Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

Growth hormone and lactogenic hormones can reduce the leptin mRNA expression in bovine mammary epithelial cells

Leptin mRNA is expressed in not only adipocytes but also mammary epithelial cells and leptin protein is present in milk. Although milk leptin is thought to influence metabolism or the immune system in neonates, there is little information about the regulation of leptin expression in mammary epithelial cells. We examined the effect of growth hormone (GH) and/or lactogenic hormone complex (DIP; dexamethasone, insulin and prolactin) on leptin mRNA expression in mammary epithelial cells. We used a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-day pregnant Holstein heifer. We confirmed that the mRNA was expressed in BMECs and the expression was significantly reduced by GH and/or DIP, when the cells were cultured on both plastic plates and cell culture inserts at days 2 and 7 after stimulation with lactogenic hormones. GH and/or DIP significantly increased level of alpha-casein mRNA in BMECs after 7 days on the cell culture inserts, but no mRNA expression was detected at day 2. GH and DIP significantly stimulated the secretion of alpha-casein from BMEC on cell culture inserts at 3.5 and 7 days. However, neither alpha-casein mRNA expression nor secretion was observed in the BMECs cultured on plastic dishes, even in the presence of GH or/and DIP. These results indicate that GH and DIP can directly reduce leptin mRNA expression in both undifferentiated and functionally differentiated bovine mammary epithelial cell.     

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.     

A proteomic analysis of the effect of growth hormone on mammary alveolar cell-T (MAC-T) cells in the presence of lactogenic hormones

The bovine mammary alveolar cell-T (MAC-T) cell line is able to uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin, and prolactin and is extensively used to study bovine mammary epithelial cell (MEC) function. Somatotropin, or growth hormone (GH), has been shown to increase milk protein synthesis both in vivo and in mammary cell models and to induce cytoskeletal rearrangement in a 3T3 fibroblast cell line and a Chinese hamster ovary cell line. To identify the nature of the effects of GH in MECs cultured with lactogenic hormones, changes in global protein expression were assessed in the MAC-T cell line with the use of two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry. Forty proteins were differentially expressed in response to GH (P < 0.05) and were related to metabolism, the cytoskeleton, protein folding, RNA and DNA processing, and oxidant stress. These widespread changes in protein expression are indicative of a global role of GH in overall cellular differentiation that may underlie the direct modulation of milk component synthesis in MEC models that have been described to date.     

Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland

During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17β-estradiol (InsHPrlE). The relative expression of β-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of β-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation.     

Bovine pituitary, kidney, uterine and mammary gland extracts contain bovine mammary epithelium growth factors that synergise with IGF-I and fetal calf serum: indication for involvement of GTP-binding proteins

Bovine mammary undifferentiated epithelial cells from young female calves, cultured in three-dimensional collagen gels in serum-free medium exhibited ultrastructural organization that resembled the in vivo situation. Extracts of bovine pituitary, kidney, uterus and mammary gland, stimulated cell proliferation in a dose-dependent manner. This mitogenic activity strongly synergised with the existant growth factors (GFs) in FCS and with IGF-I, while the addition of EGF had only minor effect. No synergistic manifestation was found with cholera toxin but pertussis toxin inhibited the growth-promoting activity of all four extracts. Other experiments indicated that this mitogenic activity does not result from prolactin, growth hormone or fibroblast growth factor. The present and former results, in which synergism between IGF-I and cholera toxin was demonstrated, suggest therefore, that the mitogenesis of normal mammary epithelial cells regulated by several tissue derived growth factors, consists of at least two pathways which are distinct from those activated by EGF and IGF-I. One of these pathways indicates involvement of pertussis toxin-sensitive GTP-binding proteins, and the other, activation of cholera toxin-sensitive adenylate cyclase.     

Effects of tripeptides and lactogenic hormones on oligopeptide transporter 2 in bovine mammary gland

This study was conducted to investigate the expression of oligopeptide transporter 2 (PepT2) and its potential function in bovine mammary gland. First, the PepT2 mRNA and protein were determined in cultured mammary epithelial cells. Then the effects of lactogenic hormones (prolactin, hydrocortisone or insulin) and substrate (threonyl-phenylalanyl-phenylalanine) on PepT2 were investigated. The PepT2 mRNA and protein were successfully detected in bovine mammary epithelial cells. PepT2 gene expression was enhanced by the addition of 50, 500 and 5000 ng/ml prolactin, 10 and 100 ng/ml hydrocortisone, and 50, 500, 5000 and 50,000 ng/ml insulin. PepT2 mRNA abundance was increased when 5, 10 and 15% of threonyl-phenylalanyl-phenylalanine was included. Responses of PepT2 to lactogenic hormones and oligopeptide inferred that it may play an important role in bovine mammary gland.     

Interactions of amino acids and hormones regulate the balance between growth and milk protein synthesis in lactating rats fed diets differing in protein content

Insulin-like growth factor-I (IGF-I), growth hormone (GH), and prolactin (PRL) play important roles in milk protein synthesis, and their plasma concentrations were reported to be affected by dietary protein intake. To investigate the relationship between circulating amino acid (AA) and concentrations of these hormones, 18 Wistar rats aged 14 wk were assigned to a low (LP; 9% protein), standard (SP; 21% protein), or high-protein (HP; 35% protein) diet from parturition through day 15 of lactation. Plasma, liver, pituitary gland, skeletal muscle, and mammary gland samples were collected at the end of treatment. Circulating and hepatic IGF-I concentrations increased linearly with elevated dietary protein concentrations (P < 0.0001). Rats receiving the HP diet had higher circulating GH (P < 0.01) and pituitary PRL concentrations (P < 0.0001) but lower pituitary GH concentration (P < 0.0001) relative to those in rats receiving the LP and SP diets. Pearson correlation test performed on composed data across treatments showed that several circulating AAs were correlated with circulating and tissue concentrations of IGF-I, GH, and PRL. Multiple linear regression analyses identified Leu, Gln, Ala, Gly, and Arg as the main AAs associated with hormone responses (R² = 0.37 ~ 0.80; P < 0.05). Rats fed the LP and HP diets had greater Igf1 and Ghr gene expression in skeletal muscle than those fed the SP diets (P < 0.01). However, LP treatment decreased Prlr mRNA abundance in mammary glands as compared with the SP and HP treatments (P < 0.05). The HP diets increased AA transporter expression (P < 0.01) but decreased mammalian target of rapamycin (P < 0.05) and 70 kDa ribosomal protein S6 kinase 1 (P < 0.01) phosphorylation in mammary glands as compared with the LP and SP diets. The results of the present study suggested that several circulating AAs mediated the effects of dietary protein supply on concentrations of IGF-I, GH, and PRL, which in turn altered the metabolism status in peripheral tissues including the lactating mammary glands.     

激素对奶牛乳腺上皮细胞乳脂肪合成的调控作用

乳脂肪是高质量的天然脂肪，其可为人类提供营养和能量，在各种膳食脂肪和油类中，是最容易被消化吸收的。乳脂肪是在乳腺中由从头合成或外源摄取的脂肪酸与甘油酯化形成的一种脂类物质，其含量的高低关系着牛奶品质的优劣和乳制品的加工特性。在奶牛的泌乳周期中，乳腺泌乳功能受多种因素影响，其中内分泌腺分泌的多种激素对奶牛乳腺上皮细胞（BMECs）乳脂的合成具有积极的调控作用。综上所述，作者介绍了氢化可的松、催乳素、胰岛素和生长激素4种泌乳相关激素对BMECs乳脂肪合成的调控机理，即从乳脂合成适宜的激素添加量、激素对乳脂球形态的影响方面初步阐释其调控作用，并从乳脂合成的关键酶及转录因子、激素对乳脂合成相关基因表达量方面深入阐释其作用机理，旨在为研究泌乳相关激素对奶牛乳腺内乳脂肪合成的调控机理提供参考。     

Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering insulin-like growth factor-I mRNA expression

Growth hormone is a major stimulator of skeletal muscle growth in animals, including cattle. In this study, we determined whether GH stimulates skeletal muscle growth in cattle by direct stimulation of proliferation or fusion of myoblasts, by direct stimulation of protein synthesis, or by direct inhibition of protein degradation in myotubes. We also determined whether these direct effects of GH are mediated by IGF-I produced by myoblasts or myotubes. Satellite cells were isolated from cattle skeletal muscle and were allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. Growth hormone at 10 and 100 ng/mL increased protein synthesis in myotubes (P < 0.05), but had no effect on protein degradation in myotubes or proliferation of myoblasts (P > 0.05). Insulin-like growth factor-I at 50 and 500 ng/mL stimulated protein synthesis (P < 0.01), and this effect of IGF-I was much greater than that of GH (P < 0.05). Besides stimulating protein synthesis, IGF-I at 50 and 500 ng/mL also inhibited protein degradation in myotubes (P < 0.01), and IGF-I at 500 ng/mL stimulated proliferation of myoblasts (P < 0.05). Neither GH nor IGF-I had effects on fusion of myoblasts into myotubes (P > 0.1). These data indicate that GH and IGF-I have largely different direct effects on bovine muscle cells. Growth hormone at 10 and 100 ng/mL had no effect on IGF-I mRNA expression in either myoblasts or myotubes (P > 0.1). This lack of effect was not because the cultured myoblasts or myotubes were not responsive to GH; GH receptor mRNA was detectable in them and the expression of the cytokine-inducible SH2-containing protein (CISH) gene, a well-established GH target gene, was increased by GH in bovine myoblasts (P < 0.05). Overall, the data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis in the muscle and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle.     

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.     

Mammary arteriovenous differences of glucose, insulin, prolactin and IGF-I in lactating sows under different protein intake levels

Mammary uptake of nutrients is dependent on their availability in the circulation but the role of hormones in that process is not known. Arteriovenous differences (AVD) of glucose and key hormones across the mammary glands were therefore determined in sows fed varying levels of protein. Sixteen lactating sows (four/dietary treatment) were fed a 7.8, 13.0, 18.2 or 23.5% crude protein (CP) isocaloric diet throughout lactation and their litters were standardized to 11 pigs within 48 h of birth. The anterior main mammary vein and a carotid artery were cannulated on day 4+/-1 of lactation and blood samples were collected every 30 min over 6h on days 10, 14, 18 and 22 of lactation to measure glucose, insulin, IGF-I, and prolactin (PRL) concentrations. Amino acid data from these sows were previously published and used here to determine residual correlations. Dietary treatments had no effect on any of the insulin or PRL variables measured (P>0.1) and, on day 18 only, IGF-I AVD was greater (P=0.05) for sows on the 23.5% compared to the 18.2% diet. On days 18 and 22, sows fed the 13% CP diet had greater arterial, venous and AVD glucose concentrations than sows fed other diets (P<0.05). Total arterial amino acid concentrations were correlated to arterial insulin (P<0.001) and PRL (P<0.05) concentrations, but not to those of IGF-I (P>0.1). Mammary AVD for total (P<0.001) and essential amino acids (P<0.05) were correlated to arterial concentrations of insulin, but not to those of IGF-I (P>0.1) or PRL (P>0.1). Mammary AVD of both total (P<0.01) and essential (P<0.05) amino acids were also correlated to mammary PRL AVD. In conclusion, dietary protein level did not affect mammary AVD and circulating lactogenic hormone concentrations. Yet, amino acid utilization by the sow mammary gland seems to be regulated via both circulating insulin concentrations and PRL binding to and uptake by porcine mammary cells.     

大豆黄酮对奶牛免疫功能和血清及乳中几种激素水平的影响

研究口服大豆黄酮对奶牛免疫功能和血清及乳中生长激素(GH)、催乳素(PRL)、生长抑素(SS)水平的影响。结果表明：1)大豆黄酮能明显提高血清及乳中特异性抗体水平,表明奶牛整体和乳腺器官的体液免疫功能明显增强。2)奶牛血清及乳中GH、PRL含量明显高于对照组,而血清SS含量显著低于对照组。本实验结果提示垂体GH、PRL和体内SS可能参与了大豆黄酮对奶牛免疫功能的调节过程。