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Liriodendrin is a lignan compound that is involved in a wide variety of physiological functions, however it is unknown whether liriodendrin plays an important role in milk production in the mammary glands. In this study, we explored the role and molecular mechanism of Liriodendrin in milk synthesis of mammary epithelial cells (MECs). Bovine MECs were treated with liriodendrin (0, 0.45, 0.9, 1.35, 1.8, and 2.25 mM) for 24 h. Liriodendrin dose-dependently increased cell number, cell cycle transition, and milk protein synthesis, as well as Cyclin D1 and mTOR phosphorylation, with the maximal effects observed at a dose of 1.35 mM. Liriodendrin increased the expression of DDX18, which mediated liriodendrin stimulation of Cyclin D1 and mTOR mRNA expression. PI3K inhibition and DDX18 knockdown experiments further confirmed that liriodendrin regulates the mRNA expression of Cyclin D1 and mTOR via the PI3K-DDX18 signaling. Mouse feeding experiment showed that liriodendrin dose-dependently promotes β-casein and DDX18 expression in mouse mammary gland. In this study, DDX18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein. These findings reveal that liriodendrin stimulates proliferation and milk protein synthesis of MECs via the PI3K-DDX18 signaling. [ABSTRACT FROM AUTHOR]

Objective Our objective was to examine the relationships of supplemental rumen-protected lysine (RPL) or lysine + methionine (RPLM) on lactational performance, plasma amino acids (AA) concentration, and nitrogen use efficiency of lactating dairy cows by using a meta-analysis approach. Methods A total of 56 articles comprising 77 experiments with either RPL or RPLM supplementation were selected and analyzed using a mixed model methodology by considering the treatments and other potential covariates as fixed effects and different experiments as random effects. Results In early lactating cows, milk yield was linearly increased by RPL (β1 = 0.013; p< 0.001) and RPLM (β1 = 0.014; p

The G protein-coupled receptors (GPCR) sensing nutritional signals (amino acids, fatty acids, glucose, etc.) are not fully understood. In this research, we used transcriptome sequencing to analyse differentially expressed genes (DEG) in mouse mammary gland tissues at puberty, lactation and involution stages, in which eight GPCR were selected out and verified by qRT-PCR assay. It was further identified the role of GPR110-mediating nutrients including palmitic acid (PA) and methionine (Met) to improve milk synthesis using mouse mammary epithelial cell line HC11. PA but not Met affected GPR110 expression in a dose-dependent manner. GPR110 knockdown decreased milk protein and fat synthesis and cell proliferation and blocked the stimulation of PA on mechanistic target of rapamycin (mTOR) phosphorylation and sterol-regulatory element binding protein 1c (SREBP-1c) expression. In summary, these experimental results disclose DEG related to lactation and reveal that GPR110 mediates PA to activate the mTOR and SREBP-1c pathways to promote milk protein and fat synthesis. [ABSTRACT FROM AUTHOR]

Objective: Our objective was to examine the relationships of supplemental rumen-protected lysine (RPL) or lysine + methionine (RPLM) on lactational performance, plasma amino acids (AA) concentration, and nitrogen use efficiency of lactating dairy cows by using a meta-analysis approach. Methods: A total of 56 articles comprising 77 experiments with either RPL or RPLM supplementation were selected and analyzed using a mixed model methodology by considering the treatments and other potential covariates as fixed effects and different experiments as random effects. Results: In early lactating cows, milk yield was linearly increased by RPL (ß1 = 0.013; p< 0.001) and RPLM (ß1 = 0.014; p<0.028) but 3.5% fat-corrected milk (FCM) and energy-corrected milk (ECM) (kg/d) was increased by only RPL. RPL and RPLM did not affect dry matter intake (DMI) but positively increased (p<0.05) dairy efficiency (Milk yield/DMI and ECM/DMI). As a percentage, milk fat, protein, and lactose were unchanged by RPL or RPLM but the yield of all components was increased (p<0.05) by feeding RPL while only milk protein was increased by feeding RPLM. Plasma Lys concentration was linearly increased (p<0.05) with increasing supplemental RPL while plasma Met increased (p<0.05) by RPLM supplementation. The increase in plasma Lys had a strong linear relationship (R2 = 0.693 in the RPL dataset and R2 = 0.769 in the RPLM dataset) on milk protein synthesis (g/d) during early lactation. Nitrogen metabolism parameters were not affected by feeding RPL or RPLM, either top-dress or when supplemented to deficient diets. Lactation performance did not differ between AA-deficient or AA-adequate diets in response to RPL or RPLM supplementation. Conclusion: RPL or RPLM showed a positive linear relationship on the lactational performance of dairy cows whereas greater improvement effects were observed during early lactation. Supplementing RPL or RPLM is recommended on deficient-AA diet but not on adequate-AA diet. [ABSTRACT FROM AUTHOR]

This study investigated the role of the mammalian target of rapamycin complex 2 (mTORC2)‐protein kinase B (AKT) signalling in methionine (Met)‐induced L‐type amino acid transporter 1 (LAT1) expression and milk protein production. Primary mammary epithelial cells (MECs) from mammary parenchymal tissues of three lactating cows and MAC‐T bovine MECs were cultured with or without 0.6 mM Met. Rapamycin‐insensitive companion of mTOR (RICTOR) siRNA, the mTORC1 inhibitor rapamycin and the AKT activator SC79 were used to evaluate the effects of mTORC2‐AKT signalling on Met‐induced LAT1 expression and function. Each experiment was performed three times. Data were analysed with a two‐sided unpaired t test or ANOVA with the Bonferroni multiple‐comparison test. Western blotting showed that Met stimulation increased RICTOR expression (~244.67%; p < 0.05; control, 0.15 ± 0.026; Met, 0.517 ± 0.109) and AKT‐S473 levels (~281.42%; p < 0.01; control, 0.253 ± 0.067; Met, 0.965 ± 0.019) in both primary MECs and MAC‐T cells. Rapamycin‐induced mTORC1 signalling inhibition decreased only Met‐induced β‐CASEIN expression by ~21.24% (p < 0.01; Met, 0.777 ± 0.01; Met and rapamycin, 0.612 ± 0.04) and did not affect Met‐stimulated AKT‐S473 levels, suggesting that mTORC2‐AKT activation upon Met stimulation also contributes to milk protein synthesis. LAT1 participates in Met‐induced β‐CASEIN expression. In dairy cow MECs, mTORC2 inhibition by RICTOR siRNA decreased LAT1 levels on the plasma membrane by ~45.13% (p < 0.01; control, 0.359 ± 0.006; siRICTOR, 0.197 ± 0.004). However, SC79‐induced AKT activation had the opposite effect (p < 0.01). In primary MECs and MAC‐T cells, Met stimulation increased cytosolic and plasma membrane LAT1 expression respectively (MECs, 113.98% and 58.43%; MAC‐T, 165.85% and 396.39%; p < 0.05). However, RICTOR siRNA significantly reduced Met‐induced plasma membrane LAT1 expression (~76.48%; Met, 0.539 ± 0.05; Met and siRICTOR, 0.127 ± 0.012; p < 0.05). Thus, Met increased LAT1 expression and function via mTORC2‐AKT signalling, upregulating milk protein synthesis in dairy cow MECs. [ABSTRACT FROM AUTHOR]

The objective of the current experiment was to elucidate the effect of peptides enzymatic hydrolysis from cottonseed protein on rumen microbial population, and hence on milking performance of lactating dairy cows. A total of 50 Holstein cows were assigned into two groups, fed diets with or without enzyme hydrolyzed cottonseed protein (ECP) supplementation. The supplemented group received 0.4% ECP in the diet. Results of rumen microbial population showed that ECP supplementation had no effect on the overall microbe diversity but an increasing trend in the population of Succiniclasticum spp. was observed with ECP supplementation (P>0.05). The results of milk production and composition showed higher lactose and milk yields in ECP supplemented diet as compared to the control group (P<0.05). However, no significant difference was observed in milk protein, milk fat, and milk urea nitrogen between the two groups (P>0.05). Dry matter, neutral detergent fiber and acid detergent fiber digestibility was also observed to be higher in the ECP group (P<0.05), while CP digestibility was not affected by ECP supplementation (P>0.05). Furthermore, an increased blood total protein and glucose and a decreased blood urea nitrogen were observed in the ECP group (P<0.05). In conclusion, it is suggested that the ECP is a rich source of peptides that can be used as a feed supplement for rumen microbial proliferation, improvement of rumen fermentation, and increase in milk yield. [ABSTRACT FROM AUTHOR]

Udder traits, influencing udder health and function, are positively correlated with lactation performance. Among them, breast texture influences heritability and impacts on the milk yield of cattle; however, there is a lack of systematic research on its underlying mechanism in dairy goats in particular. Here, we showed the structure of firm udders with developed connective tissue and smaller acini per lobule during lactation and confirmed that there were lower serum levels of estradiol (E2) and progesterone (PROG), and higher mammary expression of estrogen nuclear receptor (ER) α and progesterone receptor (PR), in dairy goats with firm udders. The results of transcriptome sequencing of the mammary gland revealed that the downstream pathway of PR, the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) signal, participated in the formation of firm mammary glands. During the culture of goat mammary epithelial cells (GMECs), high RANKL level additions promote the Inhibitor kappaB (IκB)/p65/Cyclin D1 expression related to cell proliferation and decrease the phosphorylated signal transduction and transcription activator 5 (Stat5) expression related to milk-protein synthesis of GMECs, which is consistent with electron microscope results showing that there are fewer lactoprotein particles in the acinar cavity of a firm mammary. Furthermore, co-culturing with adipocyte-like cells for 7 d is beneficial for the acinar structure formation of GMECs, while there is a slightly negative effect of high RANKL level on it. In conclusion, the results of this study revealed the structure of firm udders structure and confirmed the serum hormone levels and their receptor expression in the mammary glands of dairy goats with firm udders. The underlying mechanism leading to firm udders and a decrease in milk yield were explored preliminarily, which provided an important foundation for the prevention and amelioration of firm udders and improving udder health and milk yield. [ABSTRACT FROM AUTHOR]

Milk protein content is a key quality indicator of milk, and therefore elucidating its synthesis mechanism has been the focus of research in recent years. Suppressor of cytokine signaling 1 (SOCS1) is an important inhibitor of cytokine signaling pathways that can inhibit milk protein synthesis in mice. However, it remains elusive whether SOCS1 plays roles in the milk protein synthesis in the buffalo mammary gland. In this study, we found that the mRNA and protein expression levels of SOCS1 in buffalo mammary tissue during the dry-off period was significantly lower than those during lactation. Overexpression and knockdown experiments of SOCS1 showed that it influenced the expression and phosphorylation of multiple key factors in the mTOR and JAK2–STAT5 signaling pathways in buffalo mammary epithelial cells (BuMECs). Consistently, intracellular milk protein content was significantly decreased in cells with SOCS1 overexpression, while it increased significantly in the cells with SOCS1 knockdown. The CCAAT/enhancer binding protein α (CEBPA) could enhance the mRNA and protein expression of SOCS1 and its promoter activity in BuMECs, but this effect was eliminated when CEBPA and NF-κB binding sites were deleted. Therefore, CEBPA was determined to promote SOCS1 transcription via the CEBPA and NF-κB binding sites located in the SOCS1 promoter. Our data indicate that buffalo SOCS1 plays a significant role in affecting milk protein synthesis through the mTOR and JAK2-STAT5 signaling pathways, and its expression is directly regulated by CEBPA. These results improve our understanding of the regulation mechanism of buffalo milk protein synthesis. [ABSTRACT FROM AUTHOR]

The current study was planned to evaluate the effects of heat stress (HS) on mammary blood flow and the profile of circulating amino acids (AA) in blood and milk of lactating Holstein dairy cows. For this purpose, twenty dairy cows with similar body conditions, parity, and milk production were divided into two treatments of 10 animals each. The first treatment group was exposed to HS and reared in summer season, and second treatment group were reared in thermoneutral environment (TN) during spring season. Results showed that HS resulted in predominantly high level of total AA (TAA), glycine, serine (Ser), alanine (P<0.05) while predominant decrease concentrations of methionine (Met) and tryptophan (Trp) (P<0.05) in external pudic artery and mammary vein of experimental cows. Methionine, arginine (Arg), Trp and threonine (Thr) (P<0.05) supply to the mammary gland by pudic artery and lower amount of Met and Trp in mammary vain output (P<0.05) was observed in HS cows. Similarly, the uptake of most AA in the udder was decreased due to HS (P<0.05) and resulted in a lower (P<0.05) amount of TAA, glutamine, phenylalanine, Met, lysine, leucine, tyrosine, isoleucine, Ser, Thr, valine, and histidine in milk. A highly significant decrease (P<0.01) in amount of Arg and Trp in milk was observed in HS cows. It was concluded that during HS blood AA use in dairy animals is shifted away from milk protein synthesis by lower mammary blood flow and AA uptake by mammary gland. [ABSTRACT FROM AUTHOR]

Our previous studies found that bta-miR-106b and its corresponding target gene, CDKN1A, were differentially expressed between the mammary epithelium of lactating Holstein cows with extremely high and low milk protein and fat percentage, implying the potential role of bta-miR-106b in milk composition synthesis. In this study, with luciferase assay experiment, bta-miR-106b was validated to target the 3′-untranslated region (UTR) of bovine CDKN1A, thereby regulating its expression. Moreover, in bovine mammary epithelial cells (BMECs), over-expression of bta-miR-106b significantly down-regulated the CDKN1A expression at both mRNA and protein levels, and inhibitors of bta-miR-106b increased CDKN1A expression. Of note, we observed that bta-miR-106b accelerated cell proliferation and cell cycle, and changed the expressions of protein synthesis related pathways such as JAK-STAT and PI3K/AKT/mTOR through regulating CDKN1A expression. Our findings highlight the important regulatory role of bta-miR-106b in milk protein synthesis by targeting CDKN1A in dairy cattle. [ABSTRACT FROM AUTHOR]

The reduction of milk yield caused by heat stress in summer is the main condition restricting the economic benefits of dairy farms. To examine the impact of hyperthermia on bovine mammary epithelial (MAC-T) cells, we incubated the MAC-T cells at thermal-neutral (37°C, CON group) and hyperthermic (42°C, HS group) temperatures for 6 h. Subsequently, the cell viability and apoptotic rate of MAC-T cells, apoptosis-related genes expression, casein and amino acid transporter genes, and the expression of the apoptosis-related proteins were examined. Compared with the CON group, hyperthermia significantly decreased the cell viability (p < 0.05) and elevated the apoptotic rate (p < 0.05) of MAC-T cells. Moreover, the expression of heat shock protein (HSP)70, HSP90B1, Bcl-2-associated X protein (BAX), Caspase-9, and Caspase-3 genes was upregulated (p < 0.05). The expression of HSP70 and BAX (pro-apoptotic) proteins was upregulated (p < 0.05) while that of B-cell lymphoma (BCL)2 (antiapoptotic) protein was downregulated (p < 0.05) by hyperthermia. Decreased mRNA expression of mechanistic target of rapamycin (mTOR) signaling pathway-related genes, amino acid transporter genes (SLC7A5, SLC38A3, SLC38A2, and SLC38A9), and casein genes (CSNS1, CSN2, and CSN3) was found in the heat stress (HS) group (p < 0.05) in contrast with the CON group. These findings illustrated that hyperthermia promoted cell apoptosis and reduced the transport of amino acids into cells, which inhibited the milk proteins synthesis in MAC-T cells.

Udder traits, influencing udder health and function, are positively correlated with lactation performance. Among them, breast texture influences heritability and impacts on the milk yield of cattle; however, there is a lack of systematic research on its underlying mechanism in dairy goats in particular. Here, we showed the structure of firm udders with developed connective tissue and smaller acini per lobule during lactation and confirmed that there were lower serum levels of estradiol (E2) and progesterone (PROG), and higher mammary expression of estrogen nuclear receptor (ER) α and progesterone receptor (PR), in dairy goats with firm udders. The results of transcriptome sequencing of the mammary gland revealed that the downstream pathway of PR, the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) signal, participated in the formation of firm mammary glands. During the culture of goat mammary epithelial cells (GMECs), high RANKL level additions promote the Inhibitor kappaB (IκB)/p65/Cyclin D1 expression related to cell proliferation and decrease the phosphorylated signal transduction and transcription activator 5 (Stat5) expression related to milk-protein synthesis of GMECs, which is consistent with electron microscope results showing that there are fewer lactoprotein particles in the acinar cavity of a firm mammary. Furthermore, co-culturing with adipocyte-like cells for 7 d is beneficial for the acinar structure formation of GMECs, while there is a slightly negative effect of high RANKL level on it. In conclusion, the results of this study revealed the structure of firm udders structure and confirmed the serum hormone levels and their receptor expression in the mammary glands of dairy goats with firm udders. The underlying mechanism leading to firm udders and a decrease in milk yield were explored preliminarily, which provided an important foundation for the prevention and amelioration of firm udders and improving udder health and milk yield.

Studies on promoting milk protein yield by supplementation of amino acids have been globally conducted. Nevertheless, there is a lack of knowledge of what pathways affected by individual amino acid in mammary epithelial cells that produce milk in practice. Phenylalanine (PHE) and valine (VAL) are essential amino acids for dairy cows, however, researches on mammary cell levels are still lacking. Thus, the aim of this study was conducted to evaluate the effects of PHE and VAL on milk protein synthesis-related and energy-mediated cellular signaling in vitro using immortalized bovine mammary epithelial (MAC-T) cells. To investigate the effects of PHE and VAL, the following concentrations were added to treatment medium: 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. The addition of PHE or VAL did not adversely affect cell viability compared to control group. The concentrations of cultured medium reached its maximum at 0.9 mM PHE and 0.6 mM VAL (p < 0.05). Therefore, aforementioned 2 treatments were analyzed for proteomics. Glucose transporter 1 and mammalian target of rapamycin mRNA expression levels were up-regulated by PHE (166% and 138%, respectively) (p < 0.05). Meanwhile, sodium-dependent neutral amino acids transporter type 2 (ASCT2) and β-casein were up-regulated by VAL (173% in ASCT2, 238% in and 218% in β-casein) (p < 0.05). A total of 134, 142, and 133 proteins were detected in control group, PHE treated group, and VAL treated group, respectively. Among significantly fold-changed proteins, proteins involved in translation initiation or energy metabolism were detected, however, expressed differentially between PHE and VAL. Thus, pathway analysis showed different stimulatory effects on energy metabolism and transcriptional pathways. Collectively, these results showed different stimulatory effects of PHE and VAL on protein synthesis-related and energy-mediated cellular signaling in MAC-T cells.

Milk protein content is a key quality indicator of milk, and therefore elucidating its synthesis mechanism has been the focus of research in recent years. Suppressor of cytokine signaling 1 (SOCS1) is an important inhibitor of cytokine signaling pathways that can inhibit milk protein synthesis in mice. However, it remains elusive whether SOCS1 plays roles in the milk protein synthesis in the buffalo mammary gland. In this study, we found that the mRNA and protein expression levels of SOCS1 in buffalo mammary tissue during the dry-off period was significantly lower than those during lactation. Overexpression and knockdown experiments of SOCS1 showed that it influenced the expression and phosphorylation of multiple key factors in the mTOR and JAK2–STAT5 signaling pathways in buffalo mammary epithelial cells (BuMECs). Consistently, intracellular milk protein content was significantly decreased in cells with SOCS1 overexpression, while it increased significantly in the cells with SOCS1 knockdown. The CCAAT/enhancer binding protein α (CEBPA) could enhance the mRNA and protein expression of SOCS1 and its promoter activity in BuMECs, but this effect was eliminated when CEBPA and NF-κB binding sites were deleted. Therefore, CEBPA was determined to promote SOCS1 transcription via the CEBPA and NF-κB binding sites located in the SOCS1 promoter. Our data indicate that buffalo SOCS1 plays a significant role in affecting milk protein synthesis through the mTOR and JAK2-STAT5 signaling pathways, and its expression is directly regulated by CEBPA. These results improve our understanding of the regulation mechanism of buffalo milk protein synthesis.

Staphylococcus aureus (S. aureus) is one of the main pathogens in cow mastitis, colonizing mammary tissues and being internalized into mammary epithelial cells, causing intracellular infection in the udder. Milk that is produced by cows that suffer from mastitis due to S. aureus is associated with decreased production and changes in protein composition. However, there is limited information on how mastitis-inducing bacteria affect raw milk, particularly with regard to protein content and protein composition. The main purpose of this work was to examine how S. aureus infection affects milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were infected with S. aureus, and milk protein and amino acid levels were determined by ELISA after S. aureus invasion. The activity of mTORC1 signaling and the transcription factors NF-κB and STAT5 and the expression of the amino acid transporters SLC1A3 and SLC7A5 were measured by western blot or immunofluorescence and RT-qPCR. S. aureus was internalized by BMECs in vitro, and the internalized bacteria underwent intracellular proliferation. Eight hours after S. aureus invasion, milk proteins were downregulated, and the level of BMECs that absorbed Glu, Asp, and Leu from the culture medium and the exogenous amino acids induced β-casein synthesis declined. Further, the activity of mTORC1 signaling, NF-κB, and STAT5 was impaired, and SLC1A3 and SLC7A5 were downregulated. Eight hours of treatment with 100 nM rapamycin inhibited NF-κB and STAT5 activity, SLC1A3 and SLC7A5 expression, and milk protein synthesis in BMECs. Thus mTORC1 regulates the expression of SLC1A3 and SLC7A5 through NF-κB and STAT5. These findings constitute a model by which S. aureus infection suppresses milk protein synthesis by decreasing amino acids uptake in BMECs.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The prolactin/STAT5 and AKT1/mTOR pathways play a key role in milk protein transcription and translation, respectively. However, the correlation between them in bovine mammary epithelial cells remains unclear. Here, mRNA and protein expression levels of AKT1, STAT5, and mTOR and the phosphorylation of these proteins were determined. Cell proliferation and viability were examined using the CASY-TT assay. Purified bovine mammary epithelial cells were cultured in differentiation media for different periods. The basic differentiation medium contained a lactogenic hormone cocktail of insulin (5 μg/mL), hydrocortisone (1 μg/mL), and prolactin (5 μg/mL). The cells cultured in this medium grew slowly and expressed higher levels of p-STAT5, p-AKT1, and p-mTOR (activated form) than those of control cells. Although the phosphorylation ratio was not increased, transcription and translation of these proteins were upregulated by the addition of insulin-like growth factor-1 or growth hormone, which further increased β-casein mRNA expression. Furthermore, the three proteins were upregulated or downregulated synchronously, even after RNA interference silencing of either Stat5 or Akt1. These findings indicate that a few hormones and other factors play lactogenic and galactogenic roles by promoting two key lactogenic signaling associated with milk protein expression. We provide evidence of prolactin/STAT5 and AKT1/mTOR synchronization, establishing a direct correlation between transcription regulation and translation regulation of milk protein in bovine mammary epithelial cells. [ABSTRACT FROM AUTHOR]

Heat stress (HS) is one of the most serious factors to negatively affect the lactation performance of dairy cows. Bovine mammary epithelial cells are important for lactation. It was demonstrated that HS decreases the lactation performance of dairy cows, partly through altering gene expression within bovine mammary epithelial tissue. However, the cellular metabolism mechanisms under HS remains largely unknown. The objective of this study was to determine whether HS induced changes in intracellular metabolites and gene transcription related to amino acid metabolism, amino acid transportation and the mTOR signaling pathway. Immortalized bovine mammary epithelial cell lines (MAC-T cells, n = 5 replicates/treatment) were incubated for 12 h at 37 °C (Control group) and 42 °C (HS group). Relative to the control group, HS led to a greater mRNA expression of heat shock protein genes HSF1, HSPB8, HSPA5, HSP90AB1 and HSPA1A. Compared with the control group, metabolomics using liquid chromatography tandem–mass spectrometry identified 417 differential metabolites with p < 0.05 and a variable importance in projection (VIP) score >1.0 in the HS group. HS resulted in significant changes to the intracellular amino acid metabolism of glutathione, phenylalanine, tyrosine, tryptophan, valine, leucine, isoleucine, arginine, proline, cysteine, methionine, alanine, aspartate and glutamate. HS led to a greater mRNA expression of the amino acid transporter genes SLC43A1, SLC38A9, SLC36A1, and SLC3A2 but a lower mRNA expression of SLC7A5 and SLC38A2. Additionally, HS influenced the expression of genes associated with the mTOR signaling pathway and significantly upregulated the mRNA expression of mTOR, AKT, RHEB, eIF4E and eEF2K but decreased the mRNA expression of TSC1, TSC2 and eEF2 relative to the control group. Compared with the control group, HS also led to greater mRNA expression of the CSN1S2 gene. Overall, our study indicates that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.

Postpartum dysgalactiae syndrome (PPDS) is one of the key issues affecting breastfeeding, usually occurring as breast swelling, a low milk yield, and at length a stop of breast milk secretion. Therefore, there is a need to investigate the effectiveness of Traditional Chinese Medicine (TCM) diet therapy in treating or preventing PPDS. This study aims to analyze the effect of soybean isoflavone (SIF), a natural estrogen found in plants, on postpartum lactation performance in mice and to evaluate its potential as a treatment for PPDS. Adult female BALB/c mice at 8 weeks of age (25 ± 3 g) are randomly divided into four groups fed with different levels of SIF and a normal diet for 14 days. SIF (0, 50, 100, 200 mg kg -1 BW) is provided via intra-gastric route to the experimental mice. Using a high-throughput sequencing of microbial diversity and mammary gland metabolites, it is found that SIF-treated mice potentially show an improved milk performance via enhanced antioxidant capacity and altered gut microbiota. SIF from plant sources at a high dosage promotes the lactation in normal postpartum mice., (© 2024 Wiley-VCH GmbH.)

Heat stress (HS) is one of the most important factors posing harm to the economic wellbeing of dairy industries, as it reduces milk yield as well as milk protein content. Recent studies suggest that HS participates in the induction of tissue oxidative stress (OS), as elevated levels of reactive oxygen species (ROS) and mitochondrial dysfunction were observed in dairy cows exposed to hot conditions. The OS induced by HS likely contributes to the reduction in milk protein content, since insulin resistance and apoptosis are promoted by OS and are negatively associated with the synthesis of milk proteins. The apoptosis in the mammary gland directly decreases the amount of mammary epithelial cells, while the insulin resistance affects the regulation of insulin on mTOR pathways. To alleviate OS damages, strategies including antioxidants supplementation have been adopted, but caution needs to be applied as an inappropriate supplement with antioxidants can be harmful. Furthermore, the complete mechanisms by which HS induces OS and OS influences milk protein synthesis are still unclear and further investigation is needed.

MiR‐24‐3p, a broadly conserved, small, noncoding RNA, is abundantly expressed in mammary tissue. However, its regulatory role in this tissue remains poorly understood. It was predicted that miR‐24‐3p targets the 3′ untranslated region (3′‐UTR) of multiple endocrine neoplasia type 1 (MEN1), an important regulatory factor in mammary tissue. The objective of this study was to investigate the function of miR‐24‐3p in mammary cells. Using a luciferase assay in mammary epithelial cells (MAC‐T), miR‐24‐3p was confirmed to target the 3′‐UTR of MEN1. Furthermore, miR‐24‐3p negatively regulated the expression of the MEN1 gene and its encoded protein, menin. miR‐24‐3p enhanced proliferation of MAC‐T by promoting G1/S phase progression. MiR‐24‐3p also regulated the expression of key factors involved in phosphatidylinositol‐3‐kinase/protein kinase B/mammalian target of rapamycin and Janus kinase/signal transducer and activators of transcription signaling pathways, therefore controlling milk protein synthesis in epithelial cells. Thus, miR‐24‐3p appears to act on MAC‐T by targeting MEN1. The expression of miR‐24‐3p was controlled by MEN1/menin, indicating a negative feedback loop between miR‐24‐3p and MEN1/menin. The negatively inhibited expression pattern of miR‐24‐3p and MEN1 was active in mammary tissues at different lactation stages. The feedback mechanism is a new concept to further understand the lactation cycle of mammary glands and can possibly to be manipulated to improve milk yield and quality. MiR‐24‐3p negatively modulates the expression of MEN1/menin in mammary epithelial cells by targeting the 3 'UTR of MEN1. MiR‐24‐3p plays regulatory role of cell proliferation and milk protein synthesis in mammary epithelial cells through cooperatively acting with menin. [ABSTRACT FROM AUTHOR]

Amino acids not only serve as precursors for protein synthesis but also function as signaling molecules that can regulate the mammalian target of rapamycin (mTOR) pathway. Methionine and Lys are the most-limiting AA for milk production and a ratio of ~3:1 Lys: Met in the metabolizable protein has been determined to be ideal. Besides Met and Lys, recent studies have evaluated Ile, Leu, Val, and Thr as potentially limiting for milk protein synthesis. The objective of this experiment was to determine if varying the ratio of Lys: Thr, Lys: Ile, Lys: Val, and Lys: Leu while maintaining an ideal ratio of Lys: Met and fixed ratio of other essential AA (IPAA) elicits changes in intracellular metabolites, gene transcription related to protein synthesis, and phosphorylation status of mTOR pathway proteins. Immortalized bovine mammary epithelial cell line (MACT) cells were incubated for 12 h (n = 5 replicates/treatment) with IPAA (2.9:1 Lys: Met; 1.8:1 Lys: Thr; 2.38:1 Lys: His; 1.23:1 Lys: Val; 1.45:1 Lys: Ile; 0.85:1 Lys: Leu; 2.08:1 Lys: Arg) or IPAA supplemented with Thr, Ile, Val, and Leu to achieve a Lys: Thr 1.3:1 (LT1.3), Lys: Ile 1.29:1 (LI1.29), Lys: Val 1.12:1 (LV1.12), or Lys: Leu 0.78:1 (LL0.78). Compared with IPAA, metabolomics via gas chromatography-mass spectrometry revealed that increases in availability of Thr, Ile, Val, and Leu led to greater concentrations of essential AA (Leu, Ile, Thr), nonessential AA (Gly, Glu, Gln, Ser, Pro, Asp), and various metabolites including uric acid, phosphoric acid, N-acetylglutamic acid, and intermediates of glycolysis and the tricarboxylic acid cycle. Compared with other treatments, LV1.12 led to greater phosphorylation status of serine/threonine kinase B (Akt), mTORC1, and ribosomal protein S6 and lower phosphorylation of α subunit of eukaryotic translation initiation factor 2. In addition, LV1.12 upregulated abundance of CSN2 and both the abundance and promoter methylation of CSN1S1. Although LI1.29 led to the second highest response in mTORC1 phosphorylation status, it resulted in the lowest phosphorylation of Akt and eEF2 and mRNA abundance of CSN2 and various AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, SLC43A1). Overall, data indicate that an increase in Val at an ideal ratio of Lys: Met could further enhance milk protein synthesis by altering intracellular concentrations of essential AA and metabolites that could play a regulatory role, increasing phosphorylation status of mTORC1 and key signaling proteins, and upregulation of AA transporters. [ABSTRACT FROM AUTHOR]

Amino acids are not only precursors for but also signaling molecules regulating protein synthesis. Regulation of protein synthesis via AA occurs at least in part by alterations in the phosphorylation status of mammalian target of rapamycin (mTOR) pathway proteins. Although the ideal profile of Lys: Met to promote milk protein synthesis during established lactation in dairy cows has been proposed to be 3:1, aside from being the most-limiting AA for milk protein synthesis, the role of Met in other key biologic pathways such as methylation is not well characterized in the bovine. The objective of this study was to determine the influence of increasing supplemental Met, based on the ideal 3:1 ratio of Lys to Met, on intracellular metabolism related to protein synthesis and mTOR pathway phosphorylation status. MAC-T cells, an immortalized bovine mammary epithelial cell line, were incubated (n = 5 replicates/ treatment) for 12 h with 3 incremental doses of Met while holding Lys concentration constant to achieve the following: Lys: Met 2.9:1 (ideal AA ratio; IPAA), Lys: Met 2.5:1 (LM2.5), and Lys: Met 2.0:1 (LM2.0). The ratios of Thr: Phe (1.05:1), Lys: Thr (1.8:1), Lys: His (2.38:1), and Lys: Val (1.23:1) were the same across the 3 treatments. Applying gas chromatography–mass spectrometry metabolomics revealed distinct clusters of differentially concentrated metabolites in response to Lys: Met. Lower Phe, branched-chain AA, and putrescine concentrations were observed with LM2.5 compared with IPAA. Apart from greater intracellular Met concentrations, further elevations in Met level (LM2.0) led to greater intracellular concentrations of nonessential AA (Pro, Glu, Gln, and Gly) compared with IPAA and greater essential AA (EAA; Met, Ile, and Leu) and nonessential AA (Pro, Gly, Ala, Gln, and Glu) compared with LM2.5. However, compared with IPAA, mRNA expression of β-casein and AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, and SLC43A1) and mTOR phosphorylation were lower in response to LM2.5 and LM2.0. Overall, the results of this study provide evidence that increasing Met while Lys and the ratios of Phe, Thr, His, and Val relative to Lys were held constant could increase the concentration and utilization of intracellular EAA, in particular branched-chain AA, potentially through improving the activity of AA transporters partly controlled by mTOR signaling. Because EAA likely are metabolized by other tissues upon absorption, a question for future in vivo studies is whether formulating diets for optimal ratios of EAA in the metabolizable protein is sufficient to provide the desired levels of these AA to the mammary cells. [ABSTRACT FROM AUTHOR]

This study was undertaken to evaluate the milk protein response when cows were supplied a balanced AA profile and to determine whether a deficiency of Leucine (Leu) or Arginine (Arg) had a negative effect on milk protein. Eight mid-lactation Holstein cows were randomly assigned to 5-day continuous jugular infusions of saline ( CTL), EAA mixture prepared on the profile of casein and supplied (in % of lysine (Lys)) 100% of Lys, 33.3% of methionine (Met), 110.2% of Leu, 43.6% of Arg, 50.8% of threonine (Thr), 81.6% of valine (Val), 69.7% of isoleucine (Ile), 61.4% of phenylalanine (Phe) and 34.2% of histidine (His) ( Casein, 160 g/d), EAA mixture excluding Leu (−Leu, 163 g/d) or EAA mixture excluding Arg (−Arg, 158 g/d) in a duplicated 4 × 4 Latin square design with four infusion periods separated by 7-day interval period. The basal diet supplied 1.6 Mcal NEL and 94.4 g MP per 1 kg DM to meet requirements for lactation. The Casein treatment provided a balanced supply (in % of MP) of 10.3% Leu and 5.3% Arg, whereas in the two subsequent −Leu and −Arg treatments, the concentration of Leu and Arg was reduced to 8.4 and 4.6% respectively. Dry matter intake (15.4 kg/day) was not affected by treatments. The Casein treatment increased milk yield (14.9%, p < 0.001), milk protein yield (120 g, p < 0.001) and milk protein efficiency (0.03, p = 0.099) than CTL treatment. However, the −Leu treatment decreased the responses of above-measured parameters by 6.25%, 70 g, 0.05 (p < 0.06) (compared with Casein). These effects of Leu were related to decreased Leu concentration and improved concentration of Ile and Val in plasma. The −Arg treatment decreased the plasma Arg concentration than the Casein treatment, whereby resulted in the decrease of milk yield (5.7%, p = 0.073), milk protein yield (60 g, p = 0.011) and milk protein efficiency (0.04, p = 0.037). In conclusion, supply of EAA profile of casein can increase the lactation production in dairy cows, and 8.6% of Leu in MP partly limits the milk protein response when the requirements of Lys, Met and His were met. The level of Arg at 4.6% MP is not deemed to an ideal profile, as evidenced by decreased milk protein efficiency. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling 3 (SOCS3) is a cytokine-induced negative feedback-loop regulator of cytokine signaling. More and more evidence has proved it to be an inhibitor of signal transducers and activators of transcription 5 (STAT5). Here, we used dairy cow mammary epithelial cells (DCMECs) to analyze the function of SOCS3 and the interaction between SOCS3 and STAT5a. The expression of SOCS3 was found in cytoplasm and nucleus of DCMECs by fluorescent immunostaining. Overexpression and inhibition of SOCS3 brought a remarkable milk protein synthesis change through the regulation of JAK2/STAT5a pathway activity, and SOCS3 expression also decreased SREBP-1c expression and fatty acid synthesis. Inhibited STAT5a activation correlated with reduced SOCS3 expression, which indicated that SOCS3 gene might be one of the targets of STAT5a activation, DCMECs treated with L-methionine (Met) resulted in a decrease of SOCS3 expression. SOCS3 could also decrease cell proliferation and viability by CASY-TT detection. Together, our findings indicate that SOCS3 acts as an inhibitor of JAK2/STAT5a pathway and disturbs fatty acid synthesis by decreasing SREBP-1c expression, which validates its involvement in both milk protein synthesis and fat synthesis. In aggregate, these results reveal that low SOCS3 expression is required for milk synthesis and proliferation of DCMECs in vitro.

Background: Interest is increasing in the role of peptide-bound amino acids (AAs) in milk protein synthesis because studies have found that the uptake of some essential AAs by the mammary gland cannot meet the requirements for milk protein synthesis. Although the role of dipeptides in milk protein synthesis is clearly established, little is known about the underlying mechanisms. Objective: The objective of this study was to determine whether small peptides can be taken up intact by the peptide transporters in mammary tissue explants and the underlying mechanisms of the effects of methionyl-methionine (Met-Met) supplementation on milk protein synthesis. Methods: Mammary tissue explants were cultured in conditional medium and then treated with different concentrations of Met-Met that replaced 0%, 5%, 10%, 15%, 20%, and 25% of free Met for another 24 h. In some experiments, explants were cultured with an optimal dose of Met-Met with or without the inhibitors of peptide transporter 2 [PepT2; diethylpyrocarbonate (DEPC), 0.1 mmol/L] and aminopeptidase N (APN; bestatin, 20 µmol/L) for 24 h. Results: The substitutions of 15% free Met with Met-Met significantly promoted α-s1 casein (αs1-CN) expression in the mammary explants (P < 0.05). The inhibition of the PepT2 by DEPC or APN by bestatin significantly decreased the Met-Met-stimulated increase of αs1-CN expression (P < 0.05). Compared with the control group (0% Met-Met), absorption of Val, Met, Leu, Phe, Lys, and His was improved, and mRNA abundance of the neutral and basic AA transporter was increased in the 15% Met-Met group (P< 0.05). In addition, the mRNA abundance of the mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 gene, eukaryotic initiation factor 4E binding protein 1 gene, Janus kinase 2 (JAK2), and signal transducer and activator of transcription 5 (STAT5) was increased in the 15% Met-Met-treated group (P < 0.05). Conclusion: Met-Met promoted αs1-CN synthesis in cultured bovine mammary gland explants, and this stimulation may be mediated by enhanced intracellular substrate availability and by activating JAK2-STAT5 and mTOR signaling pathways. [ABSTRACT FROM AUTHOR]

Essential amino acid (EAA) deficiencies and imbalances were created in lactating cows by using an infusion subtraction protocol to explore effects on milk protein yield and activity state of regulators of mRNA translation in the mammary glands. Six lactating cows on a diet of 11.2% protein were infused abomasally for 5 d with saline, 563 g/d of a complete EAA mix, or EAA without His, Met, Phe, or Trp in a 6 x 6 Latin square design. Infusion of complete and imbalanced EAA solutions increased mammalian target of rapamycin (mTOR) signaling in the mammary glands, as evidenced by higher ribosomal S6 kinase 1 (S6K1) phosphorylation compared with saline infusion. Total S6K1 abundance was decreased by imbalanced AA infusions. Except for the mixture lacking Phe, infusion of EAA, whether imbalanced or not, increased abundance of total eukaryotic initiation factor 2Bε (eIF2Bε). A correlation of 0.33 between phosphorylation state of S6K1 and total eIF2Bε abundance suggests that an mTOR-mediated upregulation of eIF2Bε translation occurred. Despite increased mTOR/eIF2Bε signaling, milk protein yields increased only with the complete EAA mixture compared with saline. Low plasma concentrations of His, Met, and Phe during their respective imbalances likely interfered with protein synthesis. Total abundance and phosphorylation state of eukaryotic initiation factor 2α were not responsible for the interference. Further study of eIF2Bε as a regulator of milk protein yield is warranted. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling 3 (SOCS3) is a cytokine-induced negative feedback-loop regulator of cytokine signaling. More and more evidence has proved it to be an inhibitor of signal transducers and activators of transcription 5 (STAT5). Here, we used dairy cow mammary epithelial cells (DCMECs) to analyze the function of SOCS3 and the interaction between SOCS3 and STAT5a. The expression of SOCS3 was found in cytoplasm and nucleus of DCMECs by fluorescent immunostaining. Overexpression and inhibition of SOCS3 brought a remarkable milk protein synthesis change through the regulation of JAK2/STAT5a pathway activity, and SOCS3 expression also decreased SREBP-1c expression and fatty acid synthesis. Inhibited STAT5a activation correlated with reduced SOCS3 expression, which indicated that SOCS3 gene might be one of the targets of STAT5a activation, DCMECs treated with L-methionine (Met) resulted in a decrease of SOCS3 expression. SOCS3 could also decrease cell proliferation and viability by CASY-TT detection. Together, our findings indicate that SOCS3 acts as an inhibitor of JAK2/STAT5a pathway and disturbs fatty acid synthesis by decreasing SREBP-1c expression, which validates its involvement in both milk protein synthesis and fat synthesis. In aggregate, these results reveal that low SOCS3 expression is required for milk synthesis and proliferation of DCMECs in vitro. [ABSTRACT FROM AUTHOR]

Simple Summary: This study mainly employed metabolomics technology to determine changes of intracellular metabolite concentrations related to milk protein synthesis induced by heat stress (HS) in bovine mammary epithelial cells. HS was associated with significant differences in intracellular amino acid metabolism resulting in an increase in the intracellular amino acid concentrations. Moreover, HS promoted amino acid transportation and the activity of the mammalian target of rapamycin (mTOR) signaling pathway, which plays an important role as a central regulator of cell metabolism, growth, proliferation and survival. Greater expression of the alpha-S2-casein gene (CSN1S2) was also observed during HS. Overall, our study indicated that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS. Heat stress (HS) is one of the most serious factors to negatively affect the lactation performance of dairy cows. Bovine mammary epithelial cells are important for lactation. It was demonstrated that HS decreases the lactation performance of dairy cows, partly through altering gene expression within bovine mammary epithelial tissue. However, the cellular metabolism mechanisms under HS remains largely unknown. The objective of this study was to determine whether HS induced changes in intracellular metabolites and gene transcription related to amino acid metabolism, amino acid transportation and the mTOR signaling pathway. Immortalized bovine mammary epithelial cell lines (MAC-T cells, n = 5 replicates/treatment) were incubated for 12 h at 37 °C (Control group) and 42 °C (HS group). Relative to the control group, HS led to a greater mRNA expression of heat shock protein genes HSF1, HSPB8, HSPA5, HSP90AB1 and HSPA1A. Compared with the control group, metabolomics using liquid chromatography tandem–mass spectrometry identified 417 differential metabolites with p < 0.05 and a variable importance in projection (VIP) score >1.0 in the HS group. HS resulted in significant changes to the intracellular amino acid metabolism of glutathione, phenylalanine, tyrosine, tryptophan, valine, leucine, isoleucine, arginine, proline, cysteine, methionine, alanine, aspartate and glutamate. HS led to a greater mRNA expression of the amino acid transporter genes SLC43A1, SLC38A9, SLC36A1, and SLC3A2 but a lower mRNA expression of SLC7A5 and SLC38A2. Additionally, HS influenced the expression of genes associated with the mTOR signaling pathway and significantly upregulated the mRNA expression of mTOR, AKT, RHEB, eIF4E and eEF2K but decreased the mRNA expression of TSC1, TSC2 and eEF2 relative to the control group. Compared with the control group, HS also led to greater mRNA expression of the CSN1S2 gene. Overall, our study indicates that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS. [ABSTRACT FROM AUTHOR]

The reduction of milk yield caused by heat stress in summer is the main condition restricting the economic benefits of dairy farms. To examine the impact of hyperthermia on bovine mammary epithelial (MAC-T) cells, we incubated the MAC-T cells at thermal-neutral (37°C, CON group) and hyperthermic (42°C, HS group) temperatures for 6 h. Subsequently, the cell viability and apoptotic rate of MAC-T cells, apoptosis-related genes expression, casein and amino acid transporter genes, and the expression of the apoptosis-related proteins were examined. Compared with the CON group, hyperthermia significantly decreased the cell viability ( p < 0.05) and elevated the apoptotic rate ( p < 0.05) of MAC-T cells. Moreover, the expression of heat shock protein ( HSP ) 70 , HSP90B1 , Bcl-2-associated X protein ( BAX ), Caspase-9 , and Caspase-3 genes was upregulated ( p < 0.05). The expression of HSP70 and BAX (pro-apoptotic) proteins was upregulated ( p < 0.05) while that of B-cell lymphoma (BCL)2 (antiapoptotic) protein was downregulated ( p < 0.05) by hyperthermia. Decreased mRNA expression of mechanistic target of rapamycin (mTOR) signaling pathway-related genes, amino acid transporter genes ( SLC7A5 , SLC38A3 , SLC38A2 , and SLC38A9 ), and casein genes ( CSNS1 , CSN2 , and CSN3 ) was found in the heat stress (HS) group ( p < 0.05) in contrast with the CON group. These findings illustrated that hyperthermia promoted cell apoptosis and reduced the transport of amino acids into cells, which inhibited the milk proteins synthesis in MAC-T cells., Competing Interests: The authors declare no conflict of interest., (© Copyright 2022 Korean Society of Animal Science and Technology.)

The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and α-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. The results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus ( S. aureus ) is one of the main pathogens in cow mastitis, colonizing mammary tissues and being internalized into mammary epithelial cells, causing intracellular infection in the udder. Milk that is produced by cows that suffer from mastitis due to S. aureus is associated with decreased production and changes in protein composition. However, there is limited information on how mastitis-inducing bacteria affect raw milk, particularly with regard to protein content and protein composition. The main purpose of this work was to examine how S. aureus infection affects milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were infected with S. aureus , and milk protein and amino acid levels were determined by ELISA after S. aureus invasion. The activity of mTORC1 signaling and the transcription factors NF-κB and STAT5 and the expression of the amino acid transporters SLC1A3 and SLC7A5 were measured by western blot or immunofluorescence and RT-qPCR. S. aureus was internalized by BMECs in vitro , and the internalized bacteria underwent intracellular proliferation. Eight hours after S. aureus invasion, milk proteins were downregulated, and the level of BMECs that absorbed Glu, Asp, and Leu from the culture medium and the exogenous amino acids induced β-casein synthesis declined. Further, the activity of mTORC1 signaling, NF-κB, and STAT5 was impaired, and SLC1A3 and SLC7A5 were downregulated. Eight hours of treatment with 100 nM rapamycin inhibited NF-κB and STAT5 activity, SLC1A3 and SLC7A5 expression, and milk protein synthesis in BMECs. Thus mTORC1 regulates the expression of SLC1A3 and SLC7A5 through NF-κB and STAT5. These findings constitute a model by which S. aureus infection suppresses milk protein synthesis by decreasing amino acids uptake in BMECs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Ma, Ji, Yang, Feng, Yao, Cheng, Li, Wang and Wang.)

Simple Summary: Heat stress (HS) of dairy cows affects milk protein synthesis partially due to a decline in appetite and dry matter intake (DMI). Several published studies indicate that HS causes oxidative stress (OS) with high levels of free radicals in tissues. Furthermore, the involvement of reactive oxygen species (ROS) in inducing apoptosis, in reducing mammary epithelial cell numbers, and in endocrine disruptions are proposed as the main mechanisms by which HS-induced OS modifies milk protein synthesis. However, challenges remain in determining the levels of apoptosis in vivo, as well as the tracking of the sources of ROS formation. Therefore, further investigations are required. Heat stress (HS) is one of the most important factors posing harm to the economic wellbeing of dairy industries, as it reduces milk yield as well as milk protein content. Recent studies suggest that HS participates in the induction of tissue oxidative stress (OS), as elevated levels of reactive oxygen species (ROS) and mitochondrial dysfunction were observed in dairy cows exposed to hot conditions. The OS induced by HS likely contributes to the reduction in milk protein content, since insulin resistance and apoptosis are promoted by OS and are negatively associated with the synthesis of milk proteins. The apoptosis in the mammary gland directly decreases the amount of mammary epithelial cells, while the insulin resistance affects the regulation of insulin on mTOR pathways. To alleviate OS damages, strategies including antioxidants supplementation have been adopted, but caution needs to be applied as an inappropriate supplement with antioxidants can be harmful. Furthermore, the complete mechanisms by which HS induces OS and OS influences milk protein synthesis are still unclear and further investigation is needed. [ABSTRACT FROM AUTHOR]

In the dairy industry, glutamine (Gln) is often used as a feed additive to increase milk yield and quality; however, the molecular regulation underneath needs further clarification. Here, with bovine mammary epithelial cells (BMECs), the effects and mechanisms of Gln on cell growth and casein synthesis were assessed. When Gln was added or depleted from BMECs, both cell growth and β-casein (CSN2) expression were increased or decreased, respectively. Overexpressing or inhibiting the mechanistic target of rapamycin (mTOR) revealed that Gln regulated cell growth and CSN2 synthesis through the mTORC1 pathway. A similar intervention of ADP-ribosylation factor 1 (Arf1) uncovered that Gln activated the mTORC1 pathway through Arf1. We next observed that both guanine nucleotide exchange factors, Cytohesin-1/2/3 (CYTH1/2/3, CYTHs) and ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1), interacted with Arf1. Inhibiting CYTHs or ARFGAP1 showed that Gln supplement or depletion activated or inactivated Arf1 through CYTHs or ARFGAP1, respectively. Collectively, this study demonstrated that Gln positively regulated cell growth and casein synthesis in BMECs, which works through the CYTHs/ARFGAP1-Arf1-mTORC1 pathway. These results greatly enhanced current understanding regarding the regulation of the mTOR pathway and provided new insights for the processes of cell growth and casein synthesis by amino acids, particularly Gln.

It is shown in physiological experiments conducted on goats and cows with the use of new methodological approaches that, with various methods of hormonal stimulation of galactopoiesis, intensification of milk protein synthesis is achieved mainly due to an increase of organ blood flow and activity of transport of metabolites into mammary secretory cells. [ABSTRACT FROM AUTHOR]

Studies on promoting milk protein yield by supplementation of amino acids have been globally conducted. Nevertheless, there is a lack of knowledge of what pathways affected by individual amino acid in mammary epithelial cells that produce milk in practice. Phenylalanine (PHE) and valine (VAL) are essential amino acids for dairy cows, however, researches on mammary cell levels are still lacking. Thus, the aim of this study was conducted to evaluate the effects of PHE and VAL on milk protein synthesis-related and energy-mediated cellular signaling in vitro using immortalized bovine mammary epithelial (MAC-T) cells. To investigate the effects of PHE and VAL, the following concentrations were added to treatment medium: 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. The addition of PHE or VAL did not adversely affect cell viability compared to control group. The concentrations of cultured medium reached its maximum at 0.9 mM PHE and 0.6 mM VAL ( p < 0.05). Therefore, aforementioned 2 treatments were analyzed for proteomics. Glucose transporter 1 and mammalian target of rapamycin mRNA expression levels were up-regulated by PHE (166% and 138%, respectively) ( p < 0.05). Meanwhile, sodium-dependent neutral amino acids transporter type 2 ( ASCT2 ) and β-casein were up-regulated by VAL (173% in ASCT2 , 238% in and 218% in β-casein) ( p < 0.05). A total of 134, 142, and 133 proteins were detected in control group, PHE treated group, and VAL treated group, respectively. Among significantly fold-changed proteins, proteins involved in translation initiation or energy metabolism were detected, however, expressed differentially between PHE and VAL. Thus, pathway analysis showed different stimulatory effects on energy metabolism and transcriptional pathways. Collectively, these results showed different stimulatory effects of PHE and VAL on protein synthesis-related and energy-mediated cellular signaling in MAC-T cells., Competing Interests: No potential conflict of interest relevant to this article was reported., (© Copyright 2020 Korean Society of Animal Science and Technology.)