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Department of Pathophysiology, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki 852, Japan
(Received 25 May 1976)
In a previous study (Yamashita, Shimizu & Waki, 1972), there was a decreased response of the adrenal medulla to acetylcholine after exposure of the adrenal area to moderate doses of X-rays. In this paper, we present the results observed on the secretory response of the adrenal medulla to exogenous insulin in dogs whose heads had been irradiated with lower doses of X-rays.
Adult mongrel dogs weighing 8·9–13·9 kg were used. The animals received 200 rad X-irradiation to their heads under pentobarbitone anaesthesia. Radiation was delivered at a rate of 60·6 rad/min, using a therapy unit (200 kV, 30 mA) with a filter comprising 1·5 mm copper– 0·5 mm aluminium. Target-to-skin distance was 37 cm. Approximately 18 h after irradiation, the animals were re-anaesthetized with pentobarbitone (25 mg/kg, injected i.v.) and the left
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In previous experiments in our laboratory, the dog testis exposed to low doses of X-rays has shown great secretory activity in response to pregnant mare serum within a few days of irradiation, and this effect continues for 30 days or so (Shimizu, Yamashita, Sugihara & Tsuchiyama, 1972). However, it has not been ascertained whether the effect occurs within a few hours of exposure. We now report the response of the testis to human chorionic gonadotrophin (HCG) 1·5 h or 24 h after exposure to 200 R of X-rays. The secretory response was determined by measuring total 17-oxosteroids in spermatic venous blood.
Eighteen adult mongrel dogs (9·0–16·3 kg) were studied. Under pentobarbitone anaesthesia (injected i.v.), X-irradiation of the scrotal area alone was administered to 12 dogs in single doses of 200 R. Radiation was delivered using a 200 kV peak therapy unit (200 kV, 20 mA) through a 0·5 mm copper—0·5
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Salmon plasma contains at least three IGF-binding proteins (IGFBPs) with molecular masses of 41, 28 and 22 kDa. The 41 kDa IGFBP is similar to mammalian IGFBP-3 in size, type of glycosylation and physiological responses. In this study, we developed an RIA for the 41 kDa IGFBP. The 41 kDa IGFBP purified from serum was used for antibody production and as an assay standard. Binding of three different preparations of tracer were examined: (125)I-41 kDa IGFBP, (125)I-41 kDa IGFBP cross-linked with IGF-I and 41 kDa IGFBP cross-linked with (125)I-IGF-I (41 kDa IGFBP/(125)I-IGF-I). Only binding of 41 kDa IGFBP/(125)I-IGF-I was not affected by added IGFs, and therefore it was chosen for the tracer in the RIA. Plasma 41 kDa IGFBP levels measured by RIA were increased by GH treatment (178.9+/-4.9 ng/ml) and decreased after fasting (95.0+/-7.0 ng/ml). The molarities of plasma 41 kDa IGFBP and total IGF-I were comparable, and they were positively correlated, suggesting that salmon 41 kDa IGFBP is a main carrier of circulating IGF-I in salmon, as is mammalian IGFBP-3 in mammals. During the parr-smolt transformation (smoltification) of coho salmon, plasma 41 kDa IGFBP levels showed a transient peak (182.5+/-10.3 ng/ml) in March and stayed relatively constant thereafter, whereas IGF-I showed peak levels in March and April. Differences in the molar ratio between 41 kDa IGFBP and IGF-I possibly influence availability of IGF-I in the circulation during smoltification.
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SUMMARY
The effect of prostaglandin E2 (PGE2) on the secretion of adrenaline and noradrenaline by the adrenal gland and the interaction between PGE2 and acetylcholine in the adrenal medulla were examined in anaesthetized dogs. In splanchnicotomized dogs, i.v. injection of PGE2 failed to induce any secretion of catecholamines from the adrenal gland, whereas administration of PGE2 into the lumboadrenal artery resulted in a slight, approximately dose-dependent increase in catecholamine secretion within 2 min of the injection. This effect of PGE2 was unaffected by i.v. administration of atropine. Intravenous administration of acetylcholine 1 min after the administration of PGE2 into the lumboadrenal artery of splanchnicotomized atropine-treated dogs had a markedly greater effect on adrenal catecholamine secretion; the resultant output was about twice that evoked by acetylcholine in the absence of PGE2. The effect was more than additive, since the response to acetylcholine was at least one order of magnitude greater than that to PGE2. This indicates that PGE2 and acetylcholine may act synergistically in the adrenal medulla.
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The rate of secretion of 17-oxosteroids by the testes of anaesthetized dogs in vivo was used as an index of LH secretion. Intracarotid injection of luteinizing hormone releasing hormone (LH-RH, 1, 5 or 10 μg/kg body wt) resulted in an increase in the testicular 17-oxosteroid secretion which was roughly proportional to the dose administered and which reached a maximum 60 min after the injection. Testicular output of 17-oxosteroids was unaffected by administration of melatonin (10 or 100 μg/kg body wt) into the carotid artery. When LH-RH (5 μg/kg) was injected into the carotid artery 3 h after intracarotid injection of melatonin (10 or 100 μg/kg), the testicular response to LH-RH was considerably diminished. Pretreatment with melatonin (100 μg/kg) did not alter the testicular response to human chorionic gonadotrophin (20 i.u./kg body wt) given i.v.
It is concluded that melatonin may act directly on the anterior pituitary gland in dogs to inhibit the LH-RH-induced release of LH.
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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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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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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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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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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
