Using thyroid follicles in suspension culture, in which thyroid function is maintained for a couple of weeks, while preserving Wolff-Chaikoff effect and responding to physiological concentrations of TSH (0.1μU/ml), we have partly elucidated the pathophysiology of increased blood flow in Graves' thyroid gland. Furthermore, we are investigating the mechanism by which iodide inhibits thyroid blood flow.
Since administration of a large dose of iodide prior to subtotal thyroidectomy decreases thyroid blood flow and reduces operative morbidity and mortality in patients with Graves' disease, it is important to elucidate the mechanism by which thyroid blood flow is regulated. Furthermore, this research field will be clinically important to develop new strategies for the treatment of hypervascular and aggressive thyroid carcinoma, particularly anaplastic thyroid carcinoma. A recent report that anti-VEGF antibody reduced cancer growth in nude mice transplanted with human thyroid carcinoma is an indication this field holds for future studies [56].

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Inhibition of insulin release by norepinephrine has been attributed to activation of ATP-sensitive K⁺ channels, inactivation of voltage-dependent Ca²⁺ channels, and inhibition of adenylyl cyclase. However, direct inhibitory action of norepinephrine at a distal site of stimulus-secretion coupling has also been suggested. To obtain more direct evidence for norepinephrine inhibition of insulin release at a distal site, we performed experiments in intact, non-permeabilized β cells. In rat pancreatic islets, a combination of glucose, phorbol ester and forskolin under stringent Ca²⁺-free conditions was used as a trigger of insulin exocytosis at a distal site. Norepinephrine inhibited this Ca²⁺-independent insulin release in a concentration-dependent manner, with an IC₅₀ of 50nM. The inhibition was complete, reversible, and pertussis toxin-sensitive, and not associated with any reduction of cAMP content in the islet cells. In conclusion, norepinephrine strongly, yet reversibly, inhibits insulin release in intact β cells at a late step of exocytosis, through pertussis toxin-sensitive, G protein-mediated mechanism(s).

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We investigated the long-term estrogenic influence of genistein on the male reproductive system in mice. Newborn ICR male mice were treated with genistein (10, 100, or 1000μg/mouse) for neonatal 5 days. As positive control, administration of diethylstilbestrol (0.5-50μg/mouse) was carried out. In mice exposed to genistein, we examined weight of testes, sperm counts, sperm motility, and mRNA expression levels of estrogen receptor α (ERα) and androgen receptor (AR) at 4, 8 or 12 weeks after birth. Moreover, at 12 weeks of age, we evaluated protein level of ERα. In our conventional reproductive-toxicological study (weight of testes, sperm counts and sperm motility), neonatal transient exposure to genistein did not show adverse effects on the male reproductive system in 4, 8 or 12 week old mice. However, in mice treated with genistein mRNA expression levels of ERα and AR were reduced at 8 weeks. This reduction was recovered at 12 weeks in mice treated with a lower dose (10μg) of genistein but not in those with higher doses (100μg and 1000μg). In addition, ERα protein levels tended to decrease in 12 weeks of adulthood. Our results exhibited that the disruption of gene expression continued for long term such as 3 months after administration of genistein, even if no effect was found at conventional reproductive-toxicological level. We have shown that neonatal administration of weak estrogenic compound (genistein) affects male reproductive organs at molecular levels in adulthood.

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Standard therapy for ACTH-independent bilateral macronodular adrenocortical hyperplasia (AIMAH), a rare form of Cushing's syndrome, is bilateral adrenalectomy. Patients with AIMAH are usually elderly, with a variety of complications, and at risk for surgery. Postoperatively, they must receive lifelong corticosteroids and spend the remainder of their lives avoiding adrenal crisis. Therapy using metyrapone, a potent inhibitor of steroidogenesis, provides the advantages of avoiding the surgery. Its effectiveness is further anticipated because adrenal steroidogenic enzymes are reportedly weak in AIMAH. Treatment with metyrapone thus appears a good therapy for AIMAH, but its effectiveness has not, to our knowledge, been studied. We treated a 59-year-old man with AIMAH with metyrapone. At a low dose of metyrapone (500 to 750mg/day), his plasma cortisol levels decreased to the normal range, and hypertension and diabetes mellitus were ameliorated. Therapy using metyrapone thus appears effective in treating AIMAH, and can be recommended for high risk AIMAH patients as an alternative therapy.

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Since oxidative stress is related to autoimmune thyroid disease, we studied the production of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and cytochrome c by culture of mononuclear cells from patients with Graves' disease and Hashimoto's thyroiditis. In patients with untreated Graves' disease, 8-OHdG and cytochrome c levels in culture supernatant of mononuclear cells were significantly higher than those of healthy control subjects, while the Cytochrome c levels were significantly higher in patients with untreated Graves' disease and Hashimoto's thyroiditis than those of control subjects. Significant correlations between 8-OHdG and FT4i and cytochrome c were found. These results indicated that thyroid function has a potent influence on oxidative stress.

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A 49-year-old man was referred to our hospital for the treatment of gallstones in 1993. Bilateral adrenal nodular masses were detected incidentally by abdominal computed tomography. He had no clinical signs of Cushing's syndrome such as central obesity, striae of skin and diabetes mellitus. We performed cholecystectomy and partial adrenalectomy of right adrenal gland as a biopsy, and diagnosed him as preclinical Cushing's syndrome due to adrenocorticotropin-independent bilateral adrenal macronodular hyperplasia (AIMAH) based on endocrinological and histological examinations. We followed him up for 7 years. During the observation period, the sizes of both adrenal glands increased gradually, and finally serum cortisol level increased beyond normal range, and he showed a Cushingoid appearance such as moon face and central obesity. His skin became atrophic and very fragile, and the bone mineral density of his lumbar spine was extremely low. Serum cortisol level was elevated, and plasma ACTHlevel was always suppressed. Urinary excretion of 17-hydroxycorticosteroid and free cortisol were increased. Diurnal rhythm of cortisol and ACTH was completely lost and high dose (8mg/day) dexamethasone did not suppress urinary 17-hydroxycorticosteroid excretion. He became clinically overt Cushing's syndrome. We recommended total adrenalectomy, but he refused it. It is important to know the natural history of preclinical Cushing's syndrome due to AIMAH when choosing an adequate treatment.

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We investigated the relationships among serum testosterone levels, body fat and muscle mass distribution in women with polycystic ovary syndrome (PCOS). Subjects were 67 women with PCOS(mean age±standard deviation, 28.8±6.6 years). Baseline characteristics included age and height. Trunk-leg fat ratio and trunk-leg muscle ratio were assessed with dual-energy x-ray absorptiometry. Serum testosterone and dehydroepiandrosterone sulfate levels were measured with radioimmunoassays. Relationships among serum testosterone levels, body fat and muscle mass distribution were investigated using Pearson and partial correlation tests. Serum testosterone levels were positively correlated with trunk-leg fat ratio (r=0.398, P<0.01), but were inversely correlated with trunk-leg muscle ratio (r=-0.332, P<0.05). Trunk-leg muscle ratio was inversely correlated with trunk-leg fat ratio (r=-0.360, P<0.01). Serum testosterone levels were still correlated with trunk-leg fat ratio (r=0.500, P<0.001) and trunk-leg muscle ratio (r=-0.286, P<0.05), after adjusting for age and height. Trunk-leg fat ratio was still correlated with trunk-leg muscle ratio, after adjusting for age, height, and serum testosterone levels. Based on these results, we concluded that higher serum testosterone levels may contribute to the upper body fat distribution and peripheral muscle mass distribution. In addition, peripheral muscle mass distribution may also contribute to the upper body fat distribution.

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Spontaneous massive retroperitoneal hemorrhage from an adrenal gland is a rare event. A thoughtful and meticulous approach to such a patient, with appropriate diagnostic studies, ICU and surgical care are essential for patient survival. In patients with active bleeding, angiographic embolization is a valuable adjunct to achieve hemostasis, to allow for further work-up of the adrenal tumor, and an improved subsequent oncologic resection. Hemodynamically unstable patients, however, may require supportive transfusions in the intensive care unit, potential embolization if deemed feasible, or urgent surgical exploration. If possible, however, the acute surgical removal of an adrenal tumor within a large retroperitoneal hematoma should be avoided, as under such conditions a proper oncologic resection may not be possible. The possibility of a pheochromocytoma must always be entertained. Early recognition and treatment of patients with presumed adrenal insufficiency may decrease patient morbidity and mortality.

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Initial investigations of a 70-year-old woman with clinical Cushing's syndrome, including overnight dexamethasone suppression test, CRH test, and pituitary MRI, suggested the presence of ectopic ACTH production. Thoracic computed tomography (CT) scan revealed a mass measuring 7mm in the right lung, but it was thought to be an incidental opacity, leaving the source of ectopic ACTH undetermined for several years. During this period, although the size of the lung opacity did not change remarkably, serum cortisol levels became elevated to 43μg/dl, and the patient's symptoms worsened. T1-201 SPECT demonstrated intense accumulation in the right lung. The mass was surgically resected using thoracoscopy to investigate it as the focus of ACTH production. Histological and immunohistochemical examination confirmed that the area of intense T1-201 uptake was an ACTH-producing bronchial carcinoid. Plasma ACTH and cortisol levels decreased immediately after the surgery. In conclusion, this case demonstrated T1-201 scintigraphy as a useful tool in identifying the location of an ACTH-producing bronchial carcinoid.

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We previously developed a radioreceptor assay which is presumably specific for detection of blocking-type anti-TSH receptor (TSHR) antibodies using unsolubilized porcine TSHR. Employing this assay, we measured blocking TSH-binding inhibitory immunoglobulin (TBII) in the sera from 30 untreated Graves' patients and compared the results with those of a bioassay measuring thyroid stimulation blocking antibodies (TSBAb). Blocking TBII was positive in 9 of 30 sera (30%) and the blocking TBII activity was correlated with the total TBII value, which was measured by the radioreceptor assay using solubilized porcine TSHR. On the other hand, TSBAb determined with the conventional bioassay was positive in only 2 sera (6.7%), and no correlation was observed with the TSAb activity. In some cases in which the TSAb activity was rather high, TSBAb could not be detected by bioassay, whereas blocking TBII was positive. There was no correlation of blocking TBII activity with goiter size, thyroid hormone level, or proptosis. However, there was a tendency for anti-thyroid therapy to require a shorter time for FT₄ normalization in blocking TBII subjects, suggesting that blocking-type anti-TSHR antibody plays some role in the pathophysiology of Graves' disease. In conclusion, blocking type anti-TSHR antibodies are often found in the sera of Graves' patients when the blocking-specific radioreceptor assay is applied.

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An elevation in follicle-stimulating hormone (FSH) levels is considered to reflect lowered ovarian function, resulting in poor fecundity in infertile women. However, it remains to be clarified whether or not the significance of FSH levels applies equally to all women irrespective of age. The objective of the present study is to compare basal FSH levels in infertile women who conceived or not after stratification by age. A total of 144 infertile women between ages 25 and 45 who underwent infertility treatment due to unexplained infertility in the University of Tokyo Hospital were included in the retrospective study. Subjects were divided by age into two groups, <38 (n=98) vs_??_⊗38 (n=46) years, with ages ranging from 25 to 37, and from 38 to 45, respectively. Blood samples were collected in early follicular phase (day 4-6) for assessment of basal levels of LH, FSH, and PRL. In the older group, pregnant cases had significantly lower FSH levels (6.07±2.83mIU/ml) than nonpregnant cases (9.60±3.67mIU/ml), whereas no difference in basal FSH levels was observed between pregnant and nonpregnant cases in the younger group. Basal FSH levels of pregnant cases in the older group were significantly lower than those of pregnant cases in the younger group (8.26±2.95mIU/ml). Basal LH and PRL levels were not related to fecundity in either group. Thus, an increase in basal FSH levels as a predictor of fecundity should be considered in the context of age.

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We report three cases of patients exhibiting a false elevation of serum free triiodothyronine (FT3) as a result of a cross-reaction with diclofenac. The first case is a 66-yr-old woman with a long history of rheumatoid arthritis (RA). The patient was receiving diclofenac for the treatment of her RA. The patient was subsequently diagnosed as having thyroid papillary adenocarcinoma and received a subtotal thyroidectomy. After the operation, the patient exhibited postoperative hypothyroidism except for a gradual elevation of FT3. The other two patients also exhibited an elevated serum FT3 level after the administration of diclofenac. Serum FT3 levels in these patients decreased to normal or below normal after diclofenac administration was discontinued. In the first case, the elimination of immunoglobulin from the sera using polyethylene glycol precipitation did not reduce the FT3 level. In our hospital, Vitros ECi (enhanced chemiluminescence enzyme immunoassay) system and Vitros FT3 kit were used for FT3 assay. The patient's FT3 levels were normal or below normal when they were measured using other FT3 kits. FT3 was also detected when diclofenac was dissolved in a phosphate buffered saline. Therefore, we concluded that a cross-reaction between FT3 and diclofenac was the mechanism causing the false elevation of FT3 in these patients.

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Since glucocorticoid exerts its biological effects by binding to its receptor, the expression efficiency of the glucocorticoid receptor (GR) gene could influence glucocorticoid sensitivity. We found a polymorphism of cytosine/adenine (-22C/A) in the upstream region of the GR gene. There was no difference in the allelic frequency between normal and type 2 diabetic subjects. The promoter activity determined by luciferase assay was significantly lower in the -22A allele than in the -22C allele in both HepG2 (A allele, 4.19±0.15; C allele, 6.07±0.27, p<0.001) and human embryonic kidney 293 cell lines (A allele, 0.93±0.16; C allele, 1.51±0.32, p<0.001). This polymorphism is associated with transcription of the OR gene, which could be related to glucocorticoid sensitivity through an alteration in tissue OR number.

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