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K. Inoue, T. Sakai, and M. Hattori


Both native and recombinant basic fibroblast growth factor (FGF) exerted a marked effect on the morphology of a pituitary clonal somatotroph-like cell line (MtT/S) in vitro. Addition of 10 pg–20 ng bovine basic FGF/ml caused the cells to flatten and spread out over the culture dish, the cells showing strong surface contact. Those in contact with the culture dish were epithelial in appearance, being polygonal, closely apposed and forming a pavement-like monolayer. Basic FGF inhibited cell proliferation in a dose-dependent manner at the higher concentrations tested (0·5–20 ng/ml). This cell-adhesive effect of basic FGF was also observed in normal dispersed pituitary cells. The observed stimulation of pituitary cell adhesion is the first report of a morphological effect of basic FGF on pituitary cells and could explain the physiological significance of basic FGF and its high concentration in the pituitary gland.

Journal of Endocrinology (1991) 130, 381–386

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M Imae, Y Inoue, Z Fu, H Kato, and T Noguchi

Hepatocyte nuclear factor-3 (HNF-3) belongs to a large family of forkhead transcription factors and is made up of three members (HNF-3alpha, -3beta and -3gamma). It has been shown that HNF-3 regulates a number of metabolically important genes. However, the mechanisms underlying this regulation of HNF-3 activity by hormones and nutrition have not yet been well elucidated. In attempting to explore the regulation of gene expression of HNF-3 members by physiological status, we analyzed the effects of insulin, dexamethasone and protein malnutrition on the hepatic mRNA level of each member. Male Wistar rats were fed on a 12% casein diet, 12% gluten diet (deficient in lysine and threonine) or a protein-free diet for 1 week. The protein-free diet and gluten diet caused a 3. 7-fold increase in HNF-3g mRNA in the liver and did not affect the mRNA level of either HNF-3alpha or HNF-3beta. Daily administration of dexamethasone caused the mRNA levels of HNF-3alpha and HNF-3beta to increase (2.3- and 1.4-fold, respectively), but had no effect on the HNF-3gamma mRNA level. In diabetic rats that had been injected with streptozotocin, an elevation of the hepatic mRNA levels of HNF-3beta and HNF-3gamma was observed (1.6-and 1.9-fold, respectively). Insulin replacement in the diabetic rats decreased both mRNA levels in a dose-dependent manner. HNF-3alpha mRNA was not affected by insulin status. These results show that the genes of the three members of the HNF-3 family respond differently to hormonal and nutritional factors suggesting that the activities of HNF-3 members are regulated, at least in part, by the levels of their gene expression.

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T Adachi, M Inoue, H Hara, E Maehata, and S Suzuki

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein located in blood vessel walls at high levels and may be important in the antioxidant capability of vascular walls. The aim of this study was to assess plasma levels of EC-SOD and to evaluate the relationship of the EC-SOD level with insulin resistance in type 2 diabetic patients. We determined plasma EC-SOD in 122 patients and found for the first time that the EC-SOD level was strongly and positively related to adiponectin (r=0.503, P < 0.001), and significantly and inversely related to fasting plasma glucose (FPG) (r=-0.209, P=0.022), body-mass index (BMI) (r=-0.187, P=0.040) and homeostasis model assessment-insulin resistance index (HOMA-R) (r=-0.190, P=0.039). Stepwise-multiple regression analysis also showed a significant influence of adiponectin (F=33.27) on the EC-SOD level. Administration of pioglitazone to 19 diabetic patients significantly increased the plasma levels of EC-SOD (69.9+/-19.3 ng/ml to 97.4+/-25.9 ng/ml; P < 0.0001) and adiponectin, while it decreased tumor necrosis factor-alpha (TNF-alpha). The present observations suggest that factors related to the pathogenesis of insulin resistance play an important role in the regulation of the plasma EC-SOD concentration. It is possible that the increase in the EC-SOD level by pioglitazone administration in diabetic patients is due to a decline of TNF-alpha, which is known to suppress EC-SOD expression.

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M Okita, A Inui, T Inoue, H Mizuuchi, K Banno, S Baba, and M Kasuga

We have previously reported that corticotropin-releasing factor (CRF) is a potent stimulator of adrenocorticotropic hormone and cortisol secretion in the dog. Therefore, in the present study, we investigated the extrahypophysiotropic actions of CRF in this species. When CRF was injected into the third cerebral ventricle, it failed to inhibit food intake significantly at doses of 1.19, 3.57, and 11.9 nmol. This is in sharp contrast with the results in rodents. At the 3.57 and 11.9 nmol doses, CRF markedly stimulated the secretion of pancreatic polypeptide (PP), a hormone under vagal control, and at the highest dose CRF increased plasma glucose levels. These results suggest species differences in the feeding response to CRF and activation of the parasympathetic nervous system in the dog.

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C Mogi, H Goda, K Mogi, A Takaki, K Yokoyama, M Tomida, and K Inoue

In order to study GH cell differentiation, we used the clonal cell lines called MtT/E and MtT/S cells, which were derived from a rat mammotrophic pituitary tumor. Although MtT/E cells are non-hormone-producing ones, Pit-1 protein is present in their nuclei, which suggests that MtT/E cells are progenitor cells of the Pit-1 cell lineage and have the potential to differentiate into hormone-producing cells. On the other hand, MtT/S cells produce GH; however, the responsiveness to GH-releasing hormone (GHRH) is weak and only a small number of secretory granules are present in their cytoplasm, which suggests that MtT/S cells are premature GH cells. In order to differentiate into GH cells from MtT/E cells as a progenitor cell, we examined several differentiation factors and found that retinoic acid (RA) induced the differentiation of MtT/E cells into GH-producing cells. RA-induced GH cells partially matured with the glucocorticoid treatment; however, the responsiveness to GHRH on GH secretion was incomplete. In order to elucidate the mechanism underlying full differentiation of GH cells, we used MtT/S cells. We treated MtT/S cells with glucocorticoid and found that they differentiated into mature GH cells with many secretory granules in their cytoplasm and they responded well to GHRH. These results suggested that MtT/E and MtT/S cells are progenitor or premature GH cells, and show different responses to differentiation factors. Our data also suggested that GH cells differentiate from their progenitor cells through multistep processes.

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A. Nakano, M. Terasawa, M. Watanabe, K. Okazaki, S. Inoue, M. Kato, Y. Nimura, N. Usuda, T. Morita, and H. Hidaka


Neurocalcin (molecular weight 23 000 and 24 000) is a Ca2+-binding protein with three putative Ca2+-binding domains and is present in large amounts in nervous tissues. Neurocalcin isoproteins separated by C18 reverse-phase column chromatography are insoluble in buffer solution and it is impossible to determine the dissociation constant of neurocalcin with Ca2+. To overcome this difficulty, recombinant neurocalcin was synthesized, based on one of the cDNAs of the neurocalcin isoproteins. Stoichiometric titration experiments, using recombinant neurocalcin, indicated that this protein bound 2 mol Ca2+/mol protein and that the apparent dissociation constant for Ca2+ was 2·2 μmol/l, suggesting that neurocalcin plays a physiological role in cellular function. Immunoblotting showed that neurocalcin is present in the bovine adrenal gland in addition to the nervous tissues. Neurocalcin, identified by immunoblotting, was purified from the bovine adrenal gland. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of neurocalcin from the bovine brain showed 23 kDa and 24 kDa double bands, while SDS-PAGE of neurocalcin from the adrenal gland showed a single band of apparently 24 kDa, suggesting that the expression of neurocalcin isoproteins differs from tissue to tissue. The content of neurocalcin in the adrenal gland was 10 μg protein/100 g wet tissue. Immunohistochemical analysis showed the occurrence of neurocalcin in zona glomerulosa and adrenal medulla but not in zona fasciculata or zona reticularis. The restricted localization of neurocalcin in the adrenal gland suggests that a similar Ca2+ signal pathway may be present in zona glomerulosa and the adrenal medulla.

Journal of Endocrinology (1993) 138, 283–290

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Y Takei, H Hashimoto, K Inoue, T Osaki, K Yoshizawa-Kumagaye, M Tsunemi, T X Watanabe, M Ogoshi, N Minamino, and Y Ueta

Adrenomedullin 5 (AM5) is a new member of the calcitonin gene-related peptide (CGRP) family identified in teleost fish. Although its presence was suggested in the genome database of mammals, molecular identity and biological function of AM5 have not been examined yet. In this study, we cloned a cDNA encoding AM5 in the pig and examined its cardiovascular and renal effects. Putative mature AM5 was localized in the middle of prohormone and had potential signals for intermolecular ring formation and C-terminal amidation. The AM5 gene was expressed most abundantly in the spleen and thymus. Several AM5 genes were newly identified in the database of mammals, which revealed that the AM5 gene exists in primates, carnivores, and undulates but could not be identified in rodents. In primates, nucleotide deletion occurred in the mature AM5 sequence in anthropoids (human and chimp) during transition from the rhesus monkey. Synthetic mature AM5 injected intravenously into rats induced dose-dependent decreases in arterial pressure at 0.1–1 nmol/kg without apparent changes in heart rate. The decrease was maximal in 1 min and AM5 was approximately half as potent as AM. AM5 did not cause significant changes in urine flow and urine Na+ concentration at any dose. In contrast to the peripheral vasodepressor action, AM5 injected into the cerebral ventricle dose-dependently increased arterial pressure and heart rate at 0.1–1 nmol. The increase reached maximum more quickly after AM5 (5 min) than AM (15–20 min). AM5 added to the culture cells expressing calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of the receptor activity-modifying proteins (RAMPs), the combination of which forms major receptors for the CGRP family, did not induce appreciable increases in cAMP production in any combination, although AM increased it at 10 10–10 9 M when added to the CLR and RAMP2/3 combination. These data indicate that AM5 seems to act on as yet unknown receptor(s) for AM5, other than CLR/CTR+RAMP, to exert central and peripheral cardiovascular actions in mammals.