Calcitriol promotes M2 polarization of tumor-associated macrophages in 4T1 mouse mammary gland cancer via the induction of proinflammatory cytokines.

Our research found that vitamin D3 (VD3) treatment increased lung metastasis in mice with 4T1 murine breast cancer (BC). This study aims to investigate the impact of VD3 on the activation of tumor-associated macrophages (TAMs) in BC. Mice bearing 4T1, E0771, 67NR BC cells, and healthy mice, were fed diets with varying VD3 contents (100-deficient, 1000-normal, and 5000 IU/kg-elevated). Some mice in the 1000 and 100 IU/kg groups received calcitriol. We studied bone metastasis and characterized TAMs and bone marrow-derived macrophages (BMDMs). 4T1 cells had higher bone metastasis potential in the 5000 IU/kg and calcitriol groups. In the same mice, an elevated tumor osteopontin level and M2 polarization of TAMs (MHCIIlow CD44high phenotype) were observed. Gene expression analysis confirmed M2 polarization of 4T1 (but not 67NR) TAMs and BMDMs, particularly in the 100 IU + cal group (increased Mrc1, Il23, and Il6). This polarization was likely due to COX-2/PGE2 induction in 4T1 calcitriol-treated cells, leading to increased proinflammatory cytokines like IL-6 and IL-23. Future studies will explore COX-2/PGE2 as a primary mediator of calcitriol-stimulated inflammation in the BC microenvironment, especially relevant for BC patients with VD3 deficiency and supplementation.

Pharmacodynamic structure of deer antler base protein and its mammary gland hyperplasia inhibition mechanism by mediating Raf-1/MEK/ERK signaling pathway activation.

Mammary gland hyperplasia (MGH) is a common mammary disease whose main pathogenesis is the disruption of estradiol (E2) and progesterone (P) secretion, thereby causing overproliferation of mammary epithelial cells and mammary gland tissue hyperplasia. Deer antler base is a traditional Chinese medicine that has been used for many years to treat MGH. However, its pharmacological mechanism and pharmacodynamic material basis are unclear. In this study, we for the first time used the graded salting method to classify deer antler base protein (CNCP) as CNCP-A, CNCP-B, and CNCP-C and explored the pharmacological mechanism of the anti-MGH properties of CNCP. We found that CNCP could regulate the hormonal levels of E2, P, and follicle stimulating hormone (FSH) and improve the histopathological condition. The potential mechanism might be related to the recombinant C-Raf proto oncogene serine/threonine protein kinase/mitogen-activated protein/extracellular regulated protein kinase (Raf-1/MEK/ERK) signaling pathway. By upregulating the protein expression of the follicle stimulating hormone receptor (FSHR), cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) inhibited the activation of the downstream Raf-1/MEK/ERK signaling pathway, which in turn inhibited the proliferation of mammary epithelial cells. We analyzed the physicochemical properties of CNCP-A, CNCP-B, and CNCP-C and obtained CNCP-C-I by column chromatographic purification of the best pharmacophore protein CNCP. Using high-performance liquid gel filtration chromatography (HPGFC), we determined the molecular weight of CNCP-C-I and identified it by high-performance liquid tandem mass spectrometry (LC-MS/MS) to obtain the first match for a high confidence protein KRT1. This study provides a theoretical basis for the development of effective traditional Chinese medicines with low toxicity levels for the prevention and treatment of mammary gland diseases.

Evaluation of Curcumin Therapeutic Effects on Histological Subtypes of Canine Mammary Gland Tumours.

Canine mammary gland tumors (CMGTs) are the most frequent types of cancer in bitches and proposed as a model of human breast cancer. The anticancer effect of curcumin on human breast cancer has been extensively studied. The aim of this study was to evaluate the therapeutic effect of curcumin in two different histologies (simple carcinoma [SC] and squamous cell carcinoma [SCC]) of CMGTs. Primary canine mammary cells were isolated from the tumoral tissues surgically resected from two bitches (Case 1 and Case 2). Cell viability, apoptotic percentage, cell cycle progression and the changes in the cell morphology were evaluated. Curcumin inhibited the growth of both SC (Case 1) and SCC (Case 2) cells. However, Case 1 cells (43.48% ± 3.87% at 0.5 µM) were more sensitive to curcumin than Case 2 cells (59.36% ± 2.09% at 0.5 µM). Curcumin induced total apoptotic cell death through G0/G1 arrest, and this effect was more profound in Case 1 cells. Furthermore, cytoplasmic vacuolization, apoptotic bodies and membrane blebbing were observed in both cells following curcumin treatment. Our findings provide a novel approach for the effects of curcumin as a natural compound on CMGTs. Further investigations should be performed to investigate the molecular mechanisms of the differences in curcumin efficacy for different histological subtypes.

Anticancer potential of (-)-epicatechin in a triple-negative mammary gland model.

OBJECTIVES: The main aim of this work was to analyse the potential tumour growth inhibition effects of (-)-epicatechin (EC). Triple-negative breast cancer (TNBC) is an invasive form of cancer characterized by the absence of progesterone receptor, estrogen receptor and human epidermal growth factor receptor 2. Doxorubicin (DOX) is widely used for its anti-tumour activity. EC belongs to the flavanol subfamily and is a candidate molecule for the adjuvant treatment of cancer due to its antiproliferative activities. METHODS: Evaluation of EC effects and pathways involved in a model of TNBC. KEY FINDINGS: EC inhibited tumour growth as efficiently as DOX (inhibition rates of 74% and 79% for EC and DOX, respectively). The evaluation of adenosine monophosphate-activated protein kinase (AMPK) and Akt phosphorylation and mTOR expression indicates that EC modulates these pathways, resulting in the inhibition of cell proliferation. Additionally, we found an increase in the survival of EC-treated animals compared with control-treated animals. This effect was similar to the effects induced by DOX (survival rates of 44% and 30% for EC and DOX, respectively). CONCLUSION: EC has antiproliferative properties and increases survival in a model of TNBC. These effects may occur through the modulation of deregulated AMPK and Akt/mTOR signalling pathways.

Effects of diets high in corn oil or in extra virgin olive oil on oxidative stress in an experimental model of breast cancer.

Experimental evidence highlights the importance of dietetic factors on breast cancer. In this work we aimed to analyze the effects two oils, corn oil (rich in n-6 polyunsaturated fatty acids -PUFA-) and extra virgin olive oil (EVOO), on oxidative stress in an animal model of breast carcinogenesis. Female rats were fed a low-fat control, a high-corn oil, or a high-EVOO diet from weaning or after induction with 7,12-dimethylbenz[a]anthracene at 53 days. Animals were euthanized at 36, 51, 100 and 246 days of age. We analyzed antioxidant enzymes (mRNA and activity of superoxide dismutase, glutathione peroxidase and catalase), non-enzymatic capacity (oxidized and reduced glutathione) and DNA damage (8-oxo-dG) in tumors and mammary gland at different ages. We also analyzed lipid peroxidation (isoprostanes in serum and lipofuscin in liver). Results indicated a decrease in the enzymatic antioxidant capacity and increased oxidative stress in mammary gland of healthy young animals after a short period of high-fat diets intake, followed by an adaptation to chronic dietary intervention. After induction both diets, especially the one high in n-6 PUFA, increased the oxidized glutathione. In tumors no clear effects of the high-fat diets were observed, although in the long-term lipofuscin and 8-oxo-dG suggested greater oxidative damage by effect of the n-6 PUFA-rich diet. Considering the differential effects of these diets on mammary carcinogenesis that we have previously reported, this study suggests that these high-fat diets could have an effect on oxidative stress that would lead to different signaling pathways.

Extracellular Spermine Activates DNA Methyltransferase 3A and 3B.

We first demonstrated that long-term increased polyamine (spermine, spermidine, putrescine) intake elevated blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. Because gene methylation status is closely associated with aging-associated conditions and polyamine metabolism is closely associated with regulation of gene methylation, we investigated the effects of extracellular spermine supplementation on substrate concentrations and enzyme activities involved in gene methylation. Jurkat cells and human mammary epithelial cells were cultured with spermine and/or D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Spermine supplementation inhibited enzymatic activities of adenosylmethionine decarboxylase in both cells. The ratio of decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine increased by DFMO and decreased by spermine. In Jurkat cells cultured with DFMO, the protein levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were not changed, however the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine, DNMT 3A and especially 3B were activated. We show that changes in polyamine metabolism dramatically affect substrate concentrations and activities of enzymes involved in gene methylation.

Parenteral administration of l-arginine to twin-bearing Romney ewes during late pregnancy is associated with reduced milk somatic cell count during early lactation.

Maternal milk is the primary source of nutrition for suckling mammals, and its yield and composition are important determinants of survival during the early neonatal period. The objective of this study was to examine whether parenteral administration of l-Arg to twin-bearing ewes, during mid to late pregnancy, influenced prepartum maternal mammary gland development and subsequent lactation performance in the early postpartum period (14 d). At 80 d of pregnancy, multiparous Romney ewes were housed indoors in group pens, split into 2 cohorts, and fed a lucerne-based pellet diet, formulated to meet 100% of National Research Council-recommended requirements for twin-bearing pregnant ewes, once a day. Cohort 1 was administered l-Arg (72.7 mg/kg of live weight via i.v, 3 times a day) from d 100 of pregnancy until d 140. At d 140, ewes were euthanized and maternal mammary tissues were collected for analysis of the biochemical indices total DNA, RNA, protein, protein synthetic efficiency (protein:RNA), cell size (protein:DNA), transcriptional efficiency (RNA:DNA), and the abundance of mammalian target of rapamycin (mTOR) and mTORSer2448 protein. Cohort 2 was administered an identical l-Arg regimen as cohort 1, but from d 100 until parturition. Milk was collected over a 14-d period (d 1, 4, 7, 10, and 14) to assess milk yield and composition. In cohort 1, total mammary DNA (cell number) tended to be higher in l-Arg ewes, with no change in total mammary RNA or protein content, biochemical indices of protein synthetic efficiency, cell size or transcriptional efficiency, or mTOR protein abundance or phosphorylation. In cohort 2, milk composition analysis from l-Arg ewes showed lower (d 7-14) milk somatic cell counts, greater crude protein percentage from d 7 to 10 but lower at d 14, and altered absolute concentrations of some free AA (d 7 and 14) compared with controls. We propose that parenteral administration of l-Arg during late pregnancy is associated with increased mammary gland cellular content and decreased somatic cell counts during early lactation.

Metabolic Profiling of Dietary Polyphenols and Methylxanthines in Normal and Malignant Mammary Tissues from Breast Cancer Patients.

SCOPE: Dietary polyphenols may protect against breast cancer. However, it is unknown whether polyphenols reach human malignant breast tumors in molecular forms and(or) at concentrations likely to act against cancer. METHODS AND RESULTS: Ninteen breast cancer patients consumed three capsules daily from biopsy-confirmed diagnosis to surgery (6 ± 2 days). The capsules contained pomegranate, orange, lemon, olive, cocoa, and grapeseed extracts plus resveratrol, providing 37 different phenolics (473.7 mg), theobromine and caffeine (19.7 mg). A total of 101 metabolites are identified in urine, 69 in plasma, 39 in normal (NT), and 33 in malignant (MT) tissues by UPLC-ESI-QTOF-MS. Eight control patients did not consume extracts. Phenolic-derived metabolites in MT and NT are mainly glucuronidated and(or) sulfated. Some representative metabolites detected in MT (median and range, pmol g-1 ) are urolithin-A-3-O-glucuronide (26.2; 3.2-66.5), 2,5-dihydroxybenzoic acid (40.2; 27.7-52.2), resveratrol-3-O-sulfate (86.4; 7.8-224.4), dihydroresveratrol-3-O-glucuronide (109.9; 10.3-229.4), and theobromine (715.0; 153.9-3,216). Metabolites, as detected in breast tissues, do not exert antiproliferative or estrogenic/antiestrogenic activities in MCF-7 breast cancer cells. CONCLUSION: This is the first study that describes the metabolic profiling of dietary phenolics and methylxanthines in MT and NT comprehensively. Although phase-II conjugation might hamper a direct anticancer activity, long-term tumor-senescent chemoprevention cannot be discarded.

Possible role of phytoestrogens in breast cancer via GPER-1/GPR30 signaling.

Estrogens generated within endocrine organs and the reproductive system act as ligands for at least three types of estrogen receptors. Estrogen receptors α (ERα) and ß (ERß) belong to the so-called classical family of estrogen receptors, whereas the G protein-coupled receptor GPR30, also known as GPER-1, has been described as a novel estrogen receptor sited in the cell membrane of target cells. Furthermore, these receptors are under stimulation of a family of exogenous estrogens, known as phytoestrogens, which are a diverse group of non-steroidal plant compounds derived from plant food consumed by humans and animals. Because phytoestrogens are omnipresent in our daily diet, they are becoming increasingly important in both human health and disease. Recent evidence indicates that in addition to classical estrogen receptors, phytoestrogens also activate GPER-1 a relevant observation since GPER-1 is involved in several physiopathological disorders and especially in estrogen-dependent diseases such as breast cancer.The first estrogen receptors discovered were the classical ERα and ERß, but from an evolutionary point of view G protein-coupled receptors trace their origins in history to over a billion years ago suggesting that estrogen receptors like GPER-1 may have been the targets of choice for ancient phytoestrogens and/or estrogens.This review provides a comprehensive and systematic literature search on phytoestrogens and its relationship with classical estrogen receptors and GPER-1 including its role in breast cancer, an issue still under discussion.

A Computational-Based Approach to Identify Estrogen Receptor α/ß Heterodimer Selective Ligands.

The biologic effects of estrogens are transduced by two estrogen receptors (ERs), ERα and ERß, which function in dimer forms. The ERα/α homodimer promotes and the ERß/ß inhibits estrogen-dependent growth of mammary epithelial cells; the functions of ERα/ß heterodimers remain elusive. Using compounds that promote ERα/ß heterodimerization, we have previously shown that ERα/ß heterodimers appeared to inhibit tumor cell growth and migration in vitro. Further dissection of ERα/ß heterodimer functions was hampered by the lack of ERα/ß heterodimer-specific ligands. Herein, we report a multistep workflow to identify the selective ERα/ß heterodimer-inducing compound. Phytoestrogenic compounds were first screened for ER transcriptional activity using reporter assays and ER dimerization preference using a bioluminescence resonance energy transfer assay. The top hits were subjected to in silico modeling to identify the pharmacophore that confers ERα/ß heterodimer specificity. The pharmacophore encompassing seven features that are potentially important for the formation of the ERα/ß heterodimer was retrieved and subsequently used for virtual screening of large chemical libraries. Four chemical compounds were identified that selectively induce ERα/ß heterodimers over their respective homodimers. Such ligands will become unique tools to reveal the functional insights of ERα/ß heterodimers.

Individual-specific variation in the respiratory activities of HMECs and their bioenergetic response to IGF1 and TNFα.

Metabolic reprograming is a hallmark of cancer cells. However, the roles of pre-existing differences in normal cells metabolism toward cancer risk is not known. In order to assess pre-existing variations in normal cell metabolism, we have quantified the inter-individual variation in oxidative metabolism of normal primary human mammary epithelial cells (HMECs). We then assessed their response to selected cytokines such as insulin growth factor 1 (IGF1) and tumor necrosis factor alpha (TNFα), which are associated with breast cancer risk. Specifically, we compared the oxidative metabolism of HMECs obtained from women with breast cancer and without cancer. Our data show considerable inter-individual variation in respiratory activities of HMECs from different women. A bioenergetic parameter called pyruvate-stimulated respiration (PySR) was identified as a key distinguishing feature of HMECs from women with breast cancer and without cancer. Samples showing PySR over 20% of basal respiration rate were considered PySR+ve and the rest as PySR-ve . By this criterion, HMECs from tumor-affected breasts (AB) and non-tumor affected breasts (NAB) of cancer patients were mostly PySR-ve (88% and 89%, respectively), while HMECs from non-cancer patients were mostly PySR+ve (57%). This suggests that PySR-ve/+ve phenotypes are individual-specific and are not caused by field effects due to the presence of tumor. The effects of IGF1 and TNFα treatments on HMECs revealed that both suppressed respiration and extracellular acidification. In addition, IGF1 altered PySR-ve/+ve phenotypes. These results reveal individual-specific differences in pyruvate metabolism of normal breast epithelial cells and its association with breast cancer risk.

Elucidating fish oil-induced milk fat depression in dairy sheep: Milk somatic cell transcriptome analysis.

In this study, RNA sequencing was used to obtain a comprehensive profile of the transcriptomic changes occurring in the mammary gland of lactating sheep suffering from fish oil-induced milk fat depression (FO-MFD). The milk somatic cell transcriptome analysis of four control and four FO-MFD ewes generated an average of 42 million paired-end reads per sample. In both conditions, less than 220 genes constitute approximately 89% of the total counts. These genes, which are considered as core genes, were mainly involved in cytoplasmic ribosomal proteins and electron transport chain pathways. In total, 117 genes were upregulated, and 96 genes were downregulated in FO-MFD samples. Functional analysis of the latter indicated a downregulation of genes involved in the SREBP signaling pathway (e.g., ACACA, ACSL, and ACSS) and Gene Ontology terms related to lipid metabolism and lipid biosynthetic processes. Integrated interpretation of upregulated genes indicated enrichment in genes encoding plasma membrane proteins and proteins regulating protein kinase activity. Overall, our results indicate that FO-MFD is associated with the downregulation of key genes involved in the mammary lipogenesis process. In addition, the results also suggest that this syndrome may be related to upregulation of other genes implicated in signal transduction and codification of transcription factors.

Mechanisms of Traditional Chinese Medicine in the Treatment of Mammary Gland Hyperplasia.

Mammary gland hyperplasia (MGH) occurs with high frequency among middle-aged women and is closely related to breast cancer. The treatment of this disease has become a research hotspot. Many patients with MGH are worried about the potential side effects of the synthetic drugs they are on. Thus, they seek alternative therapy, such as traditional Chinese medicine (TCM). In recent years, along with the Chinese herbs and its active ingredients, TCM compounds have been widely accepted and implemented in the treatment of MGH, whose mechanism hitherto is not completely clear. In this paper, we elaborate the mechanism of TCM in the treatment of MGH from the perspectives of sexual hormone levels, the expression of ER and PR, hemorheology, free radical activity and lipid peroxidation, VEGF and BFGF, cell proliferation activities, anti-apoptosis gene BcL-2, promoting apoptosis gene Bax, ERK, and tumor suppressor gene. In conclusion, TCM appears to be promising for MGH treatment. This paper will provide an overview of the mechanism of TCM in the treatment of MGH. In the near term, a better understanding of TCM will be achieved through comprehensive studies of its molecular mechanism.

Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development.

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother's milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Adding Agnus Castus and Magnolia to Soy Isoflavones Relieves Sleep Disturbances Besides Postmenopausal Vasomotor Symptoms-Long Term Safety and Effectiveness.

The effectiveness for vasomotor symptoms and sleep disorders plus the long-term safety of a nutraceutical combination of agnus-castus and magnolia extracts combined with soy isoflavones (SI) and lactobacilli were assessed in postmenopausal women. A controlled study was carried out in menopausal women comparing this nutraceutical combination (ESP group) with a formulation containing isoflavones alone (C group) at the dosage recommended. The Kuppermann index, The Pittsburgh Sleep Quality Index (PSQI), and Short Form 36 (SF-36) were determined at baseline, three, six and 12 months. Endometrial thickness, mammary density and liver function were evaluated at baseline and after 12 months. One hundred and eighty women were enrolled in the study (100 in the ESP group and 80 in the C group). At the end of the treatment, mammary density, endometrial thickness, and hepatic function did not show substantial differences between groups. The Kuppermann index and particularly the tendency for hot flashes progressively and significantly decreased in frequency and severity during ESP versus C treatment. At the same time, a significant increase in sleep quality and psychophysical wellness parameters was observed in the ESP versus C groups. No adverse events were observed. Agnus-castus and magnolia, combined with SI + lactobacilli, can effectively and safely be used in symptomatic postmenopausal women, mainly when quality of sleep is the most disturbing complaint. The endometrium, mammary glands and liver function were unaffected after 12 months of treatment.

The Effects of Petroselinum Crispum on Estrogen Receptor-positive Benign and Malignant Mammary Cells (MCF12A/MCF7).

BACKGROUND: Phytoestrogens have controversial effects on hormone-dependent tumors. Herein we investigated the effects of parsley root extract (PCE) on DNA synthesis performance, metabolic activity and cytotoxicity in malignant and benign breast cells. MATERIALS AND METHODS: The PCE was prepared and analyzed by mass spectrometry. MCF7 and MCF12A cells were incubated with various concentrations of PCE and analyzed for DNA synthesis performance, metabolic activity and cytotoxicity by BrdU proliferation, MTT and LDH assays, respectively. RESULTS: PCE was found to contain a substantial ratio of lignans. At a concentration range of 0.01 µg/ml-100 µg/ml the LDH assay analysis showed no significant cytotoxicity of PCE in both cell lines. However, at 500 µg/ml PCE's cytotoxicity was well over 70% of total cell population in both cell lines. According to the BrdU proliferation assay analysis, PCE demonstrated significant DNA synthesis inhibition of up to 80% at concentrations of 10, 50, 100 and 500 µg/ml in both cell lines. Based on the MTT assay analysis, only at a concentration of 500 µg/ml, PCE demonstrated a statistically significant inhibition of cellular metabolic activity of 63% in MCF7 and 75% in MCF12A of their respective normal capacity. CONCLUSION: PCE showed antiproliferative effects in MCF7 and MCF12A cells. Further investigation is required to determine whether this effect can be solely attributed to its phytoestrogens.

Transcriptome adaptation of the bovine mammary gland to diets rich in unsaturated fatty acids shows greater impact of linseed oil over safflower oil on gene expression and metabolic pathways.

BACKGROUND: Nutritional strategies can decrease saturated fatty acids (SFAs) and increase health beneficial fatty acids (FAs) in bovine milk. The pathways/genes involved in these processes are not properly defined. Next-generation RNA-sequencing was used to investigate the bovine mammary gland transcriptome following supplemental feeding with 5% linseed oil (LSO) or 5% safflower oil (SFO). Holstein cows in mid-lactation were fed a control diet for 28 days (control period) followed by supplementation with 5% LSO (12 cows) or 5% SFO (12 cows) for 28 days (treatment period). Milk and mammary gland biopsies were sampled on days-14 (control period), +7 and +28 (treatment period). Milk was used to measure fat(FP)/protein(PP) percentages and individual FAs while RNA was subjected to sequencing. RESULTS: Milk FP was decreased by 30.38% (LSO) or 32.42% (SFO) while PP was unaffected (LSO) or increased (SFO). Several beneficial FAs were increased by LSO (C18:1n11t, CLA:10t12c, CLA:9c11t, C20:3n3, C20:5n3, C22:5n3) and SFO (C18:1n11t, CLA:10t12c, C20:1c11, C20:2, C20:3n3) while several SFAs (C4:0, C6:0, C8:0, C14:0, C16:0, C17:0, C24:0) were decreased by both treatments (P < 0.05). 1006 (460 up- and 546 down-regulated) and 199 (127 up- and 72 down-regulated) genes were significantly differentially regulated (DE) by LSO and SFO, respectively. Top regulated genes (≥ 2 fold change) by both treatments (FBP2, UCP2, TIEG2, ANGPTL4, ALDH1L2) are potential candidate genes for milk fat traits. Involvement of SCP2, PDK4, NQO1, F2RL1, DBI, CPT1A, CNTFR, CALB1, ACADVL, SPTLC3, PIK3CG, PIGZ, ADORA2B, TRIB3, HPGD, IGFBP2 and TXN in FA/lipid metabolism in dairy cows is being reported for the first time. Functional analysis indicated similar and different top enriched functions for DE genes. DE genes were predicted to significantly decrease synthesis of FA/lipid by both treatments and FA metabolism by LSO. Top canonical pathways associated with DE genes of both treatments might be involved in lipid/cholesterol metabolism. CONCLUSION: This study shows that rich α-linolenic acid LSO has a greater impact on mammary gland transcriptome by affecting more genes, pathways and processes as compared to SFO, rich in linoleic acid. Our study suggest that decrease in milk SFAs was due to down-regulation of genes in the FA/lipid synthesis and lipid metabolism pathways while increase in PUFAs was due to increased availability of ruminal biohydrogenation metabolites that were up taken and incorporated into milk or used as substrate for the synthesis of PUFAs.

1,25-Dihydroxyvitamin D induces the glutamate transporter SLC1A1 and alters glutamate handling in non-transformed mammary cells.

Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3, is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters--SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1)--is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D-treated cells. Taken together, these data identify 1,25D as a physiological regulator of multiple membrane glutamate transporters that impacts on overall cellular glutamate handling.

Investigating the effect of arachidonate supplementation on the phosphoinositide content of MCF10a breast epithelial cells.

Phosphoinositides in primary mammalian tissue are highly enriched in a stearoyl/arachidonyl (C38:4) diacylgycerol backbone. However, mammalian cells grown in culture typically contain more diverse molecular species of phosphoinositides, characterised by a reduction in arachidonyl content in the sn-2 position. We have analysed the phosphoinositide species in MCF10a cells grown in culture by mass spectrometry. Under either serum or serum starved conditions the most abundant species of PI, PIP, PIP2 and PIP3 had masses which corresponded to C36:2, C38:4, C38:3, C38:2 and C36:1 diacylglycerol backbones and the relative proportions of each molecular species were broadly similar between each phosphoinositide class (approx. 50%, 25%, 10%, 10% and 10% respectively, for the species listed above). Supplementing the culture medium with BSA-loaded arachidonic acid promoted a rapid increase in the proportion of the C38:4 species in all phosphoinositide classes (from approx. 25%-60% of total species within 24 h), but the total amount of all combined species for each class remained remarkably constant. Stimulation of cells, cultured in either normal or arachidonate-enriched conditions, with 2 ng/ml EGF for 90 s caused substantial activation of Class I PI3K and accumulation of PIP3. Despite the increased proportion of C38:4 PIP3 under the arachidonate-supplemented conditions, the total amount of all combined PIP3 species accumulating in response to EGF was the same, with or without arachidonate supplementation; there were however small but significant preferences for the conversion of some PIP2 species to PIP3, with the polyunsaturated C38:4 and C38:3 species being more favoured over other species. These results suggest the enzymes which interconvert phosphoinositides are able to act on several different molecular species and homoeostatic mechanisms are in place to deliver similar phosphoinositide pool sizes under quite different conditions of arachidonate availability. They also suggest enzymes regulating PIP3 levels downstream of growth factor stimulation (i.e. PI3Ks and PIP3-phosphatases) show some acyl selectivity and further work should be directed at assessing whether different acyl species of PIP3 exhibit differing signalling potential.

Lactogenic Activity of an Enzymatic Hydrolysate from Octopus vulgaris and Carica papaya in SD Rats.

The traditional Chinese medicine theory believes that octopus papaya soup can stimulate milk production in lactating women. The objective of this study was to determine whether dietary supplementation with an enzymatic hydrolysate of Octopus vulgaris and Carica papaya (EHOC) could increase milk production and nutritional indexes in Sprague Dawley (SD) rats. Female SD rats (n = 24) were fed a control diet (n = 8), EHOC-supplemented diet, or a positive control diet (Shengruzhi) from day 10 of pregnancy to day 10 of lactation. Maternal serum, mammary gland (day 10 of lactation), milk, and pup weight (daily) were collected for analysis. Results showed that the EHOC diet obviously elevated daily milk yield and pup weight compared to the control group (P < .05). The EHOC diet was found to increase the concentration of prolactin (PRL), progesterone (P), estradiol (E2), and growth hormone (GH) significantly in the circulation and mammary gland. Mammary glands of EHOC-treated dams showed clear lobuloalveolar development and proliferation of myoepithelial cells, but no striking variations were observed among the groups. Furthermore, the nutrition content and immune globulin concentration in the milk of EHOC-supplemented dams were higher than those of the control group, especially the cholesterol, glucose, and IgG were higher by 44.98% (P < .001), 42.76% (P < .01), and 42.23% (P < .01), respectively. In conclusion, this article demonstrates that EHOC administration has beneficial effects on milk production in the dams and on performance of the dam and pup. These results indicate that EHOC could be explored as a potentially lactogenic nutriment for lactating women.