The present study was undertaken to investigate whether human chorionic gonadotropin (hCG) beta-core fragment (hCG beta cf) was directly produced by gestational trophoblastic tumors. Immunoreactivity of hCG beta cf was demonstrated in the extracts as well as in the culture media of hydatidiform mole tissues. It was also present in the extracts of choriocarcinoma tissues, and its molar concentration exceeded that of intact hCG. The presence of hCG beta cf was then confirmed by gel chromatography and Western blot analysis. Immunohistochemistry showed localization of hCG beta cf immunoreactivity to the syncytiotrophoblasts and scattered cells in the stroma of mole tissue, and to syncytiotrophoblastic cells in choriocarcinoma. Immunoreactivity of hCG beta cf was also detected in the sera of the patients with gestational trophoblastic disease, although the hCG beta cf/hCG ratio was less than one hundredth of that in the tissue extracts. Serial measurement of serum hCG beta cf levels after mole evacuation showed that they declined much more rapidly than those of hCG and became undetectable in the patients with subsequent spontaneous resolution, while hCG beta cf remained or became detectable before the rise of hCG was observed in the patients with subsequent persistent trophoblastic disease. Taken together, these results suggest that hCG beta cf is directly produced by gestational trophoblastic tumors, and monitoring of hCG beta cf in the serum after mole evacuation may be useful for early prediction of subsequent development of postmolar persistent trophoblastic disease.
H. Sugimoto, M. Suzuki, T. Takeuchi and K. Ishikawa
To investigate the effect of cell density on rat somatotrophs, dispersed adult rat anterior pituitary cells were plated at several densities (0·6, 1·2, 2·4 and 6·0 × 105 cells/cm2) or at two densities (6·07 × 105 and 1·21 × 105 cells/cm2) as high and low densities respectively. A static incubation system was used to study the release of GH and peptidyl glycine α-amidating enzyme (PAM; a component of secretory granules) and the cellular concentration of cyclic AMP (cAMP) in basal and stimulated cells. In addition, a perifusion system was used to characterize the sensitivity to GH-releasing factor (GRF) at both high and low densities. The high density of cells in both the static incubation and perifusion systems caused a low basal secretion rate of GH and PAM. When the cultured cells were stimulated with human GRF (hGRF) in perifusion, GH secretion from cells at high density was markedly higher than that from cells at low density. The cAMP content in the static incubation system showed a similar tendency to that observed for basal and stimulated GH secretion; that is, the basal cAMP content was increased as the cell density decreased. The cellular concentration of cAMP was increased by about threefold by 1 nmol hGFR/1 and by more than tenfold by 10 nmol hGRF/1. When the medium from cells cultured at low density was replaced by that from the cells at high density, there was no change in the basal cellular cAMP content of the cells at low density. These data suggest that cell-to-cell contact in dispersed pituitary cells seems to be important in the maintenance of their cellular integrity to secrete GH.
Journal of Endocrinology (1991) 131, 237–244
T Matsuwaki, M Suzuki, K Yamanouchi and M Nishihara
We have previously reported that tumor necrosis factor-alpha (TNF-alpha) suppressed pulsatile secretion of luteinizing hormone (LH) in adrenalectomized (ADX) rats, which was restored by replacement of glucocorticoid. In the present study, we examined the role of glucocorticoid in inducing the preovulatory LH surge under conditions of infectious stress. Intravenous injection of TNF-alpha (1 microg) into the proestrous rats at 1300 h attenuated the LH surge and decreased the number of oocytes ovulated. The inhibitory effect of TNF-alpha on the LH surge was blocked by pretreatment with indomethacin, suggesting that the effects of TNF-alpha were mediated by prostaglandins (PGs). On the other hand, ADX markedly enhanced the inhibitory effect of TNF-alpha on the LH surge and subsequent ovulation, which was almost completely restored by pretreatment with a subcutaneous injection of corticosterone (10 mg). These results suggest that glucocorticoid counteracts the inhibitory effect of the cytokines on the preovulatory LH surge by suppressing PG synthesis, and thereby helps to maintain reproductive function under infectious stress conditions.
J Ogino, K Sakurai, K Yoshiwara, Yoichi Suzuki, N Ishizuka, N Seki, Yoshifumi Suzuki, H Koseki, T Shirasawa, N Hashimoto, K Yagui and Y Saito
Several mutations of the tyrosine kinase domain of insulin receptor (IR) have been clinically reported to lead insulin resistance and insulin hypersecretion in humans. However, it has not been completely clarified how insulin resistance and pancreatic β-cell function affect each other under the expression of mutant IR. We investigated the response of pancreatic β-cells in mice carrying a mutation (P1195L) in the tyrosine kinase domain of IR β-subunit. Homozygous (Ir P1195L/P1195L) mice showed severe ketoacidosis and died within 2 days after birth, and heterozygous (Ir P1195L/wt) mice showed normal levels of plasma glucose, but high levels of plasma insulin in the fasted state and after glucose loading, and a reduced response of plasma glucose lowering effect to exogenously administered insulin compared with wild type (Ir wt/wt) mice. There were no differences in the insulin receptor substrate (IRS)-2 expression and its phosphorylation levels in the liver between Ir P1195L/wt and Ir wt/wt mice, both before and after insulin injection. This result may indicate that IRS-2 signaling is not changed in Ir P1195L/wt mice. The β-cell mass increased due to the increased numbers of β-cells in Ir P1195L/wt mice. More proliferative β-cells were observed in Ir P1195L/wt mice, but the number of apoptotic β-cells was almost the same as that in Ir wt/wt mice, even after streptozotocin treatment. These data suggest that, in Ir P1195L/wt mice, normal levels of plasma glucose were maintained due to high levels of plasma insulin resulting from increased numbers of β-cells, which in turn was due to increased β-cell proliferation rather than decreased β-cell apoptosis.
T Michigami, H Yamato, H Suzuki, Y Nagai-Itagaki, K Sato and K Ozono
In patients with humoral hypercalcemia of malignancy (HHM), serum levels of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) are generally low, although the pathophysiology of the impaired vitamin D metabolism is not fully understood. In the present study, we have investigated vitamin D metabolism in our newly developed rat model of HHM in which a human infantile fibrosarcoma producing parathyroid hormone-related protein (PTHrP), named OMC-1, was inoculated s.c. into athymic nude rats. In OMC-1-bearing rats, the serum concentration of 1,25(OH)(2)D was markedly reduced when the animals exhibited severe hypercalcemia (Ca > or =15 mg/dl), while it was rather elevated in those with mild hypercalcemia. To further examine whether serum Ca levels affect 1,25(OH)(2)D concentration, we administered bisphosphonate YM529 to OMC-1-bearing rats when they exhibited severe hypercalcemia. The restoration of the serum Ca level by administration of YM529 was accompanied by a marked increase in the 1,25(OH)(2)D level, suggesting that the serum Ca level itself plays an important role in the regulation of the 1,25(OH)(2)D level in these rats. On the other hand, when the OMC-1-bearing rats were treated with a neutralizing antibody against PTHrP, serum 1,25(OH)(2)D levels remained low despite the reduction in serum Ca levels. Expression of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) in kidney was decreased in OMC-1-bearing rats with severe hypercalcemia, and markedly enhanced after treatment with bisphosphonate. This enhancement in 1 alpha-hydroxylase expression was not observed after treatment with the antibody against PTHrP. These results suggest that PTHrP was responsible for the enhanced expression of 1 alpha-hydroxylase in YM529-treated rats, and that hypercalcemia played a role in reducing the serum 1,25(OH)(2)D level in OMC-1-bearing rats by suppressing the PTHrP-induced expression of the 1 alpha-hydroxylase gene.
T. SUZUKI, K. HIRAI, H. YOSHIO, K-I. KUROUJI and T. HIROSE
That eserine and atropine stimulate the pituitary-adrenocortical system has been proved only by indirect evaluation such as the rat adrenal ascorbic acid depletion test (Dordoni & Fortier, 1950). In the experiments of Harwood & Mason (1956) eserine was injected intravenously in a dose of 0·05 mg./kg. body weight into unanaesthetized dogs. Although a marked eosinopenia was observed, no definite rise in the level of 17-hydroxycorticosteroids (17-OHCS) in peripheral blood was found after administration of eserine. In the present study attempts were made to evaluate directly the effect of eserine and atropine on adrenal 17-OHCS secretion in unanaesthetized dogs.
Six mongrel dogs weighing between 11 and 15 kg. were used. Samples of adrenal venous blood were taken from the unanaesthetized animals by a modification of the method of Satake, Sugawara & Watanabe (1927) (see Suzuki, Hirai, Yoshio, Kurouji & Yamashita, 1963).
The observations were started about 18 hr. after completion of
Y Watanabe, K Kawai, S Ohashi, C Yokota, S Suzuki and K Yamashita
To examine the structure–activity relationships in the insulinotropic activity of glucagon-like peptide-1(7–36) amide (GLP-1(7–36)amide), we synthesized 16 analogues, including eight which were designed by amino acid substitutions at positions 10 (Ala10), 15 (Serl5), 16 (Tyr16), 17 (Arg17), 18 (Lys18), 21 (Gly21), 27 (Lys27) and 31 (Asp31) of GLP-1(7–36)amide with an amino acid of GH-releasing factor possessing only slight insulinotropic activity, and three tentative antagonists including [Glu15]-GLP-1(8–36)amide. Their insulinotropic activities were assessed by rat pancreas perfusion experiments, and binding affinity to GLP-1 receptors and stimulation of cyclic AMP (cAMP) production were evaluated using cultured RINm5F cells.
Insulinotropic activity was estimated as GLP-1(7–36)amide = Tyr16>Lys18, Lys27>Gly21>Asp31⪢Ser15,Arg17>Ala10⪢GRF>[Glu15]-GLP-1(8–36) amide. Displacement activity against 125I-labelled GLP-1 (7–36)amide binding and stimulatory activity for cAMP production in RINm5F cells correlated well with their insulinotropic activity in perfused rat pancreases.
These results demonstrate that (1) positions 10 (glycine), 15 (aspartic acid) and 17 (serine) in the amino acid sequence of GLP-1(7–36)amide, in addition to the N-terminal histidine, are essential for its insulinotropic activity through its binding to the receptor, (2) the amino acid sequences for the C-terminal half of GLP-1(7–36)amide also contribute to its binding to the receptor, although they are less important compared with those of the N-terminal half, and (3) [Glu15]-GLP-1(8–36)amide is not an antagonist of GLP-1(7–36)amide as opposed to des-His1 [Glu9]-glucagon amide which is a potent glucagon antagonist.
Journal of Endocrinology (1994) 140, 45–52
Y. Nishii, K. Hashizume, K. Ichikawa, T. Miyamoto, S. Suzuki, T. Takeda, K. Yamauchi, M. Kobayashi and T. Yamada
Changes in the amount of cytosolic 3,5,3′-tri-iodo-l-thyronine (T3)-binding protein (CTBP) and its activator during administration of l-thyroxine (T4) to thyroidectomized rats were investigated. Thyroidectomy decreased the amount of CTBP in the kidney, whereas the activator was not significantly modified by thyroidectomy. The activator was increased by administration of T4 to thyroidectomized rats. The amount of CTBP was also increased by administration of T4. The activator increased the maximal binding capacity (MBC) without changes in the affinity constant for T3 binding in CTBP. A T4-induced increase in MBC in cytosol inhibited nuclear T3 binding in vitro by competition of T3 binding between CTBP and the nuclear receptor.
These results suggest that thyroid hormone increases the capacity for cytosolic T3 binding through increasing the amount of CTBP and its activator, and that these increases play a role in regulating the amount of T3 that binds to its nuclear receptor.
Journal of Endocrinology (1989) 123, 99–104
T Okada, N Matsuzaki, K Sawai, T Nobunaga, K Shimoya, K Suzuki, N Taniguchi, F Saji and Y Murata
Chorioamnionitis has been shown to be one of the most important factors in inducing preterm delivery. The present study was undertaken to examine the effects of chorioamnionitis on placental endocrine functions. Preterm placentas with histologic chorioamnionitis produced smaller amounts of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) than those without chorioamnionitis (P < 0.001). To examine the mechanism involved in the suppression of placental endocrine functions induced by chorioamnionitis, we initially confirmed the expression of lipopolysaccharide (LPS) receptor, i.e. the CD14 molecule, on trophoblasts by Northern blot analysis and immunohistochemistry. We then stimulated purified trophoblasts with LPS, which is the major agent which induces inflammatory responses in the host via the LPS receptor. The trophoblasts stimulated with LPS produced reduced amounts of hCG, hPL, and progesterone in a time- and dose-dependent fashion in spite of the induced manganese-superoxide dismutase (SOD) synthesis. Stimulation of trophoblasts with hypoxanthine and xanthine oxidase resulted in suppressed hCG production, while the simultaneous addition of SOD into the culture medium reversed the suppression of hCG production. LPS in the placenta with chorioamnionitis might directly stimulate trophoblasts through the LPS receptor (CD14), thus reducing placental endocrine functions. Superoxide anions which exogenously act on trophoblasts might be generated by simultaneous stimulation of neutrophils and monocytes at the feto-maternal interface by LPS, and additively reduce placental endocrine functions.
W Jiang, T Miyamoto, T Kakizawa, T Sakuma, S Nishio, T Takeda, S Suzuki and K Hashizume
Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRalpha and TRbeta, which act as mediators of thyroid hormone in tissues. However, the relative expression of each TR isoform in target tissues is still elusive. Herein, we have developed an RT-PCR and restriction enzyme digestion method to determine the expression of TRalpha1 and TRbeta1. We analyzed the expression of TR isoforms in 3T3-L1 preadipocytes induced to differentiate by an adipogenic cocktail in the presence or absence of 100 nM triiodothyronine (T(3)). The TRalpha1 isoform was predominantly expressed in 3T3-L1 adipocytes, and its expression was increased at the stage of development concomitant with the emergence of lipid droplets. Little, if any, TRbeta1 mRNA was detected in adipocytes. Administration of T(3) to the differentiating 3T3-L1 cells enhanced the accumulation of triglyceride. The expression profile of TRalpha1 in T(3)-treated adipocytes was similar to that in non-treated cells. The transcripts of adipogenic factors, CCAAT/enhancer binding protein beta (C/EBPbeta) and peroxisome proliferator activated receptor gamma (PPARgamma), were not altered by T(3). Lipid binding protein, aP2, that is downstream of these transcription factors was also unaffected by T(3). In contrast, the lipogenic enzyme, glyceraldehyde-3-phosphate dehydrogenase mRNA was significantly increased in the presence of T(3). Therefore, T(3) appears to be a hormone capable of modulating the expression of lipogenic enzyme and augments the accumulation of lipid droplets. We conclude that the TRalpha isoform might play an important role in the generation and maintenance of the mature adipocyte phenotype, regulating the expression of lipogenic enzymes.