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K Ono, T Akatsu, T Murakami, M Nishikawa, M Yamamoto, N Kugai, K Motoyoshi, and N Nagata

Of various PGs, PGE1 and PGE2 are shown to be the most potent stimulators of osteoclastogenesis in vitro. PGE receptors have been classified into four subtypes, EP1-EP4. Little is known about PGE receptors functioning in bone cells. In this study, using mouse marrow culture, we investigated which PGE receptors are important in osteoclast-like cell (OCL) formation induced by PGE. 11-deoxy-PGE1 (EP2, EP3 and EP4 agonist) stimulated OCL formation potently. Butaprost (EP2 agonist) stimulated it slightly, while sulprostone (EP1 and EP3 agonist) and ONO-AP-324-01 (EP3 agonist) did not. AH23848B (EP4 antagonist) inhibited PGE2-induced OCL formation in a dose-dependent manner. The expression of EP4 mRNA in mouse bone marrow was confirmed by RT-PCR. The results indicate an important role of EP4 in PGE2-induced OCL formation in marrow cultures and suggest therapeutic potential of EP4 antagonists in some clinical conditions with accelerated bone resorption.

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N. Takasu, M. Murakami, Y. Nagasawa, T. Yamada, Y. Shimizu, I. Kojima, and E. Ogata


The cytoplasmic concentration of free calcium was measured using aequorin, a calcium-sensitive photoprotein. The Ca2+ ionophore A23187 induced a rise in cytoplasmic free calcium and iodide discharge in cultured porcine thyroid cells. The minimum dose of A23187 effecting an increase in cytoplasmic free calcium induced iodide discharge. The A23187-induced rise in cytoplasmic free calcium was followed by iodide discharge. The results indicate that A23187-induced iodide discharge is mediated by a rise in the cytoplasmic concentration of free calcium.

J. Endocr. (1987) 115, 477–480

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M Furuhashi, N Ura, H Murakami, M Hyakukoku, K Yamaguchi, K Higashiura, and K Shimamoto

We investigated the effect of fenofibrate, a peroxisome proliferator-activated receptor-alpha agonist, on insulin sensitivity including lipid metabolism in skeletal muscle. Six-week-old male Sprague-Dawley rats were divided into two groups: those fed a standard chow (control) or a fructose-rich chow (fructose-fed rats (FFRs)) for 6 weeks. FFRs were treated either with a vehicle or with 30 mg/kg per day of fenofibrate for the last 2 weeks. Insulin sensitivity (M-value) was estimated by the euglycemic hyperinsulinemic glucose clamp method. Fatty acid-binding protein (FABP) in skeletal muscle was measured by ELISA, and the expression of FABP mRNA was analyzed by semi-quantitative RT-PCR. The serum and muscle triglyceride (sTG and mTG) levels and the activity of 3-hydroxyacyl-CoA dehydrogenase (HADH), a beta-oxidation enzyme, in muscle were also determined. FFRs showed a lower M-value and higher blood pressure, sTG and mTG than did the control group. The mTG was correlated positively with sTG and negatively with the M-value. Fenofibrate treatment for 2 weeks did not change blood pressure but significantly improved the M-value, sTG and mTG. FABP content and mRNA in the soleus muscle were significantly elevated in FFRs compared with those in the control group. Fenofibrate treatment further increased FABP. The HADH activity was comparable between the control group and FFRs, but significantly increased by fenofibrate treatment. These results suggest that fenofibrate improves insulin sensitivity not only by lowering serum lipids and subsequent influx of fatty acids into muscles but also by reducing intramuscular lipid content via further induction of FABP and stimulation of beta-oxidation in muscles.

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T Hayashida, K Nakahara, MS Mondal, Y Date, M Nakazato, M Kojima, K Kangawa, and N Murakami

Ghrelin, a 28 amino acid peptide, has recently been isolated from the rat stomach as an endogenous ligand for the GH secretagogue receptor. The fact that administration of ghrelin, centrally or peripherally, stimulates both food intake and GH secretion suggests that stomach ghrelin has an important role in the growth of rats. We used immunohistochemistry and radioimmunoassay to determine the age at which ghrelin-immunostained cells begin to appear in the rat stomach. Ghrelin-immunoreactive cells were found to be expressed in the fetal stomach from pregnancy day 18. The number of ghrelin-immunoreactive cells in the fetal stomach increased as the stomach grew. The amount of ghrelin in the glandular part of the rat stomach also increased, in an age-dependent manner, from the neonatal stage to adult. Eight hours of milk restriction significantly decreased the ghrelin concentration in the stomachs of 1-week-old rats, and increased the ghrelin concentration in their plasma. Administration of ghrelin to 1- and 3-week-old rats increased plasma GH concentrations. The daily subcutaneous administration of ghrelin to pregnant rats from day 15 to day 21 of pregnancy caused an increase in body weight of newborn rats. In addition, daily subcutaneous administration of ghrelin to neonatal rats from birth advanced the day of vaginal opening from day 30.7+/-0.94 to day 27.9+/-0.05. These results suggest that ghrelin may be involved in neonatal development.

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K Tamura, N Yokoyama, Y Sumida, T Fujita, E Chiba, N Tamura, S Kobayashi, M Kihara, K Murakami, M Horiuchi, and S Umemura

This study examined whether type 1 angiotensin II receptor (AT1) and angiotensin-converting enzyme (ACE) mRNAs are regulated during dietary salt loading in angiotensinogen gene-knockout (Atg-/-) mice which are genetically deficient in endogenous production of angiotensin II. Wild-type (Atg+/+) and Atg-/- mice were fed a normal-salt (0.3% NaCl) or a high-salt (4% NaCl) diet for 2 weeks. The mRNA levels were measured by Northern blot analysis. In Atg+/+ mice, concentrations of plasma angiotensin peptides were decreased by salt loading, whereas the treatment increased the brainstem, cardiac, pulmonary, renal cortex, gastric and intestinal AT1 mRNA levels. Salt loading also enhanced renal cortex ACE mRNA levels in Atg+/+ mice. Although plasma angiotensin peptides and urinary aldosterone excretion were not detected in Atg-/- mice, salt loading increased blood pressure in Atg-/- mice. In Atg-/- mice, pulmonary, renal cortex, gastric and intestinal AT1, and renal cortex and intestinal ACE mRNA levels were higher than those in Atg+/+ mice. However, salt loading upregulated AT1 mRNA expression only in the liver of Atg-/- mice, and the treatment did not affect ACE mRNA levels in Atg-/- mice. Furthermore, although the levels of ACE enzymatic activity showed the same trend with the ACE mRNA levels in the lung, renal cortex and intestine of both Atg-/- and Atg+/+ mice, the results of radioligand binding assay showed that cardiac expression of AT1 protein was regulated differently from AT1 mRNA expression both in Atg-/- and Atg+/+ mice. Thus, expression of AT1 and ACE is regulated by salt loading in a tissue-specific manner that appears to be mediated, at least partly, by a mechanism other than changes in the circulating or tissue levels of angiotensin peptides.

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T Tsushima, M Arai, O Isozaki, Y Nozoe, K Shizume, H Murakami, N Emoto, M Miyakawa, and H Demura


Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET)-induced hormonal functions in various tissues. We have studied the interaction of endothelins with porcine thyroid cells in culture. Specific binding of 125I-labelled ET-1 was demonstrated in porcine thyroid cells. The binding was displaced equally by unlabelled ET-1 and ET-2, but receptor affinity for ET-3 was lower than that for ET-1 and -2. Scatchard analysis of the data revealed a single class of high-affinity ET-1 receptors with a K d of 0·45 nmol/l and a binding capacity of 2100 sites/cell. SDS-PAGE and autoradiography of 125I-labelled ET-1 cross-linked with thyroid cell membranes demonstrated ET-1 binding sites with an apparent molecular weight of 50 kDa. These results indicated that ET-1 receptors in thyroid cells are type A ET receptors. In association with the presence of ET-1 receptors, porcine thyroid cells responded to ET-1 and ET-2 with an increase in c-fos mRNA expression. Although ET-1 did not affect DNA synthesis stimulated by either EGF or IGF-I, it dose-dependently inhibited TSH-induced iodide uptake and also inhibited iodide uptake stimulated by forskolin and 8-bromo-cAMP. ET-1 had no effect on TSH-stimulated cAMP production. Thus, ET-1 inhibited TSH-induced iodine metabolism by acting at the steps distal to cAMP production. In agreement with a recent report, immunoreactive ET-1 was detected in medium conditioned by porcine thyroid cells. Antibody to ET-1 was found to increase TSH-induced iodide uptake. These results are compatible with the notion that ET-1 negatively regulates TSH-induced iodide uptake in an autocrine manner.

Journal of Endocrinology (1994) 142, 463–470

Free access

N Murakami, T Hayashida, T Kuroiwa, K Nakahara, T Ida, MS Mondal, M Nakazato, M Kojima, and K Kangawa

Ghrelin, a 28-amino-acid peptide, has recently been isolated from the rat stomach as an endogenous ligand for the GH secretagogue receptor. We have reported previously that central or peripheral administration of ghrelin stimulates food intake, and the secretion of GH and gastric acid in rats. In the present study, we investigated how much endogenous centrally released ghrelin is involved in the control of food intake and body weight gain. We also examined the profile of ghrelin secretion from the stomach by RIA using two kinds of anti-ghrelin antiserum, one raised against the N-terminal ([Cys(12)]-ghrelin[1-11]) region and one raised against the C-terminal ([Cys(0)]-ghrelin [13-28]) region of the peptide. The former antibody recognizes specifically ghrelin with n- octanoylated Ser 3 (acyl ghrelin), and does not recognize des-acyl ghrelin. The latter also recognizes des-acyl ghrelin (i.e. total ghrelin). Intracerebroventricular treatment with the anti-ghrelin antiserum against the N-terminal region twice a day for 5 days decreased significantly both daily food intake and body weight. Des-acyl ghrelin levels were significantly higher in the gastric vein than in the trunk. Either fasting for 12 h, administration of gastrin or cholecystokinin resulted in increase of both acyl and des-acyl ghrelin levels. The ghrelin levels exhibited a diurnal pattern, with the bimodal peaks occurring before dark and light periods. These two peaks were consistent with maximum and minimum volumes of gastric content respectively. These results suggest that (1) endogenous centrally released ghrelin participates in the regulation of food intake and body weight, (2) acyl ghrelin is secreted from the stomach, (3) intestinal hormones stimulate ghrelin release from the stomach, and (4) regulation of the diurnal rhythm of ghrelin is complex, since ghrelin secretion is augmented under conditions of both gastric emptying and filling.