Changes in the gene expression of adiponectin and glucose transporter 12 (GLUT12) in lactating and non-lactating cows

Glucose delivery and uptake by the mammary gland is a rate‐limiting step in milk synthesis. Insulin resistance is believed to increase throughout the body following the onset of lactation. To study glucose metabolism in peak‐, late‐, and non‐lactating cows we analyzed the expression of an adipokine, namely, adiponectin, decreased insulin resistance, leptin, and a novel insulin‐responsive glucose transporter (GLUT12) in the adipose tissue and mammary gland by using real‐time polymerase chain reaction. Our results demonstrated that the mRNA level of adiponectin in the adipose tissue was greater in non‐lactating cows than in peak‐lactating cows. In the adipose tissue, there were no significant differences in the abundance of GLUT12 mRNA between the peak‐, late‐, and non‐lactating cows. In contrast, in the mammary gland, the mRNA level of GLUT12 was greater in non‐lactating cows than in peak‐ and late‐lactating cows. In the adipose tissue, the mRNA level of leptin and peroxisome proliferator‐activated receptor gamma 2 (PPARγ2) was greater in non‐lactating cows than in peak‐lactating cows. The results of the present study suggest that in lactating cows adiponectin plays an important role in insulin resistance in the adipose tissue; in the mammary gland, GLUT12 expression is believed to be an important factor for insulin‐dependent glucose metabolism.     

Partial cloning and localisation of leptin and its receptor in the one-humped camel (Camelus dromedarius)

Based on the studies and results presented here, leptin and its receptor were expressed by adipose tissue, mammary alveolar epithelial cells, liver hepatocytes, and the lining epithelium of the bile duct of the one-humped camel (Camelus dromedarius). Our observations support the biological importance of leptin in the mammary gland as well as the likely local effect of leptin on the peripheral tissues. We suggest that there may be an association between hepatic leptin and the lipogenic activity of the liver in the dromedary camel.     

Expression of leptin and its receptors in various tissues of ruminants

The energy metabolism of domestic animals is under the control of hormonal factors, which include thyroid hormones and leptin. Leptin signals from the periphery to the centre. It is mostly produced in the white adipose tissue and informs the central nervous system (CNS) about the total fat depot of the body. Low and high levels of leptin induce anabolic and catabolic processes, respectively. Besides controlling the food uptake and energy expenditure leptin is also involved in regulation of the reproduction and the immune system. Leptin is produced in several tissues other than fat. In the present paper the leptin expression of ruminant (Egyptian water buffalo, cow, and one-humped camel) tissues are examined. The mammary gland produces leptin in each species investigated. The local hormone production contributes to milk leptin and most probably helps to maintain lactation. Considerable leptin receptor expression was observed in the milk-producing epithelial cells, which is the same cell type that produces most of the udder leptin. Based on the results tissues participating in production have an autoregulative mechanism through which tissues can be relatively independent of the plasma leptin levels in order to maintain the desired function.     

Preliminary report on the expression of leptin and leptin receptor (ObR) in normal, hyperplastic and neoplastic canine mammary tissues

Leptin and its receptor (ObR) expression were investigated by immunohistochemistry in normal, hyperplastic and neoplastic canine mammary tissues and related to clinical-pathological features. Leptin expression was detected in healthy mammary tissues, adenosis and in benign mammary tumours and was lower in ductal hyperplasias and malignant tumours. A high percentage of ObR-positive cells were present in adenosis, benign tumours and in complex carcinomas, while ObR expression was lower in healthy mammary tissues, in ductal hyperplasias and in a large part of invasive mammary carcinomas. Our data demonstrated that cancer cells expressed at low level leptin and ObR in canine mammary tumours with a more aggressive behaviour, as well as in lymph node metastases. Consequently, leptin and ObR expressions in this species resulted to be not associated with a reduced overall survival.     

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.     

Changes in gene expression of glucose transporters in lactating and nonlactating cows

Glucose delivery and uptake by the mammary gland are a rate-limiting step in milk synthesis. It is thought that insulin-independent glucose uptake decreases in tissues, except for the mammary gland, and insulin resistance in the whole body increases following the onset of lactation. To study glucose metabolism in peak-, late-, and nonlactating cows, the expression of erythrocyte-type glucose transporter (GLUT1) and the insulin-responsive glucose transporter (GLUT4) in the mammary gland, adipose tissue, and muscle were assessed by Western blotting and real-time PCR. Our results demonstrated that the mammary gland of lactating cows expressed a large amount of GLUT1, whereas the mammary gland of nonlactating cows did not (P < 0.05). On the other hand, adipose tissue of late and nonlactating cows expressed a large amount of GLUT1, whereas the adipose tissue of peak-lactating cows did not (P < 0.05). There were no significant differences in the abundance of GLUT4 mRNA in adipose tissue and muscle, whereas GLUT4 mRNA was not detected in the mammary gland. The plasma insulin concentration was greater (P < 0.05) in nonlactating cows than in peak- and late-lactating cows. The results of the present study indicate that in lactation, GLUT1 expression in the mammary gland and adipose tissue is a major factor for insulin-independent glucose metabolism, and the expression of GLUT4 in muscle and adipose tissue is not an important factor in insulin resistance in lactation; however, the plasma insulin concentration may play a role in insulin-dependent glucose metabolism. Factors other than GLUT4 may be involved in insulin resistance.     

Leptin in ruminants. Gene expression in adipose tissue and mammary gland, and regulation of plasma concentration

This paper reviews data on leptin gene expression in adipose tissue (AT) and mammary gland of adult ruminants, as well as on plasma leptin variations, according to genetic, physiological, nutritional and environmental factors. AT leptin mRNA level was higher in sheep and goat subcutaneous than visceral tissues, and the opposite was observed in cattle; it was higher in fat than in lean selection line in sheep; it was decreased by undernutrition and increased by refeeding in cattle and sheep, and not changed by adding soybeans to the diet of lactating goats; it was increased by injection of NPY to sheep, and by GH treatment of growing sheep and cattle. Insulin and glucocorticoids in vitro increased AT leptin mRNA in cattle, and leptin production in sheep. Long daylength increased AT lipogenic activities and leptin mRNA, as well as plasma leptin in sheep. Mammary tissue leptin mRNA level was high during early pregnancy and was lower but still expressed during late pregnancy and lactation in sheep. Leptin was present in sheep mammary adipocytes, epithelial and myoepithelial cells during early pregnancy, late pregnancy and lactation, respectively. Plasma leptin in cattle and sheep was first studied thanks to a commercial “multi-species” kit. It was positively related to body fatness and energy balance or feeding level, and decreased by β-agonist injection. The recent development of specific RIA for ruminant leptin enabled more quantitative study of changes in plasma leptin concentration, which were explained for 35–50% by body fatness and for 15–20% by feeding level. The response of plasma leptin to meal intake was related positively to glycemia, and negatively to plasma 3-hydroxybutyrate. The putative physiological roles of changes in leptin gene expression are discussed in relation with published data on leptin receptors in several body tissues, and on in vivo or in vitro effects of leptin treatment.     

奶牛乳腺中4F2hc基因的表达模式及亮氨酸对其表达的调控研究

为了研究4F2hc在奶牛乳腺中的表达模式及调控方式，进一步明确氨基酸在奶牛乳腺上皮细胞中的跨膜转运过程，本研究采用Western blotting和实时荧光定量PCR技术检测了4F2hc在泌乳期和干奶期奶牛乳腺组织中的表达变化；在体外培养的泌乳期奶牛乳腺上皮细胞中添加亮氨酸，采用Western blotting和实时荧光定量PCR技术检测其对奶牛乳腺上皮细胞中4F2hc表达的影响；采用雷帕霉素抑制剂抑制mTOR信号通路，使用Western blotting方法检测mTOR信号抑制后奶牛乳腺上皮细胞中4F2hc表达以及乳蛋白合成的变化。结果显示，在泌乳期的奶牛乳腺组织中4F2hc的mRNA和蛋白表达水平均显著或极显著高于干奶期（P<0.05，P<0.01）；在体外培养的奶牛乳腺上皮细胞中添加亮氨酸可以极显著提高乳腺上皮细胞中4F2hc的mRNA和蛋白质表达水平（P<0.01）；亮氨酸刺激可以激活细胞内的mTOR信号通路（P<0.05），而雷帕霉素处理则可以显著抑制mTOR信号分子的磷酸化并极显著抑制亮氨酸诱导的4F2hc的表达（P<0.05，P<0.01），进而极显著抑制β-Casein的合成（P<0.01）。以上研究结果表明，4F2hc基因的表达与奶牛乳腺的泌乳活性之间呈正相关，亮氨酸可以通过激活mTOR信号通路来调节4F2hc基因的表达，进而影响乳蛋白的合成。     

奶牛原代乳腺上皮细胞的培养及β酪蛋白mRNA的表达

本试验旨在建立原代乳腺上皮细胞系的体外培养方法,并进行β酪蛋白mRNA的表达验证。取新鲜泌乳期的乳腺组织,采用组织块法培养纯化原代乳腺上皮细胞,利用显微镜观察细胞形态并进行细胞生长计数,采用实时荧光定量PCR技术检测β酪蛋白mRNA表达。结果显示,纯化培养的原代乳腺上皮细胞集聚成岛屿状生长,具有典型的铺路石和鹅卵石形状,细胞生长曲线呈"S"形,符合一般细胞的生长规律,并成功表达β酪蛋白mRNA。综上所述,本研究采用组织块法成功培养出具有正常生理功能的奶牛原代乳腺上皮细胞,为后续的乳腺上皮细胞功能研究提供了良好的细胞试验模型。     

Comparison between plasma and milk levels of leptin during pregnancy and lactation in cow, a relationship with beta-lactoglobulin

Leptin gene is expressed in the mammary tissue and the expression of both leptin and its receptor changes significantly during pregnancy and lactation, with high levels during the first half of pregnancy and a decrease at delivery. The aim of this work was to investigate into leptin concentration in plasma and in milk during pregnancy and the first week after parturition in dairy cow and to analyze the correlation between leptin and beta-lactoglobulin (beta-LG) concentrations in plasma and in milk. The trial was conducted on six Holstein dairy cows, reared in the same environmental conditions and evaluated with similar body condition score, during the complete reproductive cycle from insemination to the delivery. Blood from the jugular vein and milk samples were collected at weekly intervals. Plasma leptin concentration showed a lower level (p < 0.05) at the beginning of pregnancy. Milk leptin concentration showed a higher level (p < 0.01) than plasma level from week 23 to week 29 of pregnancy. Plasma beta-LG concentrations were higher (p < 0.01) compared to plasma concentrations during the first part of pregnancy, then milk levels rise and become higher than plasma levels during the last weeks before dry period. A positive correlation (p < 0.01) was observed between leptin and beta-LG both in plasma and in milk profiles.     

Bovine hepatocyte growth factor and its receptor c-Met: cDNA cloning and expression analysis in the mammary gland

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine that plays a crucial role in the embryonic and postnatal development of various organs including the mammary gland. We cloned bovine HGF and its c-Met receptor cDNAs, and examined their expression during mammary gland development in dairy cows. The 2.5-kbp HGF cDNA clone contained a 2190 bp open reading frame coding a 730 amino acid protein, while the 4.8-kbp c-Met cDNA clone contained a 4152 bp open reading frame coding a 1384 amino acid protein. The bovine HGF and c-Met sequences exhibited more than 87% identity with those of other mammals. RT-PCR analysis revealed ubiquitous expression of both HGF and c-Met mRNAs in various bovine tissues tested. HGF mRNA was detected only in the inactive stage of bovine mammary gland development and not in the developing, lactating, and involuting stages, while c-Met mRNA was detected in the inactive and involuting stages. Immunohistochemical analysis demonstrated that the c-Met protein was found on mammary epithelial cells in the inactive, developing, and involuting stages, and on myoepithelial cells in all stages. These results suggest pivotal roles of HGF and c-Met in the development of bovine mammary gland.     

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.     

Expression of bovine parathyroid hormone/parathyroid hormone- related protein (PTH/PTHrP) receptor mRNA in the mammary gland of cows

To examine the PTH/PTHrP receptor in the mammary gland, molecular cloning of bovine PTH/PTHrP receptor and measurement of its mRNA expression were carried out in cows during the periparturient period. The PTH/PTHrP receptor gene was partially cloned, and expression of bovine PTH/PTHrP receptor mRNA was detected in various tissues of the cow. In the mammary gland, PTH/PTHrP receptor mRNA expression was constantly low during the periparturient period, whereas PTHrP mRNA expression dramatically increased after parturition. This suggested that expression of PTH/PTHrP receptor mRNA in the mammary gland is not affected by lactation in cows.     

Expression of parathyroid hormone-related protein (PTHrP) mRNA in mammary gland of periparturient cows

The expression of parathyroid hormone-related protein (PTHrP) mRNA was examined in mammary gland with or without lactation, and during periparturient period in a Holstein cow and a Jersey cow. In the lactating mammary gland, PTHrP was detected in alveolar epithelial cells and the lumen by immunohistochemical analysis. The relative expression levels of PTHrP mRNA in mammary gland from lactating cows were significantly higher than those from non-lactating cows (P<0.05). During periparturient period, relative PTHrP mRNA level was remarkably low before the parturition in a Jersey and a Holstein cow, however, both levels were gradually increased and reached a peak level at 5-6 weeks after the parturition. In addition, the peak level in a Jersey cow was approximately 3-fold higher than that in a Holstein cow. From these results, PTHrP was synthesized and secreted in alveolar epithelial cells in mammary gland and increased subsequently with the lactation, suggesting a possible mechanism for the regulation of local calcium homeostasis.     

Interleukin-8 expression by mammary gland endothelial and epithelial cells following experimental mastitis infection with E. coli

Epithelial and endothelial cells play a pivotal role in initiating and controlling the movement of leukocytes into tissues during inflammation through the production of cytokines and chemokines such as interleukin-8 (IL-8). In situ hybridization with an IL-8 riboprobe was used to determine IL-8 mRNA expression by mammary gland epithelial and endothelial cells in cows with experimental Escherichia coli mastitis. Epithelial cells of the gland, especially surrounding the alveoli, had increased IL-8 mRNA levels at all time points at which tissue samples were collected (8, 12, and 24h) after E. coli challenge. Levels of IL-8 expression in the epithelial cells decreased at 24h post-infection. IL-8 expression by mammary gland endothelial cells was low, but did increase slightly at 24h post-infection. Both epithelial and endothelial cells of the mammary gland can contribute to the production of IL-8 that is typically seen in coliform mastitis.