Effects of Glucose Availability on Expression of the Key Genes Involved in Synthesis of Milk Fat,Lactose and Glucose Metabolism in Bovine Mammary Epithelial Cells

As the main precursor for lactose synthesis, large amounts of glucose are required by lactating dairy cows. Milk yield greatly depends on mammary lactose synthesis due to its osmoregulatory property for mammary uptake of water. Thus, glucose availability to the mammary gland could be a potential regulator of milk production. In the present study, the effect of glucose availability on expression of the key genes involved in synthesis of milk fat, lactose and glucose metabolism in vitro was investigated. Bovine mammary epithelial cells (BMEC) were treated for 12 h with various concentrations of glucose (2.5, 5, 10 or 20 mmol/L). The higher concentrations of glucose (10–20 mmol/L) did not affect the mRNA expression of acetyl-CoA carboxylase, diacyl glycerol acyl transferase, glycerol-3 phosphate acyl transferase and α-lactalbumin, whereas fatty acid synthase, sterol regulatory element binding protein-1 and beta-1, 4-galactosyl transferase mRNA expression increased at 10 mmol/L and then decreased at 20 mmol/L. The content of lactose synthase increased with increasing concentration of glucose, with addition of highest value at 20 mmol/L of glucose. Moreover, the increased glucose concentration stimulated the activities of pyruvate kinase and glucose-6-phosphate dehydrogenase, and elevated the energy status of the BMEC. Therefore, it was deduced that after increasing glucose availability, the extra absorbed glucose was partitioned to entering the synthesis of milk fat and lactose by the regulation of the mRNA expression of key genes, promoting glucose metabolism by glycolysis and pentose phosphate pathway as well as energy status. These results indicated that the sufficient availability of glucose in BMEC may promote glucose metabolism, and affect the synthesis of milk composition.     

Induction of mammary prolactin receptors and lactose synthesis after ovariectomy in the pregnant mouse.

The development of prolactin receptors in the mammary gland after ovariectomy was investigated in pregnant KA mice. Mice were ovariectomized on day 13 of pregnancy and used for the determination of the amount of specific binding of 125I-labelled prolactin to the mammary tissue, and the contents of lactose and nucleic acids in the mammary gland 0, 8, 24, and 72 hr after the operation. The specific binding of 125I-labelled prolactin, lactose and RNA contents in the mammary gland remained low until 8 hr, sharply increased 24 hr and decreased 72 hr after ovariectomy. When ovariectomized mice were treated with 0.2 mg progesterone, pregnancy was maintained and an increase (1.5-fold) in the amount of specific binding was observed with an increase of lactose content. Five mg progesterone completely inhibited lactose synthesis. Cortisol administered with progesterone did not show any specific change at the dose used (0.5 to 10 mg). Although the amount of specific binding was also increased after hysterectomy, this increase (2-fold) did not fully cover the increase after ovariectomy (3-fold). These results suggest that the recepter site for prolactin is induced before the initiation of lactose synthesis caused by ovariectomy during pregnancy.     

Metabolic adaptations in goat mammary tissue during pregnancy and lactation

Metabolic adaptations of goat mammary tissue during pregnancy and lactation were monitored in serial biopsies of the tissue. Changes in the synthetic capacity of secretory cells were studied by combining measurements of enzyme activities with short-term culture of mammary explants to measure lactose, casein and total protein synthesis. By these criteria, the main phase of mammary differentiation began in late pregnancy and was essentially complete by Week 5 of lactation, coinciding with the achievement of peak milk yield. While milk yield declined after Week 5, the activities of key enzymes expressed per mg DNA and the rates of lactose and casein synthesis in mammary explants were maintained over a considerable period. The results suggest that changes in the synthetic capacity of epithelial cells may account for much of the rise in milk yield in early lactation, but are not responsible for the declining phase of milk production characteristic of lactation in ruminants.     

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.     

Metabolism of aminoacyl-p-nitroanilides by rat mammary tissue

We have examined the metabolism of aminoacyl-p-nitroanilides by rat mammary tissue isolated from rats during late pregnancy, peak lactation and late lactation. The rate of hydrolysis depended upon the chemical nature of the aminoacyl-p-nitroanilide compound and the physiological state of the donor animals. Thus, mammary tissue isolated from rats during late pregnancy and peak lactation hydrolysed aminoacyl-p-nitroanilides in the order L-met-p-nitroanilide=L-leu-p-nitroanilide>L-lys-p-nitroanilide>gamma- glu-p-nitroanilide. The order of activity was the same for mammary tissue taken from rats during late lactation except that L-lys-p-nitroanilide was hydrolysed at the same rate as the neutral aminoacyl-p-nitroanilides. Mammary tissue from peak lactating rats also hydrolysed alpha-L-glu-p-nitroanilide and alpha-L-asp-p-nitroanilide but to a lesser extent than the other compounds tested. The anionic aminoacyl-p-nitroanilides were able to trans-stimulate D-aspartate efflux from mammary tissue explants and the perfused mammary gland via the high-affinity anionic amino acid carrier. The clearance of gly-L-phe by the perfused mammary gland was markedly inhibited by L-phe. The results suggest that mammary tissue expresses a variety of dipeptidases at the basolateral aspect of the mammary epithelium which are capable of hydrolysing peptides extracellularly. These enzymes may be important for providing amino acids for milk protein synthesis and/or inactivating signal peptides.     

The SV40 T-antigen induces premature apoptotic mammary gland involution during late pregnancy in transgenic mice

Transgenic animals of the line 8 contain the WAP-SV-T transgene. Females of this line synthesise the SV40 T-antigen in mammary gland epithelial cells during pregnancy and the lactation period. All females are ‘milk-less’ and the offspring have to be nursed by foster mothers. The reason for this phenomenon is a premature apoptosis during late pregnancy. Nonetheless a significant number of mammary epithelial cells escape apoptosis and all transgenic females develop breast cancer after the first lactation period.     

Parity-induced mammary epithelial cells are multipotent and express cell surface markers associated with stem cells

Parity-induced mammary epithelial cells (PI-MECs) are defined as a pregnancy hormone-responsive cell population that activates the promoter of late milk protein genes during the second half of pregnancy and lactation. However, unlike their terminally differentiated counterparts, these cells do not undergo programmed cell death during post-lactational remodeling of the gland. We previously demonstrated that upon transplantation into an epithelial-free mammary fat pad, PI-MECs exhibited two important features of multipotent mammary epithelial progenitors: a) self-renewal, and b) contribution to ductal and alveolar morphogenesis. In this new report, we introduce a new method to viably label PI-MECs. Using this methodology, we analyzed the requirement of ovarian hormones for the maintenance of this epithelial subtype in the involuted mammary gland. Furthermore, we examined the expression of putative stem cell markers and found that a portion of GFP-labeled PI-MECs were part of the CD24(+)/CD49f(high) mammary epithelial subtype, which has recently been suggested to contain multipotent stem cells. Subsequently, we demonstrated that isolated PI-MECs were able to form mammospheres in culture, and upon transplantation, these purified epithelial cells were capable of establishing a fully functional mammary gland. These observations suggest that PI-MECs contain multipotent progenitors that are able to self renew and generate diverse epithelial lineages present in the murine mammary gland.     

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.     

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.     

Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G₀/G₁ arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin.     

Suckling-induced prolactin release potentiates mifepristone-induced lactogenesis in pregnant rats

Suckling, starting at 19:00 h on Day 18 of pregnancy, induced a significant increase in serum prolactin concentration at 20:00 h on Day 19 of pregnancy, but no increase in mammary gland casein or lactose content. Mifepristone (2 mg/kg) injection at 08:00 h on Day 19 of pregnancy induced significant increases in casein, but not in lactose, 24 h after administration. Mifepristone alone did not induce prolactin secretion, indicating that lactogenesis was induced by placental lactogen in the absence of progesterone action. When mifepristone was injected into suckling rats, serum prolactin concentrations were higher than in the untreated suckling rats. Casein in these rats increased significantly 12 h after mifepristone administration and lactose at 24 h after. If the suckling mifepristone-treated rats were given two injections of bromocriptine (1.5 mg/kg) at 12:00 h on Days 18 and 19 of pregnancy, serum prolactin concentrations were not increased by suckling, but casein and lactose concentrations in the mammary gland showed values similar to those obtained in the mifepristone-treated non-suckling rats. Mifepristone can therefore potentiate suckling-induced prolactin release in pregnant rats, demonstrating a direct central inhibitory action of progesterone on prolactin secretion. This suckling-induced prolactin secretion, unable to induce casein or lactose synthesis in the presence of progesterone, enhanced significantly synthesis of these milk components in the absence of progesterone action (rats treated with mifepristone). Fatty acid synthase, which is stimulated by the suckling stimulus in lactating rats, was not modified by mifepristone or suckling in pregnant rats.     

Serotonin (5-HT) Affects Expression of Liver Metabolic Enzymes and Mammary Gland Glucose Transporters during the Transition from Pregnancy to Lactation

The aim of this experiment was to demonstrate the ability of feeding serotonin (5-HT; 5-hydroxytryptamine) precursors to increase 5-HT production during the transition from pregnancy to lactation and the effects this has on maternal energy metabolism in the liver and mammary gland. Pregnant rats (n = 45) were fed one of three diets: I) control (CON), II) CON supplemented with 0.2% 5-hydroxytryptophan (5-HTP) or III) CON supplemented with 1.35% L-tryptophan (L-TRP), beginning on d13 of pregnancy through d9 of lactation (d9). Serum (pre and post-partum), milk (daily), liver and mammary gland tissue (d9) were collected. Serum 5-HT was increased in the 5-HTP fed dams beginning on d20 of gestation and remained elevated through d9, while it was only increased on d9 in the L-TRP fed dams. 5-HT levels were increased in mammary gland and liver of both groups. Additionally, 5-HTP fed dams had serum and milk glucose levels similar to the CON, while L-TRP had decreased serum (d9) and milk glucose (all dates evaluated). Feeding 5-HTP resulted in increased mRNA expression of key gluconeogenic and glycolytic enzymes in liver and glucose transporters 1 and 8 (GLUT-1, -8) in the mammary gland. We demonstrated the location of GLUT-8 in the mammary gland both in the epithelial and vascular endothelial cells. Finally, phosphorylated 5′ AMP-activated protein kinase (pAMPK), a known regulator of intracellular energy status, was elevated in mammary glands of 5-HTP fed dams. Our results suggest that increasing 5-HT production during the transition from pregnancy to lactation increases mRNA expression of enzymes involved in energy metabolism in the liver, and mRNA abundance and distribution of glucose transporters within the mammary gland. This suggests the possibility that 5-HT may be involved in regulating energy metabolism during the transition from pregnancy to lactation.     

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

小鼠乳腺上皮细胞培养体系的建立及Heregulin-α的调控作用

旨在建立完善的小鼠乳腺上皮细胞体外培养体系,探讨Heregulin-α（HRG-α）对该体系乳腺上皮细胞增殖及代谢的影响,为进一步深入研究乳腺上皮细胞的形态、结构和功能,揭示HRG-α在小鼠乳腺发育中的作用及其规律提供理论依据.通过比较不同pH和不同血清浓度配比的生长培养液对小鼠乳腺细胞生长的影响,得出pH7.4,添加10%血清的生长培养液为乳腺上皮细胞生长的最佳条件.通过运用相差显微镜技术、MTT等研究方法进行不同取材时期乳腺上皮细胞的形态、接种存活率、倍增时间、生长曲线等生物学特性比较,获得妊娠15天为较好的乳腺细胞取材时期.采用免疫组化和RT-PCR方法对细胞重要的标志蛋白角蛋白-18进行检测,鉴定所获得的细胞为乳腺上皮细胞,成功建立小鼠乳腺上皮细胞体外培养体系.利用所建立的小鼠乳腺上皮细胞体外培养体系,采用液相色谱技术及MTT方法,初步探讨了HRG-α对小鼠乳腺上皮细胞的影响.结果表明,适宜量HRG-α对小鼠乳腺上皮细胞生长和分泌总蛋白、乳糖均有显著促进作用,0.1～20ng/mL的HRG-α可促进小鼠乳腺上皮细胞的增殖和总蛋白、乳糖含量的增加,20ng/mL时达到最大值（P〈0.01）,50和100ng/mLHRG-α抑制小鼠乳腺上皮细胞的增殖,降低小鼠乳腺上皮细胞的总蛋白、乳糖含量（P〈0.05）.     

The cyclin-dependent kinase inhibitor p27 (Kip1) regulates both DNA synthesis and apoptosis in mammary epithelium but is not required for its functional development during pregnancy

Decreased expression of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is common in breast cancer and is associated with poor prognosis. p27 is also an important mediator of steroidal regulation of cell cycle progression. We have therefore investigated the role of p27 in mammary epithelial cell proliferation. Examination of the two major functions of p27, assembly of cyclin D1-Cdk4 complexes and inhibition of Cdk2 activity, revealed that cyclin D1-Cdk4 complex formation was not impaired in p27-/- mammary epithelial cells in primary culture. However, cyclin E-Cdk2 activity was increased approximately 3-fold, indicating that the CDK inhibitory function of p27 is important in mammary epithelial cells. Increased epithelial DNA synthesis was observed during pregnancy in p27-/- mammary gland transplants, but this was paralleled by increased apoptosis. During pregnancy and at parturition, development and differentiation of p27+/+ and p27-/- mammary tissue were indistinguishable. These results demonstrate a role for p27 in both the proliferation and survival of mammary epithelial cells. However, the absence of morphological and cellular defects in p27-/- mammary tissue during pregnancy raises the possibility that loss of p27 in breast cancer may not confer an overall growth advantage unless apoptosis is also impaired.     

Polyamine biogenesis in the rat mammary gland during pregnancy and lactation

Polyamines and RNA accumulate in the rat mammary gland during pregnancy, but the major increases occur after parturition. Therefore the major increases occur after the gland has obtained its maximal complement of epithelial cells. During lactation, the spermidine concentration rises above 5mm and RNA content in the lactating mammary gland reaches a value 16 times that of the unstimulated mammary gland. The ratio of spermidine/spermine, an increase of which initially signals an elevation in biosynthetic activity, is near 1 in the normal mammary gland and is greater than 10 in the lactating mammary gland. Putrescine concentration is very low during the entire course of mammary-gland development, with the exception of early pregnancy. The low putrescine concentration probably reflects the very rapid conversion of putrescine into spermidine. Both ornithine decarboxylase, the enzyme that synthesizes putrescine, and putrescine-stimulated S-adenosyl-l-methionine decarboxylase, the enzyme that synthesizes spermidine, increase in activity during middle and late pregnancy; during lactation, both enzyme activities are elevated until the 21st day of lactation, and then decline. These declines are concomitant with involution. Also, it was found that the amount of ribonuclease activity in the mammary gland was very high during lactation, almost double that in the gland during pregnancy.