Increasing the methionine (Met) supply to lactating cows will increase mammary cell proliferation. Activating transcription factor 4 (ATF4) is a regulator of cellular amino acid sensing, linking nutrient signals to cellular anabolism; however, its role in regulating bovine mammary epithelial cell (BMEC) proliferation remains largely unknown. In this study, ATF4 expression paralleled that of Cyclin D1 in mammary tissues from dairy cows at different developmental stages (puberty, lactation and dry period), suggesting that ATF4 is associated with cell proliferation. In BMECs, knockdown of ATF4 increased the number of cells arrested in the G1 phase (∼11%; P < 0.05; control) and downregulated Cyclin D1 expression (∼40%; P < 0.05; control), indicating that ATF4 is required for cell proliferation. Treatment of BMECs with increasing concentrations of Met (0, 0.3, 0.6, 0.9, 1.0 and 1.2 mM) showed that 0.6 mM Met enhanced mammalian target of rapamycin (mTOR) phosphorylation and increased protein expression of ATF4 and Cyclin D1, without activating canonical endoplasmic reticulum stress markers. Inhibition assays demonstrated that mTOR is required for Met-induced ATF4 activation. Functional studies further revealed that ATF4 is a key mediator of Met-regulated cell proliferation. Mechanistically, Met facilitated the physical interaction between ATF4 and glycogen synthase kinase-3β (GSK3β), promoting GSK3β phosphorylation at Ser9 and subsequently increasing Cyclin D1 expression. Collectively, these findings indicate that Met promotes BMEC proliferation through the mTOR-ATF4-GSK3β signalling pathway, providing a potential nutritional strategy to enhance mammary cell proliferation in dairy cows.

The high incidence of oxidative stress in mammals during lactation affects mammary gland health, milk yield, and milk quality. However, the molecular mechanisms underlying oxidative stress-induced mammary gland dysfunction remain unclear. The objective of the present study was to investigate the underlying molecular events in the decrease in milk fat production in mammary gland subjected to oxidative stress. We generated oxidative stress cell model by incubated mouse mammary epithelial cell line HC11 with 600 μM hydrogen peroxide (H2O2). H2O2 incubation increased intracellular ROS content and MDA activity, but decreased SOD and CAT activities, as well as intracellular triglyceride (TG) content. We performed RNA sequencing (RNA-seq) to identify differentially expressed genes (DEGs) between H2O2-treated and control cells. 926 DEGs were identified, which included 457 up-regulated genes and 469 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that the DEGs is involved in lipid metabolism, cell growth and death, and mitogen-activated protein kinase (MAPK) signaling pathway. Acyl-CoA synthetase long chain family member 6 (Acsl6) was positively regulated milk fat synthesis, while oxidative stress down-regulated Acsl6 expression. Ultimately, the mouse model of oxidative stress was established, and we verified the effect of oxidative stress on milk fat production in vivo. Overall, the results revealed oxidative stress activated the p38 MAPK pathway, downregulated the transcription factor CCAAT-enhancer-binding protein alpha (Cebpα), and inhibited the expression of Acsl6, thereby suppressing lipid droplets formation and reducing intracellular TG content. These findings elucidate the molecular mechanism underlying oxidative stress-mediated suppression of milk fat production, which may provide insights for the development of redox-targeted therapeutic strategies against oxidative stress-induced metabolic disorders.

Oleic acid (OA)-enriched diet significantly enhances CD36 expression and promotes milk fat production in the mammary gland. However, the mechanisms by which OA upregulates this process are unknown. Here, GC-MS analysis and molecular docking were used to assess OA-CD36 binding, while gene expression and ChIP assays clarified the OA-mediated regulation of CD36 and milk fat synthesis. The results showed that CD36 is highly expressed in the lactating mammary tissues of dairy cows and positively regulates milk fat production. OA enters dairy cow mammary epithelial cells via direct binding to CD36. OA stimulation elevates the CD36 expression and translocation. ChIP assays confirmed CD36 as a target gene of NRF2. OA also enhances NRF2 expression and nuclear translocation, which in turn upregulates CD36 expression and function. This study elucidates the NRF2-CD36 axis as a regulator of OA-driven milk fat synthesis in mammary tissue, offering practical value for dairy industry nutritional strategies.

Salivary adenoid cystic carcinoma (SACC) is a prevalent malignant tumor of the salivary glands, characterized by invasive growth and perineural invasion, resulting in high rates of local recurrence, distant metastasis and poor long-term survival. Thus, elucidating the molecular mechanisms underlying SACC invasion and identifying effective therapeutic targets are of clinical importance. Functional cellular assays including proliferation, migration, and flow cytometry, and molecular experiments, such as western blot, were conducted in vitro to explore the effects of integrin-linked kinase (ILK) knockdown on the biological behavior of SACC cells and the expression of epithelial-mesenchymal transition (EMT) markers. Transcriptome sequencing identified S100 calcium-binding protein A4 (S100A4) as a key downstream effector regulated by ILK. Additionally, 52 clinical SACC specimens were analyzed to evaluate the correlation between S100A4 expression and clinicopathological features, as well as ILK expression. Rescue experiments validated the role of S100A4 in mediating the effects of ILK. ILK knockdown significantly suppressed the malignant behavior of SACC cells, including migration and invasion, and reversed EMT phenotypes. S100A4 expression was significantly associated with clinical features such as clinical stage and perineural invasion, along with ILK expression. Overexpression of S100A4 restored Snail expression, promoted the EMT process and rescued the impaired migration and invasion capabilities of SACC cells caused by ILK knockdown. ILK may facilitate EMT-mediated invasion in SACC cells via the Glycogen synthase kinase-3 β) signaling pathway by modulating the transcription factor Snail. S100A4 may contribute to this mechanism by modulating Snail protein. These findings imply that simultaneously targeting both ILK and S100A4 represents a novel and promising therapeutic strategy to suppress SACC progression.

Milk fat synthesis is tightly regulated by hormones and growth factors. Leptin is a versatile peptide hormone that exerts pleiotropic effects on metabolic pathways. In this study, we evaluated the expression and function of leptin and its long form receptor OB-Rb in dairy cow mammary tissues from different physiological stages and in cultured mammary epithelial cells. The results showed that the expression of leptin and OB-Rb were significantly higher in the mammary tissues of lactating cows as compared with dry cows, suggesting that they are related to milk component synthesis. In cultured dairy cow mammary epithelial cells, leptin treatment significantly increased OB-Rb expression and intracellular triacylglycerol content. Transcriptome analysis identified the difference in gene expression between leptin treated cells and control cells, and 317 differentially expressed genes were identified. Gene ontology and pathway mapping showed that lipid metabolism-related gene expression increased and signal transduction pathway-related genes were the most significantly enriched. Mechanistic studies showed that leptin stimulation enhanced sterol regulatory element-binding protein 1 expression via activating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway, which in turn up-regulated the expression of genes related to milk fat synthesis. Moreover, we found that fatty acid synthesis precursors, acetate and β-hydroxybutyrate, could positively regulate the expression of leptin and OB-Rb in bovine mammary epithelial cells, thereby potentially increasing milk fat synthesis. Our study provided novel evidence in the regulation of leptin on milk fat production in mammary glands of dairy cows, as well as experimental basis for artificial regulation of milk fat.

The uptake of AA in mammary tissues is affected by prolactin (PRL). To investigate whether PRL-induced AA uptake is involved in L-type AA transporter 1 (LAT1), we analyzed the changes of AA in the medium of dairy cow mammary epithelial cells in the presence of PRL or PRL plus BCH, an inhibitor of LAT1. Then Western blot and luciferase assay were used to detect the regulation mechanism of PRL on LAT1 expression and function. Our results showed that Thr, Val, Met, Ile, Leu, Tyr, Lys, Phe, and His are LAT1 substrates and could be transported into mammary epithelial cells via LAT1. PRL stimulation increased the uptake of most AA into mammary epithelial cells of dairy cows, however, inhibition of LAT1 transport activity reduced PRL-induced AA uptake, suggesting that the effect of PRL on AA transport depends on LAT1 expression and function. PRL stimulation upregulated LAT1 expression and plasma membrane location not only in dairy cow mammary epithelial cells, but also in mouse mammary epithelial cell line HC11. Western blot showed that PI3K-AKT-mTOR signaling could be activated in PRL-stimulated mammary epithelial cells. Treatment of cells with LY294002 decreased PI3K-AKT-mTOR activation, as well LAT1 expression, that in turn decreased milk protein synthesis. Luciferase assay showed PRL treatment increased the promoter activity of LAT1 promoter fragment −419∼-86 bp. Treatment of cells with LY294002, an inhibitor of PI3K, or SC79, an activator of AKT abolished or promoted the transcriptional activity of this promoter fragment in the presence of PRL. These results suggested that the −419∼-86 bp fragment of LAT1 promoter mediates the action of PI3K-AKT-mTOR signaling on LAT1 transcription in mammary epithelial cells of dairy cows, which in turn increased LAT1 expression and AA uptake.

This research communication screened and identified differentiated expressed genes in bovine mammary epithelial cells (BMECs) upon prolactin (PRL) stimulation. PRL of 5 μg/ml increased β-casein synthesis in BMECs with milk protein synthesis capacity. RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs). A total of 375 DEGs (165 up-regulated and 210 down-regulated) were identified between PRL-stimulated group and the control group. Gene ontology enrichment analysis showed that the up-regulated genes were primarily associated with cell functions, metabolic processes, and biological regulatory processes. Pathway enrichment analysis showed that the up-regulated genes were mainly enriched in JAK-STAT, Rap1, Ras and Notch signaling pathways, which are widely involved in cell proliferation, differentiation and milk component synthesis. This study provides an initial understanding of the changes in gene expression in BMECs with PRL-stimulation, as determined by RNA-seq transcriptomic analysis, thereby enhancing our knowledge of the molecular regulation of lactation metabolism.

This study investigated the mechanism underlying acetate-induced orphan G-protein-coupled receptor 43 (GPR43) expression and milk fat production. The mammary epithelial cells of dairy cows were treated with acetate, and the effects of GPR43 on acetate uptake and the expression of lipogenesis-related genes were determined by gas chromatography and quantitative polymerase chain reaction (qPCR), respectively. RNAi, inhibitor treatment, and luciferase assay were used to determine the effect of phosphoinositide 3-kinase-protein kinase B-specificity protein 1 (PI3K-AKT-SP1) signaling on acetate-induced GPR43 expression and function. The results showed that GPR43 was highly expressed in lactating cow mammary tissues, which was related to milk fat synthesis. 12 mM acetate significantly increased the GPR43 expression in mammary epithelial cells of dairy cows. In acetate-treated cells, GPR43 overexpression significantly increased the cellular uptake of acetate, the intracellular triacylglycerol (TAG) content, and acetate-induced lipogenesis gene expression. Acetate activated PI3K-AKT signaling and promoted SP1 translocation from the cytosol into the nucleus, where SP1 bound to the GPR43 promoter and upregulated GPR43 transcription. Moreover, the activation of PI3K-AKT-SP1 by acetate facilitated the trafficking of GPR43 from the cytosol to the plasma membrane. In conclusion, acetate upregulated GPR43 expression and function via PI3K-AKT-SP1 signaling in mammary epithelial cells, thereby increasing milk fat synthesis. These results provide an experimental strategy for improving milk lipid synthesis, which is important to the dairy industry.

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.

旨在探讨中国荷斯坦奶牛的特异性蛋白1(specificity protein,SP1)基因结构特征及其对奶牛乳脂合成的影响.根据NCBI已经公布的奶牛SP1基因序列(NM_001078027.1),利用生物信息学分析其序列保守性、理化性质、蛋白亲水性、蛋白质结构及互作蛋白;采用PCR技术扩增并克隆SP1基因CDS序列.然后,选取6头健康的中国荷斯坦奶牛,分别取泌乳期和干奶期奶牛乳腺组织,运用实时荧光定量PCR和Western blot方法检测SP1基因在不同时期奶牛乳腺组织中的表达情况.分离并纯化泌乳期奶牛乳腺上皮细胞,通过SP1过表达及干扰检测其对乳脂合成的影响,分别进行3次独立试验.结果显示,SP1基因序列在不同物种间高度保守,与山羊相似度最高(98.94％).奶牛SP1基因CDS区序列长2 361 bp,编码786个氨基酸,蛋白分子质量为80 902.17 u,理论等电点为6.94.平均疏水指数为-0.438,为不稳定的亲水性蛋白.SP1序列包含3个锌指结构,SP1蛋白二级结构以无规则卷曲(52.29％)为主.STRING蛋白互作分析结果显示,SP1与转录因子AP-l(JUN)、雌激素受体α(ERα)、MYC原癌基因蛋白(MYC)、TATA盒结合蛋白(TBP)等蛋白存在相互作用.实时荧光定量PCR和Western blot结果显示,SP1的mRNA和蛋白在泌乳期的表达量显著高于干奶期(P＜0.01).在奶牛乳腺上皮细胞中过表达SP1显著增加细胞甘油三酯的合成(P＜0.01),而干扰SP1的表达,甘油三酯合成显著降低(P＜0.01).以上结果提示,SP1正向调控乳脂合成,分析SP1基因结构和功能为深入研究SP1对泌乳奶牛乳脂合成调控机制提供理论依据.

AbstractThis research communication investigated the role and the underlying mechanism of sn-1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6) in acetate-induced mTORC1 signaling activation and milk fat synthesis in dairy cow mammary epithelial cells. The data showed AGPAT6 knockdown significantly decreased acetate-induced phosphorylation of mTORC1 signaling molecules and intracellular triacylglycerol (TAG) content, whereas this inhibition effect was reversed after the addition of 16:0,18:1 phosphatidic acid (PA), suggesting that AGPAT6 could generate PA in response to acetate simulation, that in turn activates mTORC1 signaling. PPARγ is the upstream regulator of AGPAT6 upon acetate stimulation. Luciferase assay with clones containing various deletions and mutation in AGPAT6 promoter showed that there is a RXRα binding sequence located at −96 bp of AGPAT6 promoter. Acetate stimulation significantly increased the interaction between PPARγ and AGPAT6 via this RXRα binding site. Taken together, our data indicated that AGPAT6 could activate mTORC1 signaling by producing PA during acetate-induced milk fat synthesis, and PPARγ acts as a transcription factor to mediate the effect of acetate on AGPAT6 via RXRα.

溶血磷脂酸酰基转移酶(Sn-1-acylglycerol-3-phosphate O-acyltransferase 6,AGPAT6)是脂类物质合成代谢相关酶,但其在奶牛乳脂合成中调节作用尚不明确.研究采用荧光定量PCR、Western blot等方法检测奶牛泌乳循环过程中乳腺组织中AGPAT6表达变化、奶牛乳腺上皮细胞中AGPAT6对乳脂合成影响以及相关信号通路分子表达变化.结果表明,AGPAT6 mRNA和蛋白质表达在泌乳期奶牛乳腺组织中极显著高于青春期和干奶期(P<0.01).在具有乳脂合成能力的奶牛乳腺上皮细胞中,AGPAT6基因过表达极显著提高细胞中TAG含量,AGPAT6基因干扰获得相反结果.AGPAT6过表达可激活PI3K-AKT-mTOR信号通路,添加PI3K抑制剂会抑制AGPAT6诱导PI3K-AKT-mTOR信号通路激活及细胞中TAG合成(P<0.01).以上结果显示AGPAT6表达与泌乳奶牛乳脂合成呈正相关,AGPAT6通过激活PI3K-AKT-mTOR信号通路促进奶牛乳腺上皮细胞中乳脂合成.

Increasing acetate and β-hydroxybutyrate (BHB) supply to lactating cows will increase milk fat synthesis. However, the underlying molecular mechanism remains largely unknown. Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC) is a lipid droplet-associated protein that promotes intracellular triacylglycerol accumulation. In the present study, using gene overexpression and knockdown, we detected the contributions of CIDEC on milk fat synthesis in mammary epithelial cells of dairy cows in the presence of acetate and BHB. The results showed that knockdown of CIDEC decreased fatty acid synthase (FASN) expression and intracellular triacylglycerol content, whereas overexpression of CIDEC had the opposite effect. The transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) regulates cell growth and differentiation in the mammary gland. We demonstrated that the FASN promoter had a canonical C/EBPβ binding sequence. CEBPB overexpression upregulated FASN expression and milk fat synthesis, whereas CEBPB knockdown had the opposite effect. Moreover, knockdown of CEBPB attenuated the promoting effects of CIDEC on acetate- and BHB-induced FASN transcription. Taken together, our data showed that acetate and BHB induced FASN expression in mammary epithelial cells of dairy cows in a CIDEC-C/EBPβ-dependent manner, which provides new insights into the understanding of the molecular events involved in milk fat synthesis.

Acetate and β-hydroxybutyrate (BHBA) are the predominant substrates for de novo fatty acid (FA) synthesis in mammary gland of dairy cow. To investigate the nutrigenomic role of acetate and BHBA in bovine mammary epithelial cells during milk fat production, RNA sequencing (RNA-seq) transcriptomic analysis was used to identify differentially expressed genes (DEGs) between acetate- and BHBA-treated cells (high-milk fat cells) and control cells. A total of 625 DEGs (358 upregulated and 267 downregulated) were identified between the high-milk fat cells and control cells. Gene ontology enrichment analysis revealed that the upregulated genes in high-milk fat cells were mainly involved in lipid biosynthetic process, steroid biosynthetic process, oxidation-reduction process, receptor binding, and vesicle and small molecule biosynthetic process. The downregulated genes were mainly associated with immune response, cytokine production, negative regulation of biological process, and peptidyl-threonine modification. Pathway analysis indicated that FA metabolism and steroid biosynthesis were significantly enriched for the upregulated genes in the high-milk fat cells, while apoptosis was enriched for the downregulated genes. This work provides a profile of gene expression changes that occur during acetate- and BHBA-induced milk fat synthesis in bovine mammary epithelial cells, which furthers our understanding of the molecular regulation of lipid metabolism.

The l-type amino acid transporter 1 (LAT1; also known as SLC7A5) is a transporter that allows the uptake of large neutral amino acids into mammalian cells. In dairy cows, LAT1 is highly expressed in lactating mammary tissues and involved in milk protein synthesis. Prolactin (PRL) has a lactogenic role and is capable of inducing milk production in ruminants. However, the relationship between PRL stimulation and LAT1 expression in dairy cow mammary gland has not been well understood. In this study, we showed that PRL stimulation increased expression of LAT1 and β-casein in mammary epithelial cells of dairy cows. The stimulatory effect of PRL on milk protein production was inhibited by LAT1-specific inhibitor or LAT1 knockdown, suggesting that PRL-induced milk protein production is involved in LAT1 expression. To determine whether the PRL signaling pathway participates in regulation of LAT1 expression, PRLR (PRL receptor) or STAT5 (signal transducer and activator of transcription 5) was knocked down by short interfering (si)RNA in mammary epithelial cells of dairy cows. Western blot results showed that knockdown of PRLR or STAT5 with siRNA markedly decreased PRL-stimulated LAT1 expression. In addition, we observed a marked increase in plasma membrane expression of LAT1 in PRL-stimulated cells compared with control cells. These observations indicated that PRL signaling can regulate LAT1 expression and activity in mammary epithelial cells of dairy cows, contributing to increased amino acid availability and milk protein synthesis in mammary gland of dairy cow.

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.

In this research communication we used digital gene expression (DGE) analysis to identify differences in gene expression in the mammary glands of dairy cows between early lactation and the mid-dry period. A total of 741 genes were identified as being differentially expressed by DGE analysis. Compared with their expression in dry cows, 214 genes were up-regulated and 527 genes were down-regulated in lactating cow mammary glands. Gene Ontology analysis showed that lactation was supported by increased gene expression related to metabolic processes and nutrient transport and was associated with decreased gene expression related to cell proliferation. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes showed that 579 differentially expressed genes had pathway annotations related to 204 pathways. Metabolic pathway-related genes were the most significantly enriched. Genes and pathways identified by the present study provide insights into molecular events that occur in the mammary gland between early lactation and mid-dry period, which can be used to facilitate further investigation of the mechanisms underlying lactation and mammary tissue remodeling in dairy cows.

Amino acids are required for the mammalian target of rapamycin (mTOR) signaling pathway and milk synthesis in bovine mammary epithelial cells (BMECs). However, the mechanism through which amino acids activate this pathway is largely unknown. Here we show that glycyl‐tRNA synthetase (GlyRS) mediates amino acid‐induced activation of the mTOR‐S6K1/4EBP1 pathway, and milk protein and fat synthesis in BMECs. Among 19 aminoacyl‐tRNA synthetases, only the mRNA expression of GlyRS and Leucyl‐tRNA synthetase (LeuRS) were significantly increased by several amino acids including Met and Leu. We then observed that GlyRS knockdown abolished the stimulation of Met on milk protein and fat synthesis in BMECs, whereas GlyRS overexpression led to more significantly increased milk synthesis in cells treated with Met. By western blotting and qualitative real time‐polymerase chain reaction analysis (qRT‐PCR) analysis, we next revealed that GlyRS is required for amino acid‐induced activation of the mTOR‐S6K1/4EBP1 pathway. Thus, this study establishes that GlyRS mediates amino acid‐induced activation of the mTOR pathway, thereby regulating milk protein and fat synthesis.

The mammary gland requires the uptake of AA for milk protein synthesis during lactation. The L-type amino acid transporter 1 (LAT1, encoded by SLC7A5), found in many different types of mammalian cells, is indispensable as a transporter of essential AA to maintain cell growth and protein synthesis. However, the function of LAT1 in regulating milk protein synthesis in the mammary gland of the dairy cow remains largely unknown. For the current study, we characterized the relationship between LAT1 expression and milk protein synthesis in lactating dairy cows and investigated whether the mammalian target of rapamycin complex 1 (mTORC1) signaling controls the expression of LAT1 in their mammary glands. We found that LAT1 and the heavy chain of its chaperone, 4F2, were expressed in mammary tissues of lactating cows, with the expression levels of LAT1 and the 4F2 heavy chain being significantly greater in lactating mammary tissues with high-milk protein content (milk yield, 33.8 ± 2.1 kg/d; milk protein concentration >3%, wt/vol,; n = 3) than in tissues from cows with low-milk protein content (milk yield, 33.7 ± 0.5 kg/d; milk protein concentration <3%, wt/vol; n = 3). Immunofluorescence staining of sectioned mammary tissues from cows with high and low milk protein content showed that LAT1 was located on the whole plasma membrane of alveolar epithelial cells, suggesting that LAT1 provides essential AA to the mammary gland. In cultured mammary epithelial cells from the dairy cows with high-milk protein content, knockdown of LAT1 expression decreased cell viability and β-casein expression; in contrast, overexpression of LAT1 had the opposite effect. Inhibition of mTORC1 by rapamycin attenuated the phosphorylation of molecules related to mTORC1 signaling and caused a marked decrease in LAT1 expression in the cultured cells; expression of β-casein also decreased significantly. These results suggest that LAT1 is involved in milk protein synthesis in the mammary glands of lactating dairy cows and that the mTORC1 signaling pathway might be a control point for regulation of LAT1 expression, which could ultimately be used to alter milk protein synthesis.