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H. SHIMIZU and YUKIKO OKAZAKI

Administration of progesterone causes a significant increase in body weight in adult female rats (Hervey, 1964; Hervey & Hervey, 1964) and mice (Dewar, 1964). Chlormadinone acetate (17α-acetoxy-6-chloropregna-4,6-diene-3,20-dione = CAP) seems to have a similar effect in cattle (Takeuchi, Shimizu, Toyoda, Kawai & Adachi, 1966) and rats (Shimizu & Ishibashi, 1965, unpublished results). The weight gains of adult female rats treated with CAP was therefore compared with that of controls and progesterone-treated rats.

Nulliparous adult rats of the Wistar strain, aged about 3 months, were used. They had free access to a pelleted diet containing 20% crude protein; green vegetables were given once a week. Vaginal smears were examined daily between 9.00 and 10.00 hr. Four groups of 13 rats were set up: untreated controls, controls injected with the vehicle only, CAP-treated rats and progesterone-treated rats. Both steroids were given in doses of 5 mg., s.c. once daily, as a 10 mg./ml.

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K. YAMASHITA, T. SHIMIZU, M. MIENO, A. AMANO and H. KOBA

Department of Pathophysiology, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki, Japan

(Received 16 December 1974)

We have reported the inhibitory effect of methylenedianiline, a diaminodiphenyl derivative related to amphenone B, on the testicular 17-oxosteroid secretion stimulated by human chorionic gonadotrophin (HCG) (Yamashita, 1967). Unlike amphenone B (Rosenfeld & Bascom, 1956) and methylenedianiline (Tullner, 1960), Metopirone has been shown to be a relatively selective inhibitor of 11β-hydroxylase in the adrenal cortex (Liddle, Island, Lance & Harris, 1958). However, since it also is an amphenone analogue (Chart, Sheppard, Allen, Bencze & Gaunt, 1958), the present study was undertaken to see whether it exerts an amphenone-like effect on androgen synthesis and release by the testis.

Spermatic venous blood was collected from 15 mongrel dogs, weighing between 7·9 and 13·1 kg, by the method described previously (Yamashita, 1966). In each dog the left spermatic vein was exposed by the lumbar route under

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M Shimizu, J T Dickey, H Fukada and W W Dickhoff

Western ligand blotting of salmon serum typically reveals three insulin-like growth factor (IGF) binding proteins (IGFBPs) at 22, 28 and 41 kDa. Physiologic regulation of the 22 kDa IGFBP is similar to that of mammalian IGFBP-1; it is increased in catabolic states such as fasting and stress. On the other hand, its molecular mass on Western ligand blotting is closest to mammalian IGFBP-4. The conflict between physiology and molecular mass makes it difficult to determine the identity of the 22 kDa IGFBP. This study therefore aimed to identify the 22 kDa IGFBP from protein and cDNA sequences. The 22 kDa IGFBP was purified from chinook salmon serum by a combination of IGF-affinity chromatography and reverse-phase chromatography. The N-terminal aminoacid sequence of the purified protein was used to design degenerate primers. Degenerate PCR with liver template amplified a partial IGFBP cDNA, and full-length cDNA was obtained by 5′- and 3′-rapid amplification of cDNA ends (RACE). The 1915-bp cDNA clone encodes a 23.8 kDa IGFBP, and its N-terminal amino-acid sequence matched that of purified 22 kDa IGFBP. Sequence comparison with six human IGFBPs revealed that it is most similar to IGFBP-1 (40% identity and 55% similarity). These findings indicate that salmon 22 kDa IGFBP is IGFBP-1. Salmon IGFBP-1 mRNA is predominantly expressed in the liver, and its expression levels appear to reflect circulating levels. The 3′-untranslated region of salmon IGFBP-1 mRNA contains four repeats of the nucleotide sequence ATTTA, which is involved in selective mRNA degradation. In contrast, amino-acid sequence analysis revealed that salmon IGFBP-1 does not have an Arg-Gly-Asp (RGD) integrin recognition sequence nor a Pro, Glu, Ser and Thr (PEST)-rich domain (a segment involved in rapid turnover of protein), both of which are characteristic of mammalian IGFBP-1. These findings suggest that association with the cell surface and turnover rate may differ between salmon and mammalian IGFBP-1.

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H. Shimizu, Y. Uehara, Y. Tanaka, Y. Shimomura and I. Kobayashi

ABSTRACT

Evidence is accumulating that adrenal steroids may be involved in the metabolic effects of cytokines. We evaluated the possible involvement of glucocorticoids in the inhibition of pancreatic insulin secretion by interleukin-1β (IL-1β), one of the cytokines produced by inflammatory cells. In the first group of experiments, adrenalectomized rats showed a significant reduction in basal and glucose (0·5 g/kg, i.v.)-stimulated immunoreactive insulin (IRI) levels after injection of IL-1β (1·0 μg/kg), but intact rats did not. Pretreatment with IL-1β increased plasma glucose levels 2 and 15 min after an i.v. bolus of glucose in adrenalectomized rats. In the second group of experiments, dexamethasone supplement (0·1 mg/kg) given to adrenalectomized rats cancelled the reduction in plasma glucose levels by IL-1β, and rats treated with 1·0 mg dexamethasone/kg showed a significant increase in basal IRI levels and enhanced serum IRI levels after IL-1β injection. However, 1·0 mg deoxycorticosterone/kg given daily for 7 days failed to cancel the effect of IL-1β on the reduction of serum IRI levels, although it attenuated the weight loss after adrenalectomy. The data suggested that withdrawal of glucocorticoids after adrenalectomy potentiates the effect of IL-1β on the reduction of serum IRI levels. Glucocorticoids may have a protective action against the reduction of serum IRI levels by IL-1β.

Journal of Endocrinology (1992) 132, 419–423

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K Ohtani, H Shimizu, Y Tanaka, N Sato and M Mori

Abstract

Pioglitazone hydrochloride (AD-4833), one of the thiazolidinedione analogs, is a new anti-diabetic agent which improves peripheral insulin resistance in diabetic patients. We determined the direct effect of AD-4833 on insulin secretion in HIT-T 15 cells. The effects of AD-4833 (10−7 m to 10−5 m) on insulin secretion were examined in 3 and 7 mm glucose-containing F-12 K media. The addition of 10−5 m AD-4833 significantly increased insulin secretion in both media, but its stimulatory effect was more potent in the medium containing 7 mm glucose. The addition of 10−5 m AD-4833 caused an immediate, significant increase in cytosolic free Ca2+ concentration ([Ca2+]i). Nifedipine at all concentrations from 10 to 1000 nm significantly attenuated insulin secretion by 10−5 m AD-4833. In addition, 10−5 m AD-4833 failed to stimulate insulin secretion in the Ca2+-free Kreb's-Ringer bicarbonate buffer. These data indicated that AD-4833 stimulates in vitro insulin secretion in HIT-T 15 cells, perhaps by inducing Ca2+ influx.

Journal of Endocrinology (1996) 150, 107–111

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H Tamada, Y Shimizu, T Inaba, N Kawate and T Sawada

It is well known that progesterone and estrogen are essential hormones for maintaining pregnancy in most mammals. Some specific roles of progesterone for the maintenance of pregnancy have been clarified, but the role of estrogen is not well known. This study examines the effects of the aromatase inhibitor, fadrozole hydrochloride (Fad), on fetuses, uterine physical properties and the mRNA expression of the uterine enzymes that are related to collagen metabolism during late pregnancy in rats. Continuous s.c. infusion with 300 micro g/day Fad from day 14 of pregnancy (day 1=the day of sperm detection) reduced the concentration of plasma estradiol-17beta (E(2)), and did not change that of plasma progesterone, compared with controls. The treatment increased the intrauterine pressure and reduced the size and compliance of the uterine tissue framework. It also caused injuries (hematomata on the extremities) in about one-quarter of fetuses by day 20. The collagen content of the uterine ampullae was not changed by the treatment. Uterine mRNA expressions of matrix metalloproteinase-1 (MMP-1), which degrades collagens, and of lysyl oxidase (LO), which is necessary for the formation of intra- and inter-molecular cross-links of collagen, were examined by quantitative RT-PCR. The treatment with Fad had no effect on the expression of MMP-1 mRNA and increased that of LO mRNA. Daily s.c. injection with 0.2 micro g E(2) restored the changes in uterine physical properties and the mRNA expression of LO caused by the Fad treatment, and prevented fetal injury, indicating that the influences of Fad treatment are due to estrogen deficiency but not to toxicological effects of Fad. These results imply that estrogen deficiency during late pregnancy in rats obstructs development of the uterine tissue framework so as to cause fetal injury. It is possible that an increase in the uterine expression of LO gene may be involved in this obstruction.

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H Shimizu, K Ohtani, Y Kato and M Mori

Interleukin (IL)-6, one of the cytokines released from inflammatory cells, stimulates insulin secretion in a physiological concentration (1-100 pg/ml), but the exact mechanism is still unknown. The present studies were undertaken to investigate the mechanism of IL-6-induced stimulation of insulin secretion in HIT-T 15 cells. The effects of the addition of nifedipine on the IL-6 (100 pg/ml)-induced stimulation of insulin secretion were investigated. We also examined the possibility that IL-6 (1-100 pg/ml) may stimulate insulin messenger ribonucleic acid (mRNA) expression, using the reverse transcription-polymerase chain reaction method. The addition of 100 and 1000 nM nifedipine significantly attenuated the stimulatory effects of 100 pg/ml IL-6 on insulin secretion. The addition of 1-100 pg/ml IL-6 dose-dependently increased preproinsulin mRNA expression relative to beta-actin mRNA. IL-6 increased insulin gene promoter activity of fragments A (-2188 to +337 bp) and B (-1782 to +270 bp) but not fragments C (-1275 to +270 bp), D (-1138 to +270 bp), E (-880 to +236 bp) or F (-356 to +252 bp). The addition of 10 nM nifedipine completely abolished the stimulatory effect of 10-100 pg/ml IL-6 on relative preproinsulin mRNA expression. These data raised the possibility that IL-6 increased preproinsulin mRNA expression via the stimulation of Ca(2+) influx which enhances insulin gene expression.

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H. KOBA, T. SHIMIZU, M. MIENO, A. AMANO and K. YAMASHITA

Department of Pathophysiology, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki, Japan

(Received 17 February 1975)

The mechanism underlying the depletion of ascorbic acid content of steroid-producing tissues in response to trophic hormones is poorly understood. Previous work (Koba, Kawao & Yamashita, 1971) has shown that, in the dog, the discharge of ascorbic acid as well as 17-oxosteroids into spermatic venous blood by testicular tissue stimulated with human chorionic gonadotrophin (HCG) can be suppressed by the prior administration of methylenedianiline, an amphenone analogue capable of inhibiting testicular steroidogenesis (Yamashita, 1967). Since oestrogens have been shown to be effective inhibitors of the glucose-6-phosphate dehydrogenase necessary for corticosteroid synthesis in the rat adrenal (McKerns, Coulomb, Kaleita & De Renzo, 1958), we have studied male dogs pretreated with oestradiol-17β.

Eight adult male mongrel dogs weighing 9·6–15·1 kg were used. Four animals were given 20 μg oestradiol-17β (dissolved in 0·2 ml sesame oil)/

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H Kadokawa, M Matsui, K Hayashi, N Matsunaga, C Kawashima, T Shimizu, K Kida and A Miyamoto

This study was conducted to estimate the effects of kisspeptin-10 on blood concentrations of LH and GH in prepubertal dairy heifers. Heifers received a single injection of 1 mg kisspeptin-10 (n=5) or saline (n=5) intravenously, and serial blood samples were collected at 15-min intervals to analyze the response curves of both LH and GH after injection. Peak-shaped responses were observed for concentrations of LH and GH, and the peaks were observed at 27±3 and 75±9 min, respectively, after injection, only in heifers injected with kisspeptin-10. These data suggest various possible important links among kisspeptin, the reproductive axis, and also the somatotropic axis in prepubertal Holstein heifers.

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H Shimizu, Y Shimomura, Y Nakanishi, T Futawatari, K Ohtani, N Sato and M Mori

Abstract

The decrease in estrogen in menopausal women increases body fat. The present studies were undertaken to investigate the involvement of estrogen in leptin production in vivo. In the first study, expression of ob gene mRNA in white adipose tissue was measured at 2 and 8 weeks after ovariectomy in rats. In the second, serum leptin concentration was measured in total body fat of 87 weight-matched human subjects (29 men, 29 premenopausal and 29 postmenopausal women). In the third, changes in serum leptin concentration with the menstrual cycle were determined, ob gene expression decreased in subcutaneous and retroperitoneal white adipose tissue of ovariectomized rats 8 weeks after the operation, while ovariectomy increased ob gene expression in mesenteric white adipose tissue. Serum leptin concentration was decreased by ovariectomy. Estradiol supplement reversed the effect of ovariectomy on ob gene expression and circulating leptin levels. In humans, serum leptin concentration was higher in premenopausal women than in men, and in postmenopausal women it was lower than in premenopausal women, but still higher than in men. In 13 premenopausal women, serum leptin levels were significantly higher in the luteal phase than in the follicular phase. The present studies strongly indicate that estrogen regulates leptin production in rats and human subjects in vivo. Regional variation in the regulation of ob gene expression by estrogen was found.

Journal of Endocrinology (1997) 154, 285–292