Stearic acid promotes lipid synthesis through CD36/Fyn/FAK/mTORC1 axis in bovine mammary epithelial cells.

Stearic acid (C18:0, SA) is a saturated long-chain fatty acid (LCFA) that has a prominent function in lactating dairy cows. It is obtained primarily from the diet and is stored in the form of triacylglycerol (TAG) molecules. The transmembrane glycoprotein cluster of differentiation 36 (CD36) is also known as fatty acid translocase, but whether SA promotes lipid synthesis through CD36 and FAK/mTORC1 signaling is unknown. In this study, we examined the function and mechanism of CD36-mediated SA-induced lipid synthesis in bovine mammary epithelial cells (BMECs). SA-enriched supplements enhanced lipid synthesis and the FAK/mTORC1 pathway in BMECs. SA-induced lipid synthesis, FAK/mTORC1 signaling, and the expression of lipogenic genes were impaired by anti-CD36 and the CD36-specific inhibitor SSO, whereas overexpression of CD36 effected the opposite results. Inhibition of FAK/mTORC1 by TAE226/Rapamycin attenuated SA-induced TAG synthesis, inactivated FAK/mTORC1 signaling, and downregulated the lipogenic genes PPARG, CD36, ACSL1, SCD, GPAT4, LIPIN1, and DGAT1 at the mRNA and protein levels in BMECs. By coimmunoprecipitation and yeast two-hybrid screen, CD36 interacted directly with Fyn but not Lyn, and Fyn bound directly to FAK; FAK also interacted directly with TSC2. CD36 linked FAK through Fyn, and FAK coupled mTORC1 through TSC2 to form the CD36/Fyn/FAK/mTORC1 signaling axis. Thus, stearic acid promotes lipogenesis through CD36 and Fyn/FAK/mTORC1 signaling in BMECs. Our findings provide novel insights into the underlying molecular mechanisms by which LCFA supplements promote lipid synthesis in BMECs.

Enhancement of PPARα-Inhibited Leucine Metabolism-Stimulated ß-Casein Synthesis and Fatty Acid Synthesis in Primary Bovine Mammary Epithelial Cells.

Leucine, a kind of branched-chain amino acid, plays a regulatory role in the milk production of mammalian mammary glands, but its regulatory functions and underlying molecular mechanisms remain unknown. This work showed that a leucine-enriched mixture (LEUem) supplementation increased the levels of milk protein and milk fat synthesis in primary bovine mammary epithelial cells (BMECs). RNA-seq of leucine-treated BMECs indicated alterations in lipid metabolism, translation, ribosomal structure and biogenesis, and inflammatory response signaling pathways. Meanwhile, the supplementation of leucine resulted in mTOR activation and increased the expression of BCKDHA, FASN, ACC, and SCD1. Interestingly, the expression of PPARα was independently correlated with the leucine-supplemented dose. PPARα activated by WY-14643 caused significant suppression of lipogenic genes expression. Furthermore, WY-14643 attenuated leucine-induced ß-casein synthesis and enhanced the level of BCKDHA expression. Moreover, promoter analysis revealed a peroxisome-proliferator-response element (PPRE) site in the bovine BCKDHA promoter, and WY-14643 promoted the recruitment of PPARα onto the BCKDHA promoter. Together, the present data indicate that leucine promotes the synthesis of ß-casein and fatty acid and that PPARα-involved leucine catabolism is the key target.

Effects of dietary folic acid supplementation on lactation performance and mammary epithelial cell development of dairy cows and its regulatory mechanism.

The experiment investigated the impacts of FA on the proliferation of bovine mammary gland epithelial cells (BMECs) and to investigate the underlying mechanisms. Supplementation of 10 µM FA elevated the mRNA expression of proliferating cell nuclear antigen (PCNA), cyclin A2 and cyclin D1, and protein expression of PCNA and Cyclin A1. The mRNA and protein expression of B-cell lymphoma-2 (BCL2) and the BCL2 to BCL2 associated X 4 (BAX4) ratio elevated, while that of BAX, Caspase-3 and Caspase-9 reduced by FA. Both Akt and mTOR signaling pathways were activated by FA. Moreover, the stimulation of BMECs proliferation, the alteration of proliferative genes and protein expression, the change of apoptotic genes and protein expression, and the activation of mTOR signaling pathway caused by FA were obstructed by Akt inhibitor. Suppression of mTOR with Rapamycin reversed the FA-modulated promotion of BMECs proliferation and change of proliferous genes and protein expression, with no impact on mRNA or proteins expression related to apoptosis and FA-activated Akt signaling pathway. Supplementation of rumen-protected FA in cow diets evaluated milk yields and serum insulin-like growth factor-1 and estradiol levels. The results implied that the proliferation of BMECs was stimulated by FA through the Akt-mTOR signaling pathway.

Docosahexaenoic Acid Alters Lipid Metabolism Processes via H3K9ac Epigenetic Modification in Dairy Goat.

Goat milk is increasingly recognized by consumers due to its high nutritional value, richness in short- and medium-chain fatty acids, and richness in polyunsaturated fatty acids (PUFA). Exogenous supplementation of docosahexaenoic acid (DHA) is an important approach to increasing the content of PUFA in goat milk. Several studies have reported benefits of dietary DHA in terms of human health, including potential against chronic diseases and tumors. However, the mechanisms whereby an increased supply of DHA regulates mammary cell function is unknown. In this study, we investigated the effect of DHA on lipid metabolism processes in goat mammary epithelial cells (GMEC) and the function of H3K9ac epigenetic modifications in this process. Supplementation of DHA promoted lipid droplet accumulation increased the DHA content and altered fatty acid composition in GMEC. Lipid metabolism processes were altered by DHA supplementation through transcriptional programs in GMEC. ChIP-seq analysis revealed that DHA induced genome-wide H3K9ac epigenetic changes in GMEC. Multiomics analyses (H3K9ac genome-wide screening and RNA-seq) revealed that DHA-induced expression of lipid metabolism genes (FASN, SCD1, FADS1, FADS2, LPIN1, DGAT1, MBOAT2), which were closely related with changes in lipid metabolism processes and fatty acid profiles, were regulated by modification of H3K9ac. In particular, DHA increased the enrichment of H3K9ac in the promoter region of PDK4 and promoted its transcription, while PDK4 inhibited lipid synthesis and activated AMPK signaling in GMEC. The activation of the expression of fatty acid metabolism-related genes FASN, FADS2, and SCD1 and their upstream transcription factor SREBP1 by the AMPK inhibitor was attenuated in PDK4-overexpressing GMEC. In conclusion, DHA alters lipid metabolism processes via H3K9ac modifications and the PDK4-AMPK-SREBP1 signaling axis in goat mammary epithelial cells, providing new insights into the mechanism through which DHA affects mammary cell function and regulates milk fat metabolism.

Effects of glucose availability on αS1-casein synthesis in bovine mammary epithelial cells.

Glucose has been demonstrated to affect milk protein synthesis in dairy cows. However, its potential mechanisms has not been thoroughly studied. The objective of this study was to investigate the effects of glucose availability on αS1-casein synthesis, glucose uptake, metabolism, and the expression of proteins involved in AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells (BMEC). BMEC were treated for 24 h with different concentrations of glucose (0, 7, 10.5, 14, 17.5, and 21 mM). The results showed that 10.5 and 14 mM glucose supply increased the expression of αS1-casein, glucose uptake, cellular ATP content, and the phosphorylation of mTOR and P70S6K, but repressed AMPK phosphorylation in BMEC. Compared with 10.5 and 14 mM glucose supply, 17.5 and 21 mM glucose decreased the expression of αS1-casein, P70S6K phosphorylation as well as the activity of hexokinase (HK) and pyruvate kinase (PK), but increased the activity of glucose-6-phosphate dehydrogenase (G6PD). These results indicate that 10.5 to 14 mM glucose supply is the proper range for αS1-casein synthesis, and the promotion effects may be related to the increase of glucose uptake, ATP content and the changes of key proteins' phosphorylation in AMPK/mTOR signaling pathway. However, the inhibition of the expression of αS1-casein by 17.5 and 21 mM glucose may be associated with the changes of key enzymes' activity involved in glucose metabolism.

Glucose play an important role in milk protein synthesis in dairy cows. But the effects of glucose availability on casein synthesis and its underlying mechanisms has not been thoroughly studied. To elucidate the underlying mechanisms of glucose availability affecting casein synthesis, the effects of glucose availability on αS1-casein synthesis, glucose uptake, metabolism, and the expression of proteins involved in AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells were measured. We found that the expression of αS1-casein increased with 10.5 and 14 mM glucose supplementation, which may be associated with the increase of glucose uptake, ATP content and the changes of key proteins' phosphorylation in AMPK/mTOR signaling pathway. The inhibition of αS1-casein expression with 17.5 and 21 mM glucose supplementation may be related to the changes of key enzymes' activity involved in glucose metabolism. This study provided an insight into the potential mechanisms of glucose availability affecting milk protein synthesis.

In vitro effects of 5-Hydroxy-L-tryptophan supplementation on primary bovine mammary epithelial cell gene expression under thermoneutral or heat shock conditions.

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. Exposure of dairy cows to hyperthermia during the dry period alters mammary gland involution processes leading to reduced subsequent yields. Herein, primary bovine mammary epithelial cells (pBMEC) under thermoneutral (TN, 37 °C) or heat shock (HS, 41.5 °C) conditions were cultured with either 0, 50, 200, or 500 µM 5-Hydroxy-L-tryptophan (5-HTP; 5-HT precursor) for 8-, 12- or 24-h. Expression of 95 genes involved in 5-HT signaling, involution and tight junction regulation were evaluated using a Multiplex RT-qPCR BioMark Dynamic Array Circuit. Different sets of genes were impacted by 5-HTP or temperature, or by their interaction. All 5-HT signaling genes were downregulated after 8-h of HS and then upregulated after 12-h, relative to TN. After 24-h, apoptosis related gene, FASLG, was upregulated by all doses except TN-200 µM 5-HTP, and cell survival gene, FOXO3, was upregulated by HS-50, 200 and 500 µM 5-HTP, suggesting 5-HTP involvement in cell turnover under HS. Supplementing 5-HTP at various concentrations in vitro to pBMEC modulates the expression of genes that might aid in promoting epithelial cell turn-over during involution in dairy cattle under hyperthermia.

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes.

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

Selenium and Taurine Combination Is Better Than Alone in Protecting Lipopolysaccharide-Induced Mammary Inflammatory Lesions via Activating PI3K/Akt/mTOR Signaling Pathway by Scavenging Intracellular ROS.

Mastitis is mainly induced by gram-negative bacterial infections, causing devastating economic losses to the global cattle industry. Both selenium (Se) and taurine (Tau) exhibit multiple biological effects, including reducing inflammation. However, no studies have reported the protective effect of the combined use of Se and Tau against mastitis, and the underlying mechanisms remain unclear. In this study, lipopolysaccharide (LPS), the vital virulence factor of gram-negative bacteria, was used to construct the in vivo and vitro mastitis models. The results of in vivo model showed that Se and Tau combination was more effective than either substance alone in reducing tissue hyperemia, edema, and neutrophil infiltration in the mammary acinar cavity, improving the blood-milk barrier in LPS-induced mice mastitis, and decreasing the expression of proinflammatory factors and the activity of MPO. Moreover, Se and Tau combination significantly increased the levels of LPS-induced reduction in PI3K/Akt/mTOR, but the expressions of TLRs and NLRP3 were not significantly changed in the mammary tissue. In the in vitro experiments, the effects of Se and Tau combination or alone on inflammatory factors, inflammatory mediators, MPO activity, and blood-milk barrier were consistent with those in vivo. The Se and Tau combination has also been found to increase the survival rate of BMECs compared with each substance alone via promoting cellular proliferation and inhibiting apoptosis. Also, it has been confirmed that this combination could restore the LPS-induced inhibition in the PI3K/Akt/mTOR signaling pathway. Inhibition of mTOR by Rapamycin counteracted the combined protection of SeMet and Tau against LPS-induced inflammatory damage, the inhibition of PI3K by LY294002 blocked the activation of mTOR, and the accumulation of ROS by the ROS agonist blocked the activation of PI3K. In conclusion, these findings suggested that Se and Tau combination was better than either substance alone in protecting LPS-induced mammary inflammatory lesions by upregulating the PI3K/Akt/mTOR signaling pathway.

Activation of FGFR2 Signaling Suppresses BRCA1 and Drives Triple-Negative Mammary Tumorigenesis That is Sensitive to Immunotherapy.

Fibroblast growth factor receptor 2 (FGFR2) is a membrane-spanning tyrosine kinase that mediates FGF signaling. Various FGFR2 alterations are detected in breast cancer, yet it remains unclear if activation of FGFR2 signaling initiates tumor formation. In an attempt to answer this question, a mouse model berrying an activation mutation of FGFR2 (FGFR2-S252W) in the mammary gland is generated. It is found that FGF/FGFR2 signaling drives the development of triple-negative breast cancer accompanied by epithelial-mesenchymal transition that is regulated by FGFR2-STAT3 signaling. It is demonstrated that FGFR2 suppresses BRCA1 via the ERK-YY1 axis and promotes tumor progression. BRCA1 knockout in the mammary gland of the FGFR2-S252W mice significantly accelerated tumorigenesis. It is also shown that FGFR2 positively regulates PD-L1 and that a combination of FGFR2 inhibition and immune checkpoint blockade kills cancer cells. These data suggest that the mouse models mimic human breast cancers and can be used to identify actionable therapeutic targets.

Omega-3 polyunsaturated fatty acids ameliorated inflammatory response of mammary epithelial cells and mammary gland induced by lipopolysaccharide.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), essential fatty acids for humans and animals, have been reported to play a beneficial role in a variety of inflammatory diseases. In this study, we investigated the inhibitory effects and potential molecular mechanisms of n-3 PUFAs on the inflammatory response in lipopolysaccharide (LPS)-stimulated mammary alveolar cell line (MAC-T). Results showed that n-3 PUFAs could abate LPS-induced secretions of tumor necrosis factor-α, interleukin (IL)-6 and IL-1ß in MAC-T cells through the nuclear transcription factor kappa B (NF-κB) signal pathway. Meanwhile, n-3 PUFA intervention attenuated histopathologic changes of mammary glands, the white blood cell number decrease, and the alkaline phosphatase level decrease in the serum of mice challenged by LPS. Furthermore, n-3 PUFA intervention improved the ecological structure of the flora in terms of the structural disorder of the non-significant dominant flora induced by LPS in mice. Collectively, both in vitro and in vivo experiments revealed that n-3 PUFAs have a positive effect on LPS-induced inflammatory response, which was possibly mediated by the NF-κB signaling pathway and the intestinal microbiota.

Glutamine Regulates Cell Growth and Casein Synthesis through the CYTHs/ARFGAP1-Arf1-mTORC1 Pathway in Bovine Mammary Epithelial Cells.

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 ß-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.

Dietary Arginine Supplementation during Gestation and Lactation Increases Milk Yield and Mammary Lipogenesis in Rats.

BACKGROUND: Arginine, an essential amino acid during the reproductive period, has been shown to enhance lactation performances in livestock. Whether it could help mothers with breastfeeding difficulties is not known. OBJECTIVES: This study aimed to determine whether dietary arginine supplementation would enhance milk production in rat dams nursing large 12-pup litters and, if so, what mechanisms are involved. METHODS: In 3 series of experiments, differing in dam killing timing, 59 primiparous, pregnant Sprague-Dawley rats (mean ± SD weight: 254 ± 24.7 g) were randomly assigned to receive either 1) an AIN-93G diet supplemented with l-arginine at 2.0% (ARG diet), through lactation and gestation (AGL group); 2) a control AIN-93G diet including at 3.5% an isonitrogenous mix of amino acids that are not essential for lactation (MA diet), during gestation and lactation (MA group); or 3) the MA diet during gestation and the ARG diet during lactation (AL group). Milk flow was measured using deuterated water enrichment between days 11 and 18. Plasma hormones and mammary expression of genes involved in lactation were measured using ELISA and qRT-PCR, respectively, at lactation days 12, 18, or 21 in the 3 experiments. Data were analyzed by ANOVA. RESULTS: Dam food intake, pup weight gain, milk flow normalized to dam weight, and milk fat concentration were 17%, 9%, 20%, and 20% greater in the AGL group than in the MA group, respectively (P < 0.05). Genes involved in lipogenesis and lipid regulation were overexpressed ≤2.76-fold in the mammary gland of AGL dams compared with MA dams (P < 0.05) and plasma leptin concentration was 39% higher (P = 0.008). Milk flow and composition and mammary gene expression of the AL group did not differ from those of the MA group, whereas milk fat concentration and flow were 26% and 37% lower than in the AGL group, respectively. CONCLUSIONS: Arginine supplementation during gestation and lactation enhances milk flow and mammary lipogenesis in rats nursing large litters.

Expression patterns of major genes in fatty acid synthesis, inflammation, oxidative stress pathways from colostrum to milk in Damascus goats.

The molecular regulation of milk secretion and quality in the transition period from colostrum to milk in goats is largely unknown. In the present study, mammary gland secretion of goats was collected in 0th, 4th, 7th, 14th and 28th days after parturition. In addition to composition and fatty acid profile of colostrum or milk, FASN, SCD, ACACA, COX-2, NRF2, TLR2, NF-kB, LTF and PTX3 genes expression patterns were determined from milk somatic cells. While somatic cell count (SCC), malondialdehyde (MDA), fat, fat-free dry matter, protein and lactose were highest as expression levels of the oxidative and inflammatory genes, freezing point and electrical conductivity were lowest in colostrum. With the continuation of lactation, most of the fatty acids, n3 ratio, and odour index increased but C14:0 and C16:0 decreased. While FASN was upregulated almost threefolds in 14th day, ACACA was upregulated more than fivefolds in 7th and 14th days. Separately, the major genes in fatty acid synthesis, inflammation and oxidative stress were significantly associated with each other due to being positively correlated. MDA was positively correlated with SCC and some of the genes related inflammation and oxidative stress. Furthermore, significant negative correlations were determined between SCC and fatty acid synthesis related genes. With this study, transition period of mammary secretion was particularly clarified at the molecular levels in Damascus goats.

Forsythiaside a plays an anti-inflammatory role in LPS-induced mastitis in a mouse model by modulating the MAPK and NF-κB signaling pathways.

Forsythiaside A, a major bioactive component extracted from Forsythiae fructus, possesses multiple biological properties, especially anti-inflammatory properties. In the present study, the anti-inflammatory effect of forsythiaside A was investigated in lipopolysaccharide (LPS)-induced acute mastitis in mice. Our results showed that the expression levels of IL-1ß, IL-6, TNF-α, p38 MAPK, IκBα, and NF-κB p65 in the LPS group were all up-regulated, and obvious pathological changes were observed by sectioning. Compared with those in the LPS group, the expression levels of the above factors were significantly reduced, and the inflammation symptoms were also significantly reduced by section observation after forsythiaside A intervention. These results indicated that forsythiaside A effectively inhibited LPS-induced mammary inflammation in mice by attenuating the activation of the NF-κB and p38 MAPK signaling pathways.

Suppressive effects of curcumin on milk production without inflammatory responses in lactating mammary epithelial cells.

BACKGROUND: Curcumin is a naturally occurring polyphenol found in Curcuma longa with multiple therapeutic properties, such as anti-inflammatory, wound healing and anti-cancer effects. Curcuma longa is also used as a galactagogue to improve milk production during lactation. PURPOSE: To assess curcumin could have therapeutic potential for breastfeeding mothers, we investigated whether and how curcumin influences milk production in lactating mammary epithelial cells (MECs) at the cellular and molecular levels. METHODS: We prepared a lactating MEC culture model that produced milk components and formed less-permeable tight junctions (TJs) to investigate the molecular mechanism of curcumin on milk production, TJs, and inflammation in vitro. RESULTS: Curcumin downregulated milk production in lactation MECs concurrently with inactivation of lactogenesis-relating signaling (STAT5 and glucocorticoid receptor). The maintenance of a less-permeable TJ barrier was also confirmed, although the TJ protein claudin-4 increased. Curcumin inactivated NFκB and STAT3 signaling, which are closely involved in inflammatory responses in weaning and mastitis mammary glands. The expression levels of IL-1ß and TNF-α were also decreased by curcumin treatment. Furthermore, curcumin blocked activation of inflammatory signaling by lipopolysaccharide treatment in MECs, similar to those in MECs that were treated with diclofenac sodium. The drastic phosphorylation of ERK was induced by curcumin treatment in the absence of EGF. U0126, an inhibitor of ERK phosphorylation, attenuated the adverse effects of curcumin on lactating MECs. CONCLUSION: The results of the present study suggests that curcumin downregulates milk production via inactivation of STAT5 and GR signaling with concurrent suppression of inflammatory responses via STAT3 and NFκB signaling in MECs. These findings provide new insights into the role of curcumin as a mild suppressor of milk production without inflammatory damages in breastfeeding mothers.

Effect of Hordei Fructus Germinatus on differential gene expression in the prolactin signaling pathway in the mammary gland of lactating rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In China, Hordei Fructus Germinatus (HFG) is the germinated and dried fruit of Hordeum vulgare L, which is commonly used in clinical Chinese medicine. Traditional Chinese Medicine (TCM) theory holds that HFG can be both medicinal and edible, which means that it is derived from food medicine. Raw HFG and roasted HFG are used to treat hypogalactia, hyperprolactinemia and indigestion. In recent years, the lactogenic and galactophygous effects of HFG have attracted increasing attention. Nevertheless, there is much confusion over the use of raw and processed HFG, and the mechanism of its lactogenic effect seems remains poorly understood. AIM OF THE STUDY: This study aimed to explore the lactogenic effect of raw HFG and roasted HFG on rats with overloaded lactation and to reveal the underlying molecular mechanism. MATERIALS AND METHODS: Raw and processed HFG water decoctions were given to overloaded lactation model rats at a dose of 1.7800 g kg-1·d-1, and the control group was given the same volume of water. The lactogenic effect of raw and processed HFG was evaluated by measuring daily lactation, body weight and pup body weight, serum PRL, E2, and GH contents after parturition, and the pathological characteristics of mammary tissue sections. cDNA microarrays can be used to screen diverse gene expression patterns and signaling pathways related to prolactin. The expression of relevant differentially expressed genes was verified by real-time PCR and western blotting. RESULTS: In vivo experiments demonstrated that the raw HFG water decoction stimulated mammogenesis, accelerated the transformation of the lobular acinar system, resulted in denser mammary epithelial cells and thicker glandular ducts that were full of milk and facilitated the secretion of milk. Moreover, HFG increased PRL, E2, and GH levels, pup body weight, daily lactation and the body weight of lactating rats. Following gene chip identification, KEGG pathway enrichment analysis revealed genes that were highly related to prolactin in the prolactin signaling pathway and JAK-STAT signaling pathway, and the main differentially expressed genes were Jak2 (down), Stat5α (up), cyclin D1 (up), SOCS1 (up), CISH (down) and PRLR (up). Compared with the control group, RT-PCR results indicated that Jak2 and CISH were downregulated and that Stat5α, cyclin D1, SOCS1 and PRLR were upregulated. Western blot assays showed that PRLR, STAT5α and cyclin D1 levels in the mammary glands of the raw HFG water decoction group were significantly increased, which was consistent with the results of cDNA microarray screening. CONCLUSION: The present study reveals that raw HFG effectively enhances lactation in rats, possibly by influencing the prolactin/JAK-STAT signaling pathway.

Pistacia lentiscus extract enhances mammary epithelial cells' productivity by modulating their oxidative status.

We assessed the potential of phenolic compounds from Pistacia lentiscus (lentisk) to enhance production of milk constituents in bovine mammary epithelial cells (MEC). MEC were exposed to 0 (control), 1 or 10 ppm of polyphenols from lentisk ethanolic extract (PLEE) for 24 h. PLEE were absorbed by the MEC plasma membrane, but also penetrated the cell to accumulate in and around the nucleus. PLEE increased triglyceride content in the cell and its secretion to the medium, and significantly increased intracellular lipid droplet diameter. Compared to control, PLEE increased dose-dependently the lactose synthesis, secretion of whey proteins, and contents of casein. To evaluate mitochondrial activity under pro-oxidant load, MEC were preincubated with PLEE and exposed for 2 h to H2O2. Exposure to H2O2 increased the proportion of cells with impaired mitochondrial membrane potential twofold in controls, but not in PLEE-pre-treated cells. Accordingly, proton leakage was markedly decreased by PLEE, and coupling efficiency between the respiratory chain and ATP production was significantly enhanced. Thus, lentisk polyphenols divert energy to production of milk fat, protein and lactose, with less energy directed to cellular damage control; alternatively, PLEE enables MEC to maintain energy and oxidative status under extreme metabolic rate required for milk production and secretion, and reduces the limitation on energy required to support production.

Nutritional combinatorial impact on the gut microbiota and plasma short-chain fatty acids levels in the prevention of mammary cancer in Her2/neu estrogen receptor-negative transgenic mice.

Breast cancer is the second leading cause of cancer-related mortality in women. Various nutritional compounds possess anti-carcinogenic properties which may be mediated through their effects on the gut microbiota and its production of short-chain fatty acids (SCFAs) for the prevention of breast cancer. We evaluated the impact of broccoli sprouts (BSp), green tea polyphenols (GTPs) and their combination on the gut microbiota and SCFAs metabolism from the microbiota in Her2/neu transgenic mice that spontaneously develop estrogen receptor-negative [ER(-)] mammary tumors. The mice were grouped based on the dietary treatment: control, BSp, GTPs or their combination from beginning in early life (BE) or life-long from conception (LC). We found that the combination group showed the strongest inhibiting effect on tumor growth volume and a significant increase in tumor latency. BSp treatment was integrally more efficacious than the GTPs group when compared to the control group. There was similar clustering of microbiota of BSp-fed mice with combination-fed mice, and GTPs-fed mice with control-fed mice at pre-tumor in the BE group and at pre-tumor and post-tumor in the LC group. The mice on all dietary treatment groups incurred a significant increase of Adlercreutzia, Lactobacillus genus and Lachnospiraceae, S24-7 family in the both BE and LC groups. We found no change in SCFAs levels in the plasma of BSp-fed, GTPs-fed and combination-fed mice of the BE group. Marked changes were observed in the mice of the LC group consisting of significant increases in propionate and isobutyrate in GTPs-fed and combination-fed mice. These studies indicate that nutrients such as BSp and GTPs differentially affect the gut microbial composition in both the BE and LC groups and the key metabolites (SCFAs) levels in the LC group. The findings also suggest that temporal factors related to different time windows of consumption during the life-span can have a promising influence on the gut microbial composition, SCFAs profiles and ER(-) breast cancer prevention.

Fenugreek Stimulates the Expression of Genes Involved in Milk Synthesis and Milk Flow through Modulation of Insulin/GH/IGF-1 Axis and Oxytocin Secretion.

We previously demonstrated galactagogue effect of fenugreek in a rat model of lactation challenge, foreshadowing its use in women's breastfeeding management. To assess longitudinal molecular mechanisms involved in milk synthesis/secretion in dams submitted to fenugreek supplementation, inguinal mammary, pituitary glands and plasma were isolated in forty-three rats nursing large 12 pups-litters and assigned to either a control (CTL) or a fenugreek-supplemented (FEN) diet during lactation. RT-PCR were performed at days 12 and 18 of lactation (L12 and L18) and the first day of involution (Inv1) to measure the relative expression of genes related to both milk synthesis and its regulation in the mammary gland and lactogenic hormones in the pituitary gland. Plasma hormone concentrations were measured by ELISA. FEN diet induced 2- to 3-times higher fold change in relative expression of several genes related to macronutrient synthesis (Fasn, Acaca, Fabp3, B4galt1, Lalba and Csn2) and energy metabolism (Cpt1a, Acads) and in IGF-1 receptor in mammary gland, mainly at L12. Pituitary oxytocin expression and plasma insulin concentration (+77.1%) were also significantly increased. Altogether, these findings suggest fenugreek might extend duration of peak milk synthesis through modulation of the insulin/GH/IGF-1 axis and increase milk ejection by activation of oxytocin secretion.

Short communication: Effect of supplemental zinc source with and without evaporative cooling on systemic and mammary metabolism of lactating dairy cows during summer.

The negative effects of heat stress partly result from disturbed systemic metabolic responses and possibly altered mammary gland metabolism of lactating dairy cows. Our previous research reported that supplemental dietary Zn sources may affect milk fat synthesis of lactating cows during summer. Thus, our objective was to evaluate the systemic and mammary metabolism of cows fed 2 supplemental Zn sources under 2 environmental conditions. Multiparous lactating Holstein cows (n = 72; days in milk: 99.7 ± 13.4 d; parity: 2.9 ± 0.3) were randomly assigned to 4 treatments in a 2 × 2 factorial arrangement. Treatments included 2 different environments: cooled (CL) using fans and misters or noncooled (NC), and 2 supplemental Zn sources: 75 mg of Zn hydroxychloride/kg of DM (IOZ) or 35 mg of Zn hydroxychloride/kg of DM + 40 mg of Zn-Met complex/kg of DM (ZMC). The 168-d experiment was divided into baseline and environmental challenge phases, 84 d each. During the baseline phase, all cows were cooled and fed respective dietary treatments, and during the environmental challenge phase cows continued receiving the same diets but NC cows were deprived of cooling. Temperature-humidity index averaged 77.6 ± 3.8 and 77.8 ± 3.8 for CL and NC pens, respectively, during the environmental challenge phase. Plasma was collected before the baseline phase and at 1, 3, 5, 12, 22, 26, 41, 54, 61, 68, 75, and 81 d of the environmental challenge phase for metabolites and insulin analyses. Mammary biopsies were collected before the baseline phase and at 7 and 56 d of the environmental challenge phase to measure mRNA abundance of proteins related to mammary metabolism. Compared with CL, NC reduced plasma glucose, nonesterified fatty acids, ß-hydroxybutyrate, and triglyceride concentrations, but increased insulin concentration. Cows fed ZMC had greater plasma triglyceride concentration than IOZ. Treatments had no effect on mRNA abundance of protein related to mammary fatty acid and glucose metabolism except that NC cows had greater mammary mRNA abundance of 6-phosphogluconate dehydrogenase and ATP-dependent 6-phosphofructokinase than CL cows. In conclusion, deprivation of evaporative cooling influenced the metabolism of lactating dairy cows but dietary Zn source had no apparent effect.