Acidity-activatable dynamic hybrid nanoplatforms derived from extracellular vesicles of M1 macrophages enhance cancer immunotherapy through synergistic triple immunotherapy.

Immunotherapy exhibits considerable promise for sustained tumor reduction. However, current cancer immunotherapy methods elicit limited responses due to the inadequate immunogenicity exhibited by cancer cells. This obstacle may be addressed using nanoplatforms that can activate synergistic therapies (photodynamic therapy and ferroptosis) in response to the acidic pH of the tumor microenvironment. We previously developed an amphiphilic photosensitizer, SR780, which displays satisfactory photodynamic effects. This photosensitizer is inactivated when bound to Fe3+ (SR780Fe) but is activated upon release in mildly acidic conditions. In this study, M1 macrophage-derived extracellular vesicles (EVs) were fused with REV and SR780Fe-loaded liposomes (REV@SR780Fe@Lip) to form REV@SR780Fe@LEV hybrid nanovesicles. Further modification with the RS17 peptide for tumor targeting enabled a combination of photodynamic therapy, ferroptosis, and cGAS-STING pathway activation, resulting in enhanced antitumor efficacy through a synergistic effect. Upon laser irradiation, REV@SR780Fe@LEV-RS17 demonstrated antitumor effects in 4T1 breast cancer models, including the inhibition of lung and liver metastasis, as well as prevention of tumor recurrence.

PI3K/AKT/mTORC1 signalling pathway regulates MMP9 gene activation via transcription factor NF-κB in mammary epithelial cells of dairy cows.

Matrix metalloproteinase 9 (MMP9) plays a pivotal role in mammary ductal morphogenesis, angiogenesis and glandular tissue architecture remodeling. However, the molecular mechanism of MMP9 expression in mammary epithelial cells of dairy cows remains unclear. This study aimed to explore the underlying mechanism of MMP9 expression. In this study, to determine whether the PI3K/AKT/mTORC1/NF-κB signalling pathway participates in the regulation of MMP9 expression, we treated mammary epithelial cells with specific pharmacological inhibitors of PI3K (LY294002), mTORC1 (Rapamycin) or NF-κB (Celastrol), respectively. Western blotting results indicated that LY294002, Rapamycin and Celastrol markedly decreased MMP9 expression and P65 nuclear translocation. Furthermore, we found that NF-κB (P65) overexpression resulted in elevated expression of MMP9 protein and activation of MMP9 promoter. In addition, we observed that Celastrol markedly decreases P65-overexpression-induced MMP9 promoter activity. Moreover, the results of the promoter assay indicated that the core regulation sequence for MMP9 promoter activation may be located at -420 ∼ -80 bp downstream from the transcription start site. These observations indicated that the PI3K/AKT/mTORC1 signalling pathway is involved in MMP9 expression by regulating MMP9 promoter activity via NF-κB in the mammary epithelial cells of dairy cows.

Prolactin upregulates amino acids uptake in dairy cow mammary epithelial cells via LAT1.

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.

Potential of metagenomic next-generation sequencing in detecting infections of ICU patients.

BACKGROUND: Due to the limitations of traditional microbiological detection techniques in evaluating complicated infections in ICU patients, it is necessary to explore novel and effective methods to improve the clinical detection of ICU patients' infections. OBJECTIVE: This study aimed to evaluate the efficiency and specificity of mNGS in screening pathogens in the blood, deep phlegm, urine, and other sample types of ICU patients exploring an effective method for infection detection. METHODS: A total of 56 ICU patients with 131 samples were included in this study. The sample types included blood, deep phlegm, urine, drainage, anal swabs, and other types. Samples were analyzed by both conventional detection method and mNGS tests. The diagnosis efficiency and consistency of the two methods were compared. The distribution of the identified pathogens was analyzed. Moreover, the clinical features of patients with mNGS-positive or mNGS-negative results were compared. RESULTS: The positive rate of mNGS was 81.7% (107/131) including 3.1% (4/131) weakly positive, while the positive rate of traditional detection was only 30.5%, including 29 strong positive results and 11 weak positive results. Additionally, there were 41 patients chose to adjust anti-infection strategies according to the results of mNGS, which significantly saved treatment costs. The mNGS-positive patients showed a shorter ICU hospitalization and higher intention to adjust anti-infection strategies than the mNGS-negative patients. CONCLUSION: mNGS is of great potential for the pathogen detection of ICU patients, and has a higher detection rate than traditional detection methods. Further clinical application investigations can be carried out to expand the application of mNGS.

mTORC2-AKT-LAT1 signalling participates in methionine-induced ß-CASEIN expression in mammary epithelial cells of dairy cows.

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 ß-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 ß-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.

PPARγ-AGPAT6 signaling mediates acetate-induced mTORC1 activation and milk fat synthesis in mammary epithelial cells of dairy cows.

This 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.

Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC) regulates acetate- and ß-hydroxybutyrate-induced milk fat synthesis by increasing FASN expression in mammary epithelial cells of dairy cows.

Increasing acetate and ß-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 ß (C/EBPß) regulates cell growth and differentiation in the mammary gland. We demonstrated that the FASN promoter had a canonical C/EBPß 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/EBPß-dependent manner, which provides new insights into the understanding of the molecular events involved in milk fat synthesis.

Slc43a3 is a regulator of free fatty acid flux.

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.

Quantitative proteomics identified 3 oxidative phosphorylation genes with clinical prognostic significance in gastric cancer.

The aim of the present study was to explore the underlying mechanisms involved in gastric cancer (GC) formation using data-independent acquisition (DIA) quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in biological metabolic processes in GC. Totally, 745 differentially expressed proteins (DEPs) were found in GC tissues vs. gastric normal tissues. Despite enormous complexity in the details of the underlying regulatory network, we find that clusters of proteins from the DEPs were mainly involved in 38 pathways. All of the identified DEPs involved in oxidative phosphorylation were down-regulated. Moreover, GC possesses significantly altered biological metabolic processes, such as NADH dehydrogenase complex assembly and tricarboxylic acid cycle, which is mostly consistent with that in KEGG analysis. Furthermore the higher expression of UQCRQ, NDUFB7 and UQCRC2 were positively correlated with a better prognosis, implicating these proteins may as novel candidate diagnostic and prognostic biomarkers.

Laminin-dependent integrin ß1 signaling regulates milk protein synthesis via prolactin/STAT5 pathway in bovine mammary epithelial cells.

Successful induction of milk protein synthesis relies on prolactin/STAT5. In mice, both laminin and ß1 integrin were necessary for STAT5 activity induced by prolactin treatment, resulting in transcriptional activation of ß-casein. However, the mechanism by which ß1 integrin increases the bovine milk protein synthesis is not well known. In order to display the crosstalk between integrin signaling and lactogenic signaling, we investigated the mechanism by which laminin mediated lactogenic effects via interaction with ß1 integrin on bovine mammary epithelial cells (BMECs). Therefore, localization of ß1 integrin was examined by immunofluorescence. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting. The results showed that ß1 integrin were detected in basal mammary cells and basal membrane surface of adherent BMECs. However, basal distribution of ß1 integrin was not sufficient to increase ß-casein synthesis in the absence of integrin activation by laminin. A lactogenic hormone cocktail of insulin, hydrocortisone, and prolactin stimulated overall lactogenic effects, including upregulated expression of ß1 integrin, activation of prolactin/STAT5 signaling, and consequent increase of ß-casein synthesis. In response to a 24 h prolactin treatment, the abundance of STAT5, ß1 integrin, and ß-casein in BMECs with laminin was higher compared to that with a control substratum. Meanwhile, laminin-dependent lactogenic effects were inhibited by blocking ß1 integrin function, resulting in attenuated STAT5 activity and decreased ß-casein synthesis. These results indicated that ß1 integrin was a key mediator of the laminin-dependent prolactin/STAT5 signaling, which regulated the sustained STAT5 activity necessary for ß-casein expression in BMECs.

Prolactin regulates LAT1 expression via STAT5 (signal transducer and activator of transcription 5) signaling in mammary epithelial cells of dairy cows.

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 ß-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.

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

In this research communication, a cell model with elevated ß-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.

Wnt signaling regulates cytosolic translocation of connexin 43.

The availability of intracellular, stabilized ß-catenin, a transcription factor coactivator, is tightly regulated; ß-catenin is translocated into the nucleus in response to Wnt ligand binding to its cell membrane receptors. Here we show that Wnt signal activation in mammalian cells activates intracellular mobilization of connexin 43 (Cx43), which belongs to a gap junction protein family, a new target protein in response to extracellular Wnt signal activation. Transmission electron microscopy showed that the nuclear localization of Cx43 was increased by 8- to 10-fold in Wnt5A- and 9B-treated cells compared with controls; this Wnt-induced increase was negated in the cells where Cx43 and ß-catenin were knocked down using shRNA. There was a significant (P < 0.001) and concomitant depletion of the cell membrane and cytosolic signal of Cx43 in Wnt-treated cells with an increase in the nuclear signal for Cx43; this was more obvious in cells where ß-catenin was knocked down using shRNA. Conversely, Cx43 knockdown resulted in increased ß-catenin in the nucleus in the absence of Wnt activation. Coimmunoprecipitation of Cx43 and ß-catenin proteins with a casein kinase (CKIδ) antibody showed that Cx43 interacts with ß-catenin and may form part of the so-called destruction complex. Functionally, Wnt activation increased the rate of wound reepithelization in rat skin in vivo.

Functional analysis of the dairy cow mammary transcriptome between early lactation and mid-dry period.

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.

CaDHN5, a Dehydrin Gene from Pepper, Plays an Important Role in Salt and Osmotic Stress Responses.

Dehydrins (DHNs), as a sub-family of group two late embryogenesis-abundant (LEA) proteins, have attracted considerable interest owing to their functions in enhancing abiotic stress tolerance in plants. Our previous study showed that the expression of CaDHN5 (a dehydrin gene from pepper) is strongly induced by salt and osmotic stresses, but its function was not clear. To understand the function of CaDHN5 in the abiotic stress responses, we produced pepper (Capsicum annuum L.) plants, in which CaDHN5 expression was down-regulated using VIGS (Virus-induced Gene Silencing), and transgenic Arabidopsis plants overexpressing CaDHN5. We found that knock-down of CaDHN5 suppressed the expression of manganese superoxide dismutase (MnSOD) and peroxidase (POD) genes. These changes caused more reactive oxygen species accumulation in the VIGS lines than control pepper plants under stress conditions. CaDHN5-overexpressing plants exhibited enhanced tolerance to salt and osmotic stresses as compared to the wild type and also showed increased expression of salt and osmotic stress-related genes. Interestingly, our results showed that many salt-related genes were upregulated in our transgenic Arabidopsis lines under salt or osmotic stress. Taken together, our results suggest that CaDHN5 functions as a positive regulator in the salt and osmotic stress signaling pathways.

FABP5 is a critical regulator of methionine- and estrogen-induced SREBP-1c gene expression in bovine mammary epithelial cells.

The intracellular fatty acid-binding proteins (FABPs) are a well-conserved family that function as lipid chaperones. Ongoing studies are focused on identification of the mechanistic complexity and vast biological diversity of different isoforms of FABPs. However, the molecular mechanism of FABP5 in the regulation of milk fat synthesis in the mammary gland of dairy cows is still largely unknown. Here, we report that FABP5 acts as a critical regulator of terol response element-binding protein-1c (SREBP-1c) gene expression induced by methionine (Met) and estrogen (E2) in bovine mammary epithelial cells (BMECs). We observed that the expression of FABP5 was markedly higher in dairy cow mammary tissue during the lactating period than the puberty period and the dry period. FABP5 is located in the cytoplasm, and Met and E2 significantly increase the protein levels of FABP5 in BMECs. Using gene function study approaches, we revealed that FABP5 positively regulates SREBP-1c gene expression and promotes milk fat synthesis. We confirmed that FABP5 is required for Met- and E2-induced SREBP-1c gene expression and milk fat synthesis. We further uncovered that fatty acids are needed for FABP5-mediated SREBP-1c gene expression. Thus, our study demonstrates that FABP5 is a critical regulator of Met- and E2-induced SREBP-1c gene expression leading to milk fat synthesis.

Knockdown of fascin-1 expression suppresses cell migration and invasion of non-small cell lung cancer by regulating the MAPK pathway.

Fascin-1 is a cytoskeletal protein and it can specifically bind to F-actin, it can be abnormally expressed in a variety of solid tumors. Fascin-1 was identified as a factor for poor prognosis in non-small cell lung cancer (NSCLC). However, the relevant molecular mechanisms are not yet fully understood. In this study, the fascin-1 knockdown cells were produced by lentivirus infection, and then cell proliferation, invasion and cell migration assay were used to investigate the role of fascin-1 in NSCLC cells. The MAPK pathway related proteins were determined by western blot. In the current study, lentivirus-mediated fascin-1 knockdown significantly decreased the proliferation of NSCLC cells. Furthermore, fascin-1 silencing partly inhibited cell invasion and migration. Inhibition of fascin-1 decreased the activity of the MAPK pathway. Therefore, targeting fascin-1 may inhibit the growth and metastasis of NSCLC cells, which is a potentially effective therapeutic strategy for treating NSCLC.

The effects of L-type amino acid transporter 1 on milk protein synthesis in mammary glands of dairy cows.

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 ß-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 ß-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.

The effects of cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC) on milk lipid synthesis in mammary glands of dairy cows.

Adequate lipid synthesis by the mammary gland during lactation is essential for the survival of mammalian offspring. Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC) is a lipid droplet-associated protein and functions to promote lipid accumulation and inhibit lipolysis in mice and human adipocytes. However, the function of CIDEC in regulation of milk lipid synthesis in dairy cow mammary gland remains largely unknown. In this study, 6 multiparous Holstein cows (parity = 3) in early lactation were allocated to high-fat milk (milk yield 33.9 ± 2.1 kg/d, milk fat >3.5%, n = 3) and low-fat milk (milk yield 33.7 ± 0.5 kg/d, milk fat <3.5%, n = 3) groups according to their milk fat content. Lactating cows were slaughtered at 90 d in milk and mammary tissues were collected to detect CIDEC localization. Immunofluorescence staining of sections of lactating mammary glands with high- and low-fat milk showed that CIDEC was expressed in the cytoplasm of epithelial cells and localized to lipid droplets. Lipid droplets and CIDEC protein were also detected in isolated lactating mammary epithelial cells of dairy cows. Immunostaining of CIDEC in isolated mammary epithelial cells also confirmed its presence in the nucleus. The knockdown of CIDEC in cultured bovine mammary epithelial cells decreased milk lipid content and reduced expression of genes associated with mammary de novo fatty acid synthesis, short- and long-chain intracellular fatty acid activation, triacylglycerol synthesis, and transcription regulation. These genes included those for acetyl-CoA carboxylase (ACC, -60%), fatty acid synthase (FASN, -65%), acyl-CoA synthetase short-chain family member 2 (ACSS2, -50%), acyl-CoA synthetase long-chain family member 1 (ACSL1, -30%), diacylglycerol acyltransferase 1 (DGAT1, -60%), sterol regulatory element-binding protein 1 (SREBP1, -45%), and SREBP cleavage activating protein (SCAP, -66%). Conversely, in cells overexpressing CIDEC, triacylglycerol content was increased, and transcription of those genes involved in milk lipid synthesis was coordinately upregulated. These results suggest that CIDEC plays an important role in regulating milk lipid synthesis in dairy cow mammary gland via a mechanism involving gene expression, which provides further insight into the mechanisms regulating mammary lipogenesis in ruminants.

Effects of glucose on lactose synthesis in mammary epithelial cells from dairy cow.

BACKGROUND: Lactose, as the primary osmotic component in milk, is the major determinant of milk volume. Glucose is the primary precursor of lactose. However, the effect of glucose on lactose synthesis in dairy cow mammary glands and the mechanism governing this process are poorly understood. RESULTS: Here we showed that glucose has the ability to induce lactose synthesis in dairy cow mammary epithelial cells, as well as increase cell viability and proliferation. A concentration of 12 mM glucose was the optimum concentration to induce cell growth and lactose synthesis in cultured dairy cow mammary epithelial cells. In vitro, 12 mM glucose enhanced lactose content, along with the expression of genes involved in glucose transportation and the lactose biosynthesis pathway, including GLUT1, SLC35A2, SLC35B1, HK2, ß4GalT-I, and AKT1. In addition, we found that AKT1 knockdown inhibited cell growth and lactose synthesis as well as expression of GLUT1, SLC35A2, SLC35B1, HK2, and ß4GalT-I. CONCLUSIONS: Glucose induces cell growth and lactose synthesis in dairy cow mammary epithelial cells. Protein kinase B alpha acts as a regulator of metabolism in dairy cow mammary gland to mediate the effects of glucose on lactose synthesis.