Akihiko Hagino

In vitro differentiation of a cloned bovine mammary epithelial cell.

The aim of the study was to establish in vitro a bovine mammary epithelial cell (MEC) clone, able to respond to mitogenic growth factors and to lactogenic hormones. Mammary tissue from a 200-d pregnant Holstein cow was used as a source of MEC, from which a clone was established through a process of limiting dilution. When plated on plastic, the cells assumed a monolayer, cobblestone, epithelial-like morphology, with close contact between cells. Inclusion of IGF-1 and EGF in the media significantly increased the number of cells 5 d after plating. All cells stained strongly for cytokeratin and moderately for vimentin at young and old passage stages, indicating the epithelial nature of this cell clone. When the cells were plated at a high density on a thin layer of a commercial extracellular matrix preparation (Matrigel), lobular, alveoli-like structures developed within approximately 5 d, with a clearly visible lumen. When cells were plated onto Matrigel in differentiation media (containing lactogenic hormones), detectable quantities of alpha-casein were present in the media and particularly on the lumen side of the structures. Omission of one of the lactogenic hormones (insulin, prolactin or hydrocortisone) reduced alpha-casein release to the limit of detection of the assay used. Lactoferrin was also produced when the cells were plated on Matrigel, again principally on the lumen side of the lobules, though this was independent of the lactogenic hormones. By passage 40, the cells had senesced, and it was not possible to induce alpha-casein or lactoferrin production. This study notes the establishment of a functional bovine mammary epithelial cell clone, which is responsive to mitogenic and lactogenic hormones and an extracellular matrix.

Growth hormone acts on the synthesis and secretion of α-casein in bovine mammary epithelial cells

To study the effect of growth hormone (GH) on the functions of mammary epithelia, we examined the effect of GH on the synthesis and secretion of alpha-casein in a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-d-pregnant Holstein heifer. GH receptors (GHR) were observed in the BMEC on the membrane as well as in the cytoplasm. After BMEC were plated onto cell culture inserts, GH stimulated alpha-casein release in both the presence and absence of the lactogenic hormone complex, which included dexamethasone, insulin and prolactin (DIP). DIP enhanced the effect of GH on alpha-casein release. Although alpha(s1-) casein mRNA expression was not detected in untreated control cells, its expression was observed in BMEC in response to the GH, DIP and GH + DIP treatments. Expression was greater for GH and GH + DIP than for just DIP. Expression of GHR mRNA was increased by DIP treatment, while the mRNA expression was little changed by GH treatment. We conclude that GH acts on BMEC and induces the expression of both the alpha-casein gene and protein. GHR gene expression was shown to be regulated by DIP and GHR. GHR may be involved in a synergic effect between GH and DIP on casein secretion. These results suggest that GH, in addition to its widely accepted homeorhetic role in vivo, also can act on the mammary parenchyma, and that the effects of GH on mammary epithelial cells could partly account for the clear galactopoietic effect of recombinant bovine GH seen in lactating dairy cows.

Changes in circulating adiponectin and metabolic hormone concentrations during periparturient and lactation periods in Holstein dairy cows.

Although our previous report demonstrated that adiponectin and AdipoR1 gene expressions changed among different lactation stages in the bovine mammary gland, its in vivo kinetics remain unclear in ruminant animals. In this study, we investigated the changes in circulating concentrations of adiponectin, as well as other metabolic hormones and metabolites, (i) during the periparturient period and (ii) among different lactation stages, in Holstein dairy cows. In experiment 1, serum adiponectin concentrations increased after parturition. Serum insulin concentrations were lower in the postpartum than prepartum period, whereas serum growth hormone (GH) concentrations increased in the postpartum period. Serum nonesterified fatty acids (NEFA) levels were increased during the postpartum period and were dependent on the parity. In experiment 2, there was no significant difference in plasma adiponectin concentrations among lactational stages. Plasma insulin concentrations tended to be lower in early lactation while plasma GH levels tended to be higher. Plasma NEFA concentrations were significantly lower in mid- and late-lactation stages than non-lactation stages. These findings indicate that elevation of serum adiponectin might be involved in energy metabolism just around parturition, and might exert its action through regulation of receptor expression levels in target tissues in each lactational stage in Holstein dairy cows.

Chemerin analog regulates energy metabolism in sheep

Accumulating data suggest a relationship between chemerin and energy metabolism. Our group previously described gene cloning, expression analysis and the regulatory mechanism of chemerin and its own receptor in mice and cattle. The objective of the present study was to investigate the physiological effect of chemerin on endocrine changes and energy metabolism in sheep using a biologically stable chemerin analog. The chemerin analog was intravenously administrated (100 or 500 µg/head) to sheep, and plasma insulin and metabolites (glucose, nonesterified fatty acids (NEFA), triglyceride, total cholesterol and high-density lipoprotein (HDL) cholesterol) were analyzed. The chemerin analog dramatically increased the insulin levels, and glucose levels were decreased. NEFA levels were slightly decreased at 20 min but then increased gradually from 60 to 180 min after analog administration. In addition, injection of the chemerin analog immediately increased triglyceride and total cholesterol but not HDL levels. These results suggested that chemerin analog regulated insulin secretion related to glucose metabolism and the release of triglycerides in sheep in vivo. This study provides new information about endocrine and metabolic changes in response to chemerin in sheep.

Octanoate stimulates cytosolic triacylglycerol accumulation and CD36 mRNA expression but inhibits Acetyl coenzyme A carboxylase activity in primary cultured bovine mammary epithelial cells

Mammary epithelial cells, which express and secrete leptin into milk, accumulate triacylglycerol (TAG). We examined effects on the accumulation of cytosolic TAG of addition of short- (acetate and butyrate) or medium- (octanoate) chain fatty acids to the medium bathing bovine mammary epithelial cells (bMEC). Octanoate stimulated the accumulation of TAG in a concentration-dependent manner from 1 to 10 mM and increased lipid droplet formation and mRNA expression of CD36 (a fatty acid translocase). Additionally, expression of a peroxisome proliferator activated receptor (PPAR) gamma 2 protein that is a lipid-activated transcription factor, was increased by the addition of acetate or octanoate. However, leptin mRNA expression was significantly reduced by addition of acetate or butyrate. Both short- and medium-chain fatty acids inhibited acetyl coenzyme A carboxylase (ACC) activities, which is pivotal in lipid synthesis, but elevated expression of uncoupling protein 2 (UCP2) mRNA, which is important in energy expenditure. These results suggest that octanoate induces cytosolic TAG accumulation and the formation of lipid droplets, and that acetate and butyrate inhibit leptin expression and lipid synthesis in bMEC.

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 and localization of carbonic anhydrase in bovine mammary gland and secretion in milk

Little attention has been paid to carbonic anhydrase VI (CA VI), a secretory type isozyme, in the bovine mammary gland, although the gland is an important exocrine gland and CA VI is known to localize in exocrine glands such as salivary and lacrimal glands in various animal species. In the present study mRNA expression and protein localization of CA VI in isolated gland tissues and in cloned epithelial cells from the mammary gland of Holstein cows (Bos taurus) were observed by reverse transcript polymerase chain reaction and immunocytochemistry. Also, changes of CA VI concentrations in milk were measured for 2 months postpartum by an enzyme-linked immunosorbent assay. CA VI gene expression was detected in the gland tissues and epithelial cells, and CA VI protein was localized in the cytoplasm of the epithelial cells. Colostrum contained the highest concentration of CA VI protein (100 ng/ml), decreasing in an exponential manner (P<0.001). We conclude that bovine mammary epithelial cells synthesize and secrete CA VI in colostrum at higher concentration than in normal milk, implying its role to compensate for low CA VI secretion in neonatal calves.

Insulin response to glucose and glucose tolerance following feeding in sheep.

Sheep offered a roughage diet for 4 h daily were injected intravenously with glucose before and at various times after feeding. The insulin secretory response to glucose and the rate of disappearance of injected glucose were determined. While the basal concentration of plasma insulin was unchanged, the base-line plasma glucose concentration tended to decrease during the meal. The glucose load brought about an increase in the plasma insulin concentration at each injection, but the insulin response to glucose and the rate of glucose disposal were increased during the meal. On varying the time of feeding between 08.00 and 16.00 hours, the increase in the insulin response to glucose and in the rate of glucose disposal always appeared to be related to the giving of food, independent of the time food was offered. It is concluded that feeding increases the insulin response to an intravenous glucose load even when the increase in the basal level of plasma insulin on feeding is very modest in sheep given a roughage diet. The increased insulin response and glucose disposal rate following feeding did not appear to be related to diurnal rhythms in insulin secretory activity or glucose metabolism.

Apelin is involved in postprandial responses and stimulates secretion of arginine-vasopressin, adrenocorticotropic hormone, and growth hormone in the ruminant

Apelin and its mRNA are expressed in several tissues, including the supraoptic and paraventricular nuclei in the hypothalamus. Although apelin is reported to be involved in the regulation of fluid homeostasis, little is known about the postprandial dynamics of apelin in plasma and its regulatory effects on the anterior pituitary hormones of ruminants. Therefore, the aims of this study were to investigate the following: (1) changes in plasma apelin concentrations in response to food intake under conditions of hydration (free access to water) or dehydration (water restriction), and (2) the effects of intravenous administration of apelin on plasma concentrations of arginine-vasopressin (AVP), ACTH, GH, and insulin. In Experiment 1 with the use of goats, the postprandial plasma apelin concentration was significantly increased under the dehydration condition compared with the hydration condition, and this increase was accompanied by increased plasma concentrations of AVP and ACTH after 24 h of dehydration. In Experiment 2 with the use of sheep and hydration conditions, the intravenous administration of apelin ([Pyr(1)]-apelin-13; 0.5 mg/head) caused a tendency to increase or caused a significant increase in plasma concentrations of AVP, ACTH, GH, insulin, and glucose. On the basis of these findings, we concluded that apelin is involved in the feeding process, and it regulates endocrine functions in the anterior pituitary gland via AVP in ruminant animals.

Physiological conditions and genetic controls of phaeomelanin pigmentation in nestling barn swallows

Lay Summary Many animals possess conspicuous traits that appear to be nonfunctional for survivorship but are used in communication. Such traits often provide information about the quality of the possessors to others. However, although empirical studies have found many signal traits that are associated with individual quality, the underlying mechanisms remain to be understood. In this study, we showed that high-quality individuals could produce colourful plumage without increasing oxidative stress through genetic control.