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A Watanabe, I Uchida, K Kadota, and N Katoh

We measured the amounts of a vesicular transport factor, p115/transcytosis-associated protein (p115/TAP) and its mRNA, in mammary glands from cows in which lactation was induced hormonally. The highest level of p115/TAP mRNA, determined by Northern blotting, was detected in the developing stage. In contrast to the mRNA level, the amount of protein, determined by immunoblot analysis using anti-p115/TAP antibodies raised against a p115/TAP-derived recombinant fusion protein, was higher during the lactating stages than at other times. Immunohistochemical study showed that p115/TAP was predominantly localized in mammary epithelial cells. The p115/TAP was also detected in tissues other than the mammary gland but, in contrast to the situation in the mammary gland, the protein and its mRNA levels in those tissues were independent of the stage of lactation. The increased level of p115/TAP mRNA during the developing stage and the maintenance of p115/TAP protein during lactation suggest that the synthesis of p115/TAP is regulated during mammary development and differentiation, and also that the protein is involved in a function related to lactation.

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K Katoh, K Yoshioka, H Hayashi, T Mashiko, M Yoshida, Y Kobayashi, and Y Obara

Postprandial changes in plasma concentrations of GH, insulin, IGF-I, leptin and metabolites were compared between young Holstein bull calves fed with milk alone (control group) and with milk+5′-uridylic acid (UMP) (UMP group). UMP (2 g/day) was given with milk at 0830 h and 1530 h for 11 days from the 4th to the 14th day after birth. The perirenal fat weight was significantly lower in the UMP group than in the control group, but there was no significant difference in the weights of the liver, spleen and heart between the groups. Basal GH concentrations in the UMP group were slightly higher, but the postprandial increase in plasma insulin level and the area under the curve for insulin in the UMP group were significantly lower than those in the control group. There was no significant difference in IGF-I levels between the groups. In addition, the postprandial glucose concentrations were lower in the UMP group as reflected by the insulin level, and nonesterified fatty acid concentrations were not different. In the muscle (M. longissimus thoracis) sampled at 14 days of age, the triacylglycerol (TAG) content was significantly greater but glycogen content was significantly lower in the UMP group than in the control group. From these results, we have concluded that feeding 5′-UMP at 2 g/day for 11 days significantly alters TAG accumulation in the body and plasma concentrations of GH and insulin in young bull calves.

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K Katoh, G Furukawa, K Kitade, N Katsumata, Y Kobayashi, and Y Obara

Changes in plasma concentrations of GH and insulin in response to feeding and stimulation with GH-releasing hormone (GHRH) or GH-releasing peptide (GHRP-6, a ligand for endogenous GH secretagogue receptors) were compared between 3-week-old (milk-fed) and 12-week-old (concentrate and hay-fed) calves. Feeding of a milk-replacer diet in 3-week-old animals significantly increased the basal (prefeeding) concentrations of GH, insulin and glucose in plasma, whereas feeding of concentrate and hay in 12-week-old animals did not cause a significant change in these traits. However, in the animals maintained on a milk-replacer diet until 12 weeks of age, postprandial plasma GH concentrations and AUC (area under the curve) were not different from those in the age-matched weaned group. The venous injection of either GHRH (0.25 μg/kg) or GHRP-6 (2.5 μg/kg) significantly increased plasma GH concentrations in both 3- and 12-week-old animals, but GH AUC was significantly greater in 3-week-old than in 12-week-old animals. Insulin concentration was transiently but significantly increased by the injection of GHRP-6 only in 12-week-old animals, the AUC being greater in 12-week-old than 3-week-old animals. From these results, we conclude that postprandial levels of plasma GH and insulin concentrations are altered after weaning and by aging, and that the quality of diets or development of the neuroendocrine functions in the digestive–pituitary system may be involved in this alteration.

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K Katoh, M Yoshida, Y Kobayashi, M Onodera, K Kogusa, and Y Obara

In order to assess the biological significance of weaning and water deprivation on the control of plasma concentrations of adrenocorticotropic hormone (ACTH), cortisol, growth hormone (GH) and metabolites in response to stimulation with arginine-vasopressin (AVP) and corticotropin-releasing hormone (CRH), we carried out three experiments in which male goats before and after weaning were intravenously injected with AVP or CRH alone, or in combination with each other. In experiment 1, 17-week-old (post-weaning) goats were intravenously injected with AVP or CRH alone at the doses of 0.1, 0.3 and 1.0 nmol/kg body weight (BW). The AVP injection significantly and dose dependently increased plasma levels of ACTH, cortisol, GH and metabolites, whereas the injection with CRH did not cause significant increases in the levels of these parameters. In experiment 2, 4-week-old (pre-weaning) and 13-week-old (post-weaning) goats were injected with either AVP or CRH alone, followed by a combined injection of both secretagogues at a dose of 0.3 nmol/kg BW. Although the basal levels of the hormones and metabolites, with the exception of glucose, were greater in the 4-week-old goats, the hormone responses induced by stimulation with AVP were weaker than those induced in 13-week-old goats. Additionally, there were no responses in any hormone patterns to CRH stimulation in 4-week-old goats. In experiment 3, 13-week-old goats were injected with CRH alone followed by injection with AVP for two consecutive days of water deprivation. The animals were subjected to withdrawal of up to 20% of the total blood volume and water deprivation for up to 28 h. However, no significant differences in plasma ACTH, cortisol or GH levels were observed between days 1 and 2. Based on these results, we concluded that: (1) AVP is a more potent stimulant than CRH in terms of its ability to induce increases in plasma levels of ACTH, cortisol and GH; (2) the role of AVP as a secretagogue of hypothalamus–pituitary–adrenal hormones is strengthened, whereas the ineffective role of CRH remains unaltered, by weaning; (3) acute stress such as massive withdrawal of blood volume and subjection to water deprivation may not be sufficient burdens to alter stress-related hormone levels in young goats.

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K Katoh, T Komatsu, S Yonekura, H Ishiwata, A Hagino, and Y Obara

The present experiment was carried out to investigate the effects of exogenous adenosine 5'-triphosphate (ATP) and growth hormone (GH) on cellular H(+) efflux rate (extracellular acidification rate) and Ca(2+) concentration ([Ca(2+)](c)) in cloned bovine mammary epithelial cells (bMEC) raised from the mammary gland of a 26-day-pregnant Holstein heifer. Perifusion of 2-day cultured cells with a medium containing ATP (10, 100 and 1000 micromol/l) for 30 min caused a significant and concentration-dependent increase in the cellular H(+) efflux rate. ATP application (100 micromol/l) caused a transient and large increase in [Ca(2+)](c) in all cells. In contrast, perifusion with a medium containing bovine GH at 10, 50 and 250 ng/ml for 30 min caused a significant decrease in the cellular H(+) efflux rate in a concentration-dependent manner. However, bovine GH application (50 ng/ml) caused a small decrease followed by an increase, in some cases, in [Ca(2+)](c). In bMEC treated with lactogenic hormones (1 microgram/l prolactin, 1 nmol/ml dexamethasone and 5 microgram/ml insulin) for 2 days, the increased H(+) efflux rate induced by ATP was significantly reduced, whereas the negative response induced by GH was inversely and significantly changed to the positive. Treatment of the cells with lactogenic hormones reduced the increase in [Ca(2+)](c) induced by ATP stimulation, while it enhanced the increase in [Ca(2+)](c) induced by GH stimulation. Application of ATP or GH did not cause any significant changes in [pH](c). Treatment with lactogenic hormones enhanced GH receptor (GHR) transcription that was determined by RT-PCR. From these results, we conclude that exogenous application of ATP and GH causes prompt and significant responses in H(+) transport and [Ca(2+)](c) that were significantly changed in the opposite direction by the treatment with lactogenic hormones. The lactogenic hormone treatment also enhanced GHR transcription, which may change post-receptor signal transduction systems for both agents in the bMEC.

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T Takahashi, K Sato, S Kato, T Yonezawa, Y Kobayashi, Y Ohtani, S Ohwada, H Aso, T Yamaguchi, S G Roh, and K Katoh

Ghrelin is a multifunctional peptide that promotes an increase of food intake and stimulates GH secretion. Ghrelin secretion is regulated by nutritional status and nutrients. Although a high-protein (HP) diet increases plasma ghrelin secretion in mammals, the mechanisms and the roles of the elevated ghrelin concentrations due to a HP diet have not been fully established. To clarify the roles of elevated acylated ghrelin upon intake of a HP diet, we investigated the regulation of ghrelin concentrations in plasma and tissues in wethers fed with either the HP diet or the control (CNT) diet for 14 days, and examined the action of the elevated plasma ghrelin by using a ghrelin-receptor antagonist. The HP diet gradually increased the plasma acylated-ghrelin concentrations, but the CNT diet did not. Although the GH concentrations did not vary significantly across the groups, an injection of ghrelin-receptor antagonist enhanced insulin levels in circulation in the HP diet group. In the fundus region of the stomach, the ghrelin levels did not differ between the HP and CNT diet groups, whereas ghrelin O-acyltransferase mRNA levels were higher in the group fed with HP diet than those of the CNT diet group were. These results indicate that the HP diet elevated the plasma ghrelin levels by increasing its synthesis; this elevation strongly suppresses the appearance of insulin in the circulation of wethers, but it is not involved in GH secretion. Overall, our findings indicate a role of endogenous ghrelin action in secretion of insulin, which acts as a regulator after the consumption of a HP diet.