The present study was designed to identify the effects of metyrapone-induced corticosterone deficiency on Leydig cell steroidogenesis in adult male rats. Adult Wistar rats (200-250g body weight) were treated with metyrapone, an inhibitor of corticosterone synthesis (10mg/100g body weight, s.c., twice daily) for 10 days. Experimental animals were killed along with controls, blood was collected, and sera separated for testosterone and estradiol assays. Testes were removed and Leydig cells were isolated, purified and used for estimating the specific activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) and ¹⁴C-glucose oxidation. Serum testosterone (p<0.05), Leydig cellular ¹⁴C-glucose oxidation (p<0.001) and the specific activity of 17β-HSD (p<0.01) were significantly decreased in metyrapone treated rats. However, serum estradiol was not markedly altered compared to control. In addition to this, a set of in vitro experiments were also performed to identify the effects of metyrapone-induced corticosterone deficiency on hCG and prolactin-induced Leydig cell testosterone production. Metyrapone treatment significantly (p<0.05) decreased the Leydig cellular basal as well as hCG and its combination with prolactin stimulated testosterone production in vitro. It is concluded from the present study that the inhibitory effects of metyrapone-induced corticosterone deficiency on Leydig cell steroidogenesis are mediated through impaired glucose oxidation and 17β-HSD activity. in vitro studies showed that corticosterone deficiency impairs not only hCG action but also the potentiating effect of prolactin on Leydig cell steroidogenesis.

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To investigate the differences in leptin production by regional fat mass, 76 postmenopausal Japanese women were enrolled in this study. Age, height, weight, and body mass index (BMI, wt/ht²) were recorded. Serum leptin levels were measured by RIA. Trunk fat mass, total body fat mass, and percentage of body fat were measured by dual-energy x-ray absorptiometry (DEXA). The ratio of trunk to leg fat mass (trunk-leg fat ratio), an index of body fat distribution, was also assessed by DEXA. Relationship of leptin levels with baseline characteristics and anthropometric variables were investigated by Pearson correlation test. Serum leptin levels were positively correlated with BMI (r=0.683, p<0.0001), total body fat mass (r=0.680, p<0.0001), trunk fat mass (r=0.632, p<0.0001), and percentage of body fat (r=0.624, p<0.0001). However, no significant correlation was observed between trunk-leg fat ratio and leptin levels (r=0.181). Age and height were not correlated with leptin levels. Based on these results, we concluded that body fat distribution does not serve as a predictor of leptin levels in postmenopausal women.

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Withdrawal of estrogen by ovariectomy increases adiposity, but decreases the circulating levels of the ob gene product, leptin, which inhibits food intake. The reduction of circulating leptin levels may thus play an important role in the induction of obesity by ovariectomy. To examine this hypothesis, body weight change by ovariectomy was investigated in leptin-deficient genetically obese (ob/ob) mice with leptin supplement. Prior to the operation, obese (ob/ob) female mice were treated with intraperitoneal administration of recombinant mouse leptin (1.0 μg/g body weight/day) for 8 days. Then, half of the leptin-treated mice and their lean littermates were bilaterally ovariectomized and their body weight changes were observed for 56 days. From 16 days after the operation, a significant increase in body weight by ovariectomy was observed only in lean mice without leptin treatment. From 44 days, a significant body weight gain by ovariectomy was observed in leptin-treated obese mice. Ovariectomy significantly increased retroperitoneal white adipose tissue weight in their lean littermates, but not in leptin-treated obese mice. It was suggested that the reduction of circulating leptin levels may play an important role in the increases of acute phase body weight gain by ovariectomy, but during static phase, the direct effects of estrogen withdrawal may appear independent of leptin-mediated effects.

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Familial acromegaly (FA) is a rare inherited disease characterized by clustering of somatotrophic adenomas and acromegaly within a family without other manifestations of multiple endocrine neoplasia-type 1 (MEN-1). The genetic basis of this pituitary-specific phenotype is largely unknown, and its relationship to the MEN-1 locus on chromosome 11q13 also remains unclear. To test the hypothesis that FA results from a germline mutation of the MEN-1 locus, we performed a linkage analysis in a Japanese family with 2 members showing manifestations of acromegaly due to somatotroph adenomas. We also examined the adenoma of one patient for loss of heterozygosity (LOH) at 11q13 locus and for the presence of mutations of codon 201 and 227 in the gene for Gsα. Our results provided no evidence that either germline alterations of the MEN-1 locus, LOH at 11q13, or somatic mutation of Gsα plays a causative role in the development of somatotroph adenomas in our FA family. Together with the previous reports, these results suggest that there are at least two distinct subgroups of FA: one that results from a mutation in MEN-1 locus and the other whose causative gene is located outside the 11q13 locus.

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Molecular epidemiologic studies have reported a relationship between 1α,25 dihydroxyvitamin D3 (1,25(OH)₂D₃) and the development and progression of malignant tumors. (1,25(OH)₂D₃) exerts its biological activity by binding the vitamin D receptor (VDR), while recent studies have demonstrated that VDR gene polymorphisms affect serum levels of (1,25(OH)₂D₃). Serum levels of (1,25(OH)₂D₃) are reported to be significantly lower in patients with renal cell carcinoma (RCC) compared to non-cancer control patients. The purpose of this study was to investigate the TaqI VDR polymorphism in Japanese RCC patients and non-cancer controls in order to determine if an association exists between VDR genotype and the risk of developing RCC as well as clinical risk factors. A total of 102 RCC patients and 204 controls were genotyped for a previously described TaqI restriction fragment length polymorphism (RFLP) of the VDR gene. Products were digested into T allele or the t allele according to the absence or presence of a TaqI restriction site. Individuals were classified as TT, Tt or tt. The genotype TT was statistically more frequent among RCC patients (80.4%) compared to controls (61.8%) (OR = 2.54; 95% CI, 1.44-4.46; p = 0.0006). In addition, the occurrence of the genotype TT was significantly higher in patients with rapid-growth-type group (92.1%) compared to slow-growth-type group (73.4%) (OR = 4.22; 95% CI, 1.15-15.53; p = 0.0175). These data demonstrate that VDR genotype plays an important role in determining the risk of developing more aggressive RCC in Japanese.

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Angiogenesis and bone remodeling are closely associated, and vascular endothelial cells may have potential roles for osteoclastic bone resorption. We examined whether clonal endothelial cells established from bone, aorta and brain of Balb/c mice influenced osteoclast-like cell formation in vitro. As low as 1% conditioned media of those endothelial cells inhibited osteoclast-like cell formation in bone marrow cultures induced by 1,25-dihydroxyvitamin D₃, and did so in spleen cell cultures in the presence of soluble receptor activator of nuclear factor-κB ligand (RANKL), M-CSF and prostaglandin E₂. The level of osteoprotegerin (OPG), a decoy receptor for RANKL, secreted by endothelial cells was not high enough to inhibit osteoclastogenesis. These observations suggest that endothelial cells derived from various tissues secrete factor(s) that inhibits precursors to differentiate into osteoclasts even in the presence of optimal stimulators for osteoclastogenesis. Hence, endothelial cells in bone may inhibit recruitment of fresh osteoclasts, and those in tissues other than bone may be involved in prohibiting ectopic osteoclastogenesis.

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We established a new analytical system in which functioning cells were transplanted directly into the pancreas and liver. The retrograde transplantation of beta cell line, Min6 cells, into the streptozotocin-diabetic mice normalized plasma glucose and insulin levels. The injected cells were protected from pancreatic enzymes with enzyme inhibitor. Blood glucose decreased gradually over 10 days and the diabetic mice recovered weight at the same time. Intraperitoneal glucose tolerance test showed that the peak of plasma glucose of the transplanted mice was less than half that of the control. The insulin secretion of the transplanted mice was recovered and stimulated 4.6 times from the basal secretion. Histological analyses showed that the pancreas and liver were characterized by Min6 cell clusters dispersed throughout the organs. Min6 cells were detected near the pancreatic or bile ducts. It is suggested that the injected cells obstructed the peripheral ducts where they settled. The weight of pancreas and liver did not differ significantly in either Min6 transplanted or the control mice. The metabolic effects on the weights of these organs appeared the same in both groups. This is the first report that cells transplanted via ducts into the pancreas and liver performed their biological function. Our transplantation model makes possible the in vivo analysis of the regeneration machinery of the pancreas and liver.

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Insulin resistance is closely related to developing type 2 diabetes mellitus. Visceral fat accumulation is associated with insulin resistance, which affects the free fatty acid (FFA) metabolism. We investigated the interactions among visceral fat accumulation, FFA metabolism and insulin resistance in 20 patients with type 2 diabetes mellitus, including 11 obese and 9 non-obese subjects. Body fat distribution was estimated by measuring the areas of both subcutaneous and visceral fat mass on abdominal computed tomography at the umbilical level. Glucose infusion rate (GIR) and plasma FFA responses to insulin were determined as an index of insulin resistance and anti-lipolytic action, respectively, in a euglycemic hyperinsulinemic clamp study. There was an inverse correlation between GIR and insulin-induced decrease in plasma FFA in all diabetic patients (r = − 0.652, P<0.01). Visceral fat mass area was well correlated with GIR (r = − 0.583, P<0.01) and insulin-induced decrease in plasma FFA (r = 0.724, P<0.001), whereas subcutaneous fat mass area was not correlated either with GIR or plasma FFA decrease. These findings suggest that visceral fat accumulation results in increasing the resistance against the anti-lipolytic action of insulin, and that FFA metabolism is closely related with glucose utilization in patients with type 2 diabetes mellitus.

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Bisphenol A (BPA), a monomer of plastic used in consumer products, is abundant in the environment and enters the body by ingestion or adsorption. In order to characterize the estrogenic effect of BPA, we performed luciferase assay on three independent cell lines derived from different tissues transfected with either human ERα cDNA or ERβ cDNA. The estrogenic activities of BPA were detectable in all cell lines via both ERα and ERβ. In 293T cells and Hec-1 cells, the estrogenic activities were significantly decreased when cells expressing ERα were incubated with 10^-6 M BPA in the presence of 10^-8 M 17β-estradiol (E2) while the activities via ERβ were essentially unchanged in the same conditions. Interestingly, no reduction of estrogenic activity was detected in HOS-TE85 cells via either ERα or ERβ. Our results indicate that BPA only acts as an agonist of estrogen via ERβ whereas it has dual actions as an agonist and antagonist in some types of cells via ERα. Thus, the activity of BPA may depend on the ER subtype and the tissue involved.

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The presence of human progesterone receptor (PR) mRNA variants has been demonstrated in uterine endometrium and breast tissues as well as in cancer cells of these tissues. While exon deletions by the alternative splicing in these variants have been reported, there are few reports available on the PR mRNA variants with exon insertion. In the present study, we attempted to detect a PR mRNA variant containing the exon insertions in normal uterine endometrium. Endometrial tissues were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with PCR primers which were located in exons 3 and 8. Analysis of the RT-PCR products revealed the presence of a novel PR mRNA variant which contained a 232 bp inserted nucleotide sequence between exons 4 and 5. We termed this transcript the “i45 PR mRNA variant”. Genomic analysis indicated that the inserted sequence was derived from two novel independent exons of 123 bp and 109 bp, termed “exon i45a” and “exon i45b”, respectively, which are located between exons 4 and 5 of the human PR gene. The i45 PR mRNA variant was further detected in uterine endometrial cancer tissues as well as in the normal uterine endometrium. These results demonstrate the presence of a novel PR mRNA variant with exon insertions in the human tissue for the first time. The i45 PR variant protein, possibly transcribed from this i45 PR mRNA variant, may play physiological and/or pathological roles in the human uterine endometrium.

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Cell-to-cell interaction is required for the differentiation of osteoclast precursors as well as for osteoclast function. The present study was undertaken to determine whether 1,25 dihydroxyvitamin D₃ (1,25D), PTH, IL-1α and PGE₂ depend on cell-to-cell interactions through the intercellular adhesion molecule (ICAM)-1/leukocyte function-associated antigen (LFA)-1 pathway in osteoclast formation and bone resorption. We found that mouse osteoblasts expressed ICAM-1 and that the expression was increased by treatment with PTH, IL-1α or 1,25D, but not by PGE₂. In resorption assays measuring either ⁴⁵Ca release from bone organ cultures or pit formation in bone cell cultures, 1,25D-, PTH- and IL-1α-stimulated resorption was inhibited by anti-ICAM-1 monoclonal antibody (mAb) and/or anti-LFA-1 mAb, while basal and PGE₂-stimulated bone resorbing activities were not affected by these mAbs. Furthermore, in a mouse bone marrow culture system, stimulation of osteoclast-like (OCL) cell formation by 1,25D (10 nM), PTH (10ng/ml) or IL-1α (10ng/ml) was inhibited by the addition of anti-ICAM-1 mAb and/or anti-LFA-1 mAb. In a coculture system of murine spleen cells and osteoblasts, the ICAM-1/LFA-1 interaction was also involved in 1,25D-, PTH- and IL-1α-stimulated TRAP-positive MNC formation. However, anti-ICAM-1 mAb and anti-LFA-1 mAb did not alter either 1,25D- or PTH-stimulated receptor activator of NF-κB ligand (RANKL) mRNA transcription in bone marrow cultures. Taken together, we here propose that ICAM-1-mediated cell-to-cell adhesion of osteoblasts and osteoclast precursors is involved in RANKL-dependent osteoclast maturation stimulated by 1,25D, PTH, and IL-1α.

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We report monozygotic twins of different sexual phenotypes. One of the twins had complete female external genitalia except for a mild clitoromegaly. She had bilateral gonads consisting of the wavy stroma and scant dysgenetic seminiferous tubules. No androgen secretion was induced by gonadotrophin stimulation. The other twin had hypospadiac male genitalia. His gonads were located intrascrotally and he had good androgenic response to a stimulation test. Conventional and fluorescence in situ hybridization chromosome analysis disclosed that both twins had a 47,X,idic(Y),idic(Y)/46,X,idic(Y)/45,X and 47,X, + mar × 2.ish idic(Y)(q11.2)(DYZ3 + + × 2)/46,X, + mar.ish idic(Y)(q11.2)(DZY3 + + )/45,X. These twins were clinically monochorionic and allelotype analysis in these twins and their parents with microsatellite markers showed the affirmative probability of 0.999999994 for monozygosity. The ratio of mosaicism, gonadal histology, and testosterone productivity were reasonably correlated to the genital virilization in these monozygotic twins, showing discordant sexual phenotypes.

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The aim of this study is to determine the body fat distribution and cardiovascular disease risk factors in pre- and postmenopausal obese women matched for weight, height and body mass index (BMI). Study group consisted of 405 premenopausal overweight/obese (BMI >27 kg/m², mean 37.83 ± 6.91 kg/m²) and 405 postmenopausal overweight/obese (BMI >27 kg/m²), BMI-matched (mean 37.77 ± 6.84 kg/m²) women. None of the women were on hormone replacement therapy. Insulin resistance was evaluated by “homeostasis model assessment” (HOMA) formula. Intraabdominal fat volume was calculated according to the following formula: IAF (L) = [(0.370 × abdominal sagittal diameter) − 4.85]. Age, waist circumference, waist to hip ratio (WHR) and intraabdominal fat volume were significantly higher in postmenopausals compared with BMI-matched premenopausal women (p<0.001). Systolic and diastolic blood pressure, glucose, uric acid, cholesterol and triglyceride were significantly higher (p<0.001) and HDL-cholesterol was significantly lower (p<0.05) in postmenopausals. No significant differences were observed with respect to insulin and HOMA. When age-matched pre- and postmenopausal women were compared, only total cholesterol was significantly higher in the postmenopausal group. However, older postmenopausal women (>50 years) had significantly higher systolic blood pressure, waist circumference, WHR, glucose and uric acid concentrations compared with younger(≤50 years) postmenopausals. It is concluded that an increase in abdominal fat accumulation and unfavorable alterations in risk factors disturb postmenopausal obese women even if total body weight and BMI do not change during menopause transition. Ageing, particularly throughout the postmenopausal years, has important effects on the detrimental changes associated with menopause.

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Our recent study of the gene expression profile in thyroid carcinoma showed an overexpression of osteonectin mRNA, an extracellular matrix protein, in an anaplastic carcinoma. To confirm this, we measured the expression levels of osteonectin mRNA in 84 thyroid normal and tumor tissues, including five anaplastic carcinomas by realtime quantitative reverse-transcription PCR. Increased expression of osteonectin mRNA was observed in anaplastic carcinoma tissue. However, in five anaplastic carcinoma cell lines, no increase was observed in the expression levels of osteonectin mRNA. These findings suggest the possibility that increased expression of osteonectin mRNA in anaplastic carcinoma tissue may be due to its overexpression in stromal cells, but not in anaplastic carcinoma cells.

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We report a case in which endoscopic ultrasonography (EUS), intraductal ultrasonography (IDUS) and contrast-enhanced EUS using Levovist helped to localize insulinoma correctly. A 74-year-old woman complained of symptomatic fasting hypoglycemia with relatively high concentration of serum insulin level. Dynamic contrast-enhanced computed tomography revealed a small tumor of 8 mm diameter in the pancreatic head. Insulin secretion was strongly stimulated by calcium injection into the gastroduodenal artery. To clarify the precise localization, we performed EUS, IDUS and contrast-enhanced EUS. The tumor was enhanced clearly by Levovist, and the distance from the main pancreatic duct was more than 3 mm. Therefore, a preoperative decision could be made to use the enucleation method for resection of the tumor. The surgeon could enucleate the tumor in a brief operation according to the preoperative diagnosis, and serum glucose levels returned to normal range after the operation. Contrast-enhanced EUS using Levovist was shown to be a useful diagnostic method for precise localization of small insulinoma.

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A 46-year-old Japanese male was admitted for the evaluation of severe hypertension. He was obese and had a eunuchoidal body habitus. Chromosomal analysis revealed a 46, XY/47, XXY karyotype. Serum LH, FSH and testosterone levels were low, indicating hypogonadotropic hypogonadism. Endocrinological dynamic tests disclosed presence of hypothalamic panhypopituitarism, partial diabetes insipidus, type 2 diabetes mellitus and low renin essential hypertension. Brain computed tomography and magnetic resonance imaging revealed intra- and extrasellar masses. Histological examination of the tissue obtained at transsphenoidal surgery showed a Rathke’s cleft cyst (RCC). To the best of our knowledge, this is the first case report of mosaic Klinefelter’s syndrome accompanied by symptomatic RCC, type 2 diabetes mellitus and low renin essential hypertension.

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