) synthesis via GPR54 and its downstream signaling. First, we isolated the bovine mammary epithelial cells (bMECs) from lactating Holstein dairy cows, and treated them with different concentrations of Kp-10. Compared with the control cells, the synthesis of CSN2 is significantly increased at a concentration of 100 nM of Kp-10. In addition, the increased effect of CSN2 synthesis was blocked when the cells were pre-treated with the selective inhibitor of GPR54 Peptide-234 (P-234). Mechanistic study revealed that Kp-10 activated ERK1/2, AKT, mTOR and STAT5 in bMECs. Moreover, inhibiting ERK1/2, AKT, mTOR and STAT5 with U0126, MK2206, Rapamycin and AG490 could block the effects of Kp-10. Together, these results demonstrate that Kp-10 facilitates the synthesis of CSN2 via GPR54 and its downstream signaling pathways mTOR, ERK1/2, STAT5 and AKT.

MiR-139 suppresses \u03b2-casein synthesis and proliferation in bovine mammary epithelial cells by targeting the GHR and IGF1R signaling pathways

BackgroundMicroRNAs have important roles in many biological processes. However, the role of miR-139 in healthy mammary gland remains unclear. The objective of this study was to investigate the effects of miR-139 on lactation in dairy cows.ResultsHere, we found that miR-139 was down-regulated in mid-lactation dairy cow mammary tissues compared with mid-pregnancy tissues. Then, we prioritized two of potential target genes of miR-139 in cow, growth hormone receptor (GHR) and type I insulin-like growth factor receptor (IGF1R) for further functional studies based on their roles in lactation processes. Dual luciferase reporter assays validated direct binding of miR-139 to the 3′- untranslated region (UTR) of GHR and IGF1R. Moreover, over-expression or silencing of miR-139 affected mRNA levels of GHR and IGF1R in cultured bovine mammary epithelial cells (BMECs). Furthermore, over-expression of miR-139 decreased protein levels of β-casein, proliferation in mammary epithelial cell, and the protein levels of IGF1R and key members of the GHR or IGF1R pathways as well, whereas silencing miR-139 produced the opposite result. Among these signal molecules, signal transducer and activator of transcription-5 (STAT5), protein kinase B (also known as AKT1), mammalian target of rapamycin (mTOR), and p70-S6 Kinase (p70S6K) are involed in β-casein synthesis, and Cyclin D1 is involved in cell proliferation. In addition, silencing GHR decreased protein levels of β-casein, IGF1R, and key members of the IGF1R pathway, whereas co-silencing miR-139 and GHR rescued the expression of GHR and reversed GHR silencing effects.ConclusionsOur results demonstrate that GHR and IGF1R are target genes of miR-139 in dairy cow. MiR-139 suppresses β-casein synthesis and proliferation in BMECs by targeting the GHR and IGF1R signaling pathways.

Methionine Induces LAT1 Expression in Dairy Cow Mammary Gland by Activating the mTORC1 Signaling Pathway.

Methionine is the limiting amino acid for milk protein synthesis in dairy cows. The effect of methionine availability on milk protein synthesis is dependent on its active transport into cells through amino acid transporters. L-type amino acid transporter 1 (LAT1), which induces the transport of neutral amino acids, is highly expressed in lactating mammary gland. However, the effect of methionine on LAT1 expression and the mechanism governing this process in dairy cow mammary gland are poorly understood. In this study, we show that treatment of dairy cow mammary epithelial cells with increasing concentrations of methionine for 24 h resulted in increased expression of LAT1 and its associated protein 4F2 heavy chain (4F2hc). Maximal expression levels occurred after treatment with 0.6 mM methionine. Methionine treatment also increased cell viability and β-casein synthesis. Western blots showed that methionine induced LAT1 and 4F2hc expression by activating mammalian target of rapamycin complex 1 (mTORC1) signaling. Inhibition of mTORC1 signaling by rapamycin or raptor siRNA prevented the upregulation of LAT1 and 4F2hc. These results indicate that methionine may activate the mTORC1 signaling pathway and further increase LAT1 and 4F2hc expression in dairy cow mammary gland, thus affecting milk protein synthesis.

Milk protein synthesis is regulated by T1R1/T1R3, a G protein-coupled taste receptor, through the mTOR pathway in the mouse mammary gland.

Understanding the regulatory mechanism of milk protein synthesis is important to develop strategies to improve milk protein and enhance lactation performance. The mammalian target of rapamycin (mTOR) pathway is a crucial modulator of protein synthesis. In this study, we want to investigate if T1R1/T1R3 can regulate milk protein synthesis and mediate the mTOR pathway in the mice mammary gland in vivo. T1R1 knockout mice, WT mice, and mammary explants were used. The weigh-suckle-weigh method was used to quantify the milk yield. The expression level of β-casein and AA transporter mRNA were analyzed by qPCR. Western blot was used to analyze protein abundance of members of the mTOR pathway. As expected, the knockout of T1R1 not only reduced the total milk yield in the mice mammary glands, but also repressed β-casein synthesis. Additionally, the phosphorylation of 4EBP1 and S6K was significantly decreased in T1R1 knockout mice. The T1R1 knockout also increased the protein abundance of the AA transporter SLC3A2 and mRNA expression of SLC7A5/SLC3A2 and SLC1A5. Activation of the mTOR pathway was repressed by inhibition of T1R3 or T1R1 knockout in mammary gland explants. T1R1/T1R3 modulates the mTOR pathway to regulate milk protein synthesis in the mouse mammary gland in vivo.

Combination of histidine, lysine, methionine, and leucine promotes β-casein synthesis via the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells.

The ratio of different AA in the diets of cows is vital to improve milk protein yield. β-Casein is one of the important milk proteins with high nutritive value. However, the suitable ratio of essential amino acids (EAA) for the expression of β-casein in the immortalized bovine mammary epithelial cell line is not fully characterized. This study employed response surface methodology to determine the optimal ratio of His, Lys, Met, and Leu on β-casein expression level in vitro and clarified the effect of the 4 EAA on β-casein via the mechanistic target of rapamycin (mTOR) signaling pathway. A central composite design containing 5 axial points per EAA and 28 combinations of the 4 EAA was used in our study. The results of response surface methodology and the changes of the mTOR-related signaling proteins were determined by western blot. The results showed that β-casein level was significantly affected by all 4 EAA (R2 = 0.71). The optimum conditions for β-casein expression are found to be 5.47 mM of His, 7.48 mM of Lys, 1.17 mM of Met, and 8.21 mM of Leu (His:Lys:Met:Leu = 5:6:1:7) in the designed scope of concentration. The interaction of Leu and Met significantly affected β-casein expression (P < 0.01). The phosphorylation of mTOR (Ser2481), regulatory associated protein of target of rapamycin (Ser792), ribosomal protein S6 kinase 1 (Thr389), ribosomal protein S6 (Ser235/236), and eukaryotic elongation factor 2 (Thr56) was increased with the supplementation of either single EAA or an optimal combination of EAA. However, the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (Thr37) was decreased with the addition of Lys, Met, or Leu alone. Furthermore, the phosphorylation (P) of eIF2α (Ser51) was decreased when Met was supplemented alone. Under the optimal mixture of 4 EAA, the phosphorylation of mechanistic target of rapamycin complex 1 signaling proteins was significantly greater than the single EAA supplementations and the expression of β-casein was 98% as high as the positive control (i.e., medium with all AA). A similar trend was found with P-ribosomal protein S6 kinase 1 and P-ribosomal protein S6. In conclusion, the extracellular concentrations of His, Lys, Met, and Leu at a ratio of 5:6:1:7 maximized β-casein expression in the immortalized bovine mammary epithelial cell line may occur via activation of the mechanistic target of rapamycin complex 1 signaling pathway.

Optimal ratios of essential amino acids stimulate β-casein synthesis via activation of the mammalian target of rapamycin signaling pathway in MAC-T cells and bovine mammary tissue explants.

Amino acids are the building blocks of proteins and serve as key molecular components upstream of the signaling pathways that regulate protein synthesis. The objective of this study was to systematically investigate the effect of essential AA ratios on milk protein synthesis in vitro and to elucidate some of the underlying mechanisms. Triplicate cultures of MAC-T cells and bovine mammary tissue explants (MTE) were incubated with the optimal AA ratio (OPAA; Lys:Met, 2.9:1; Thr:Phe, 1.05:1; Lys:Thr, 1.8:1; Lys:His, 2.38:1; and Lys:Val, 1.23:1) in the presence of rapamycin (control), OPAA, a Lys:Thr ratio of 2.1:1, a Lys:Thr ratio of 1.3:1, a Lys:His ratio of 3.05:1, or a Lys:Val ratio of 1.62:1 for 12 h; the other AA concentrations were equal to OPAA. In some experiments, the cells were cultured with OPAA with or without rapamycin (100 ng/mL) or with mammalian target of rapamycin (mTOR) small interference RNA, and the MTE were exposed to OPAA with rapamycin for β-casein expression. Among the treatments, the expression of β-casein was greatest in the MTE cultured with OPAA. In MAC-T cells, the OPAA upregulated the mRNA expression of SLC1A5 and SLC7A5 but downregulated the expression of IRS1, AKT3, EEF1A1, and EEF2 compared with the control. The OPAA had no effect on the mTOR phosphorylation status but increased the phosphorylation of S6K1 and RPS6. When the MTE were treated with rapamycin in the presence of OPAA, the expression of β-casein was markedly decreased. The phosphorylation of RPS6 and 4EBP1 also was reduced in MAC-T cells. A similar negative effect on the expression of RPS6KB1 and EIF4EBP1 was detected when the cells were cultured with either rapamycin or mTOR small interference RNA. The optimal AA ratio stimulated β-casein expression partly by enhancing the transport of AA into the cells, cross-talk with insulin signaling and a subsequent enhancement of mTOR signaling, or translation elongation in both MAC-T cells and bovine MTE.

0861 Optimal combination of histidine, lysine, methionine, and leucine affect β-casein synthesis via mTOR signaling pathway in bovine mammary epithelial cells

0861 Optimal combination of histidine, lysine, methionine, and leucine affect β-casein synthesis via mTOR signaling pathway in bovine mammary epithelial cells

Perinatal exposure to bisphenol A modifies the transcriptional regulation of the β-Casein gene during secretory activation of the rat mammary gland

With the aim to analyze whether bisphenol A (BPA) modifies β-Casein (β-Cas) synthesis and transcriptional regulation in perinatally exposed animals, here, pregnant F0 rats were orally exposed to 0, 0.6 or 52 μg BPA/kg/day from gestation day 9 until weaning. Then, F1 females were bred and mammary glands were obtained on lactation day 2. Perinatal BPA exposure decreased β-Cas expression without modifying the activation of prolactin receptor. It also decreased the expression of glucocorticoid receptor in BPA52-exposed dams and β1 and α6 integrins as well as dystroglycan in both BPA groups. In addition, BPA exposure altered the expression of histone-modifying enzymes and induced histone modifications and DNA methylation in the promoter, enhancer and exon VII of the β-Cas gene. An impaired crosstalk between the extracellular matrix and lactogenic hormone signaling pathways and epigenetic modifications of the β-Cas gene could be the molecular mechanisms by which BPA decreased β-Cas expression.