Graves' disease is a common organ-specific autoimmune disease characterized by overstimulation of the thyroid gland with agonistic anti-thyrotropin (TSH) receptor autoantibodies, which leads to hyperthyroidism and diffuse hyperplasia of the thyroid gland. Several groups including us have recently established several animal models of Graves' hyperthyroidism using novel immunization approaches, such as in vivo expression of the TSH receptor by injecting syngeneic living cells coexpressing the TSH receptor, the major histocompatibility complex (MHC) class II antigen and a costimulatory molecule, or genetic immunization using plasmid or adenovirus vectors coding the TSH receptor. This breakthrough has made it possible for us to study the pathogenesis of Graves' disease in more detail and has provided important insights into our understanding of disease pathogenesis. The important new findings that have emerged include: (i) the shed A subunit being the major autoantigen for TSAb, (ii) the significant role played by dendritic cells (DCs) as professional antigen-presenting cells in initiating disease development, (iii) contribution of MHC and particularly non-MHC genetic backgrounds in disease susceptibility, and (iv) influence of some particular infectious pathogens on disease development. However, the data regarding Th1/Th2 balance of TSH receptor-specific immune response or the association of Graves' hyperthyroidism with intrathyroidal lymphocytic infiltration are rather inconsistent. Future studies with these models will hopefully lead to better understanding of disease pathogenesis and help develop novel strategies for treatment and ultimately prevention of Graves' disease in humans.

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Mastoparan, a tetradecapeptide purified from wasp venom, has been shown to stimulate glucose transport in rat adipocytes (Suzuki et al. Biochem Biophys Res Comm 189: 572–580, 1992) although the mechanism of its action has remained undefined. Here, we characterized the action of mastoparan on glucose transport in rat adipocytes. Mastoparan at a concentration of 20 μM or more caused a dose-dependent release of lactate dehydrogenase (LDH) from the cells, which closely correlated with its stimulatory effect on glucose uptake. The mastoparan-induced glucose uptake was inhibited neither by deprivation of ATP with KCN nor by addition of phloretin, a direct inhibitor of glucose transporter, suggesting that the ability of mastoparan to stimulate glucose uptake did not derive from activation of the glucose transport system (i.e. translocation or activation of GLUT4 and/or GLUT1). On the other hand, mastoparan at a lower concentration (15 μM or below), which showed an insignificant effect on LDH release, potentiated the insulin action on glucose transport and Akt phosphorylation in the presence of adenosine deaminase. The effect of mastoparan was not additive to that of phenylisopropyladenosine and was completely abolished by pretreatment of adipocytes with pertussis toxin (1 μg/ml for 2 hours). Thus, the present study disclosed duality in the action of mastoparan on glucose uptake in rat adipocytes. At a concentration of 15 μM or less, it enhances the insulin action on glucose transport by a pertussis toxin-sensitive Gi protein-dependent mechanism. At higher concentrations, however, mastoparan increases non-specific permeability of the plasma membrane, which causes LDH release as well as glucose uptake not mediated through glucose transporter.

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The purpose of this study was to determine whether the changes of sex hormone-binding globulin (SHBG) affect the pregnancy outcome in women undergoing controlled ovarian hyperstimulation (COH) for assisted reproduction. Forty-five infertile women who were undergoing pituitary desensitization and COH for in vitro fertilization (IVF) with or without intracytoplasmic sperm injection (ICSI) and 19 women with normal menstrual cycles participated in the study. Fasting blood samples of the follicular and luteal phases, including follicular fluid during oocyte retrieval, were obtained for determination of estradiol (E₂), progesterone (P₄), testosterone (T), and SHBG concentrations. The SHBG levels increased progressively during the course of COH, but remained constant throughout normal menstrual cycles. A positive correlation existed between E₂ and SHBG levels in both the follicular and luteal phases. The mean plasma SHBG concentration and E₂/T ratio were significantly higher, while the level of T and the free androgen index were significantly lower, in the luteal phase of women who conceived than in those who did not conceive following COH. The changes of follicular fluid SHBG level and E₂/T ratio were similar to those in plasma. We concluded, therefore, that increases in SHBG in the follicular and luteal phases may be a reflection of the functional state of ovarian stimulation, and further that such elevations may influence the pregnancy outcome through the modulation of circulating estrogen and androgen balance during down-regulated COH cycles for IVF/ICSI.

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We report a rare case of type 1 diabetes in a woman associated with acromegaly who was treated with surgery after pregnancy. An 18-year-old woman came to our hospital in April, 1998, complaining of thirst, polydipsia, polyuria, appetite loss, body weight loss of 8 kg in a month, and amenorrhea beginning 2 months earlier. Based on laboratory data, she was diagnosed as having type 1 diabetes mellitus. Although we suspected her of having acromegaly because of high growth hormone (GH) levels (6.9 or 8.5 ng/ml), blood levels of insulin-like growth factor 1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) were within normal range and the circadian rhythm of her blood GH levels was normally maintained. Her blood GH level was elevated to 12.6 ng/ml 15 minutes after a TRH administration. Blood GH levels were suppressed from 49 ng/ml to 1.5 ng/ml 4 hours after an oral administration of 2.5 mg of bromocriptine. A magnetic resonance images (MRIs) showed pituitary swelling, but no nodules were found in the pituitary. Therefore, we diagnosed her as having acromegaly and observed her without surgery, while prescribing diet therapy and intensive insulin therapy for diabetes. We started a treatment of oral administration of 7.5 mg of bromocriptine per day for the acromegaly from April 28, 2000, because her elevated GH was suspected of causing her diabetes to be poorly controlled. During a pregnancy from October, 2000 to September, 2001, diabetic control was improved with increased administration of insulin under a constant dose of bromocriptine. She delivered a normal full-term infant. After the bromocriptine therapy was stopped as she hoped to breastfeed, blood levels of GH and IGF-1 became elevated and her diabetic control deteriorated. As her pituitary tumor observed in pituitary MRIs became larger during the course, a transsphenoidal surgery was performed on March 8, 2002. After the surgery, blood levels of GH and IGF-1 lowered and diabetic control improved again. We concluded as follows: to rule out acromegaly in patients with poorly controlled diabetes, 1) measurements of serum GH and IGF-1 should be performed, and 2) pituitary MRIs should be performed if blood levels of GH or IGF-1 are high.

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The aim of this prospective cross-sectional study was to investigate the hypertrophic effects of endogenous subclinical hyperthyroidism on myocardium and early development of left ventricular hypertrophy (LVH) in essential hypertensive patients accompanied by endogenous subclinical hyperthyroidism. A total of 31 consecutive patients with stage I hypertension were included in the study. Sixteen of them also had endogenous subclinical hyperthyroidism that they were unaware before. The patients and the controls formed out of ten healthy subjects all underwent an investigation of thyroid functions and cardiologic evaluation. The mean wall thickness of the left ventricle in the stage I hypertensive group with endogenous subclinical hyperthyroidism (group I) was significantly increased as compared with both hypertensive patients without thyroid disease (group II) and the control subjects. The mean left ventricle mass was also significantly higher in group I than group II. Both of the patients' groups had an increased prevalence of LVH as compared with the controls. In this study, hypertensive patients with subclinical hyperthyroidism presented more increase in left ventricular mass, suggesting that subclinical hyperthyroidism may contribute to left ventricular hypertrophy forming a natural progression to hypertension. The hypertensive population should always be screened for endogenous subclinical hyperthyroidism, and should be examined for the criteria of left ventricular hypertrophy by echocardiography in early stages.

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Decreased circulating levels of adiponectin, a novel adipose-derived adipocytokine, in obesity possibly contribute to the development of insulin resistance which is a major factor in the pathogenesis of type 2 diabetes. The present study was conducted to examine whether circulating and adipose tissue adiponectin levels are modulated by chronic treatment with metformin and intensive treatment with insulin in murine models of obesity and type 2 diabetes, db/db mice with a C57BL/KsJ genetic background. Nine-week-old male db/db mice were treated with metformin, insulin, and vehicle for 4 weeks. Expectedly, metformin treatment led to inhibition of weight gain and improvement of hyperinsulinemia. Insulin treatment lowered fasting blood glucose levels to normal values, although it sustained hyperinsulinemic state. However, after 4 weeks of treatment, serum adiponectin levels were not significantly elevated in either metformin-treated or insulin-treated db/db mouse group (14.2 ± 0.7 and 16.7 ± 1.0 μg/ml, respectively) compared to vehicle-treated group (14.9 ± 0.6 μg/ml). Similarly, adipose tissue adiponectin levels determined by Western blot analysis were not increased in either metformin-treated or insulin-treated group relative to vehicle-treated group. Recent studies have shown that adiponectin possibly has the same physiological effects on lipid and glucose metabolism that metformin has. Therefore, an elevation in blood concentration of metformin following the treatment might lead to suppression in adiponectin synthesis in adipose tissue, independent of inhibition in weight gain and improvement in hyperinsulinemia by metformin treatment. The present results indicate that adiponectin is not involved in the mechanism by which metformin treatment enhances insulin sensitivity. Moreover, our results suggest that adiponectin synthesis in adipose tissue may be suppressed under hyperinsulinemic state sustained by insulin treatment, even though hyperglycemia is markedly reduced. We conclude that antidiabetic treatment with metformin and insulin does not affect circulating and adipose tissue adiponectin levels.

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A 46-year-old male with long-term treatment-resistant hypertension and past history of cerebral hemorrhage was found to have suppressed plasma renin activity (PRA) and normal plasma aldosterone concentration (PAC) with aldosterone/renin ratio of 25.3. Furosemide plus upright test did not stimulate PRA, but computed tomography scan of the abdomen revealed no abnormal lesions in either adrenal gland. Selective adrenal venous sampling (SAVS) showed that PAC in the left and the right adrenal vein were 1000 ng/dl and 230 ng/dl, respectively, which increased to 1500 ng/dl and 620 ng/dl, respectively, after ACTH stimulation. Diagnosis of primary aldosteronism due to hypersecretion of aldosterone from the left adrenal gland was made, and laparoscopic left adrenalectomy was performed. Pathological examination of the ‘apparently normal’ adrenal tissue resected revealed the presence of poorly encapsulated multiple adrenocortical micronodules which showed positive immunoreactivity for 3β-hydroxysteroid dehydrogenase by immunohistochemical study, but negative immunoreactivity in the hyperplastic zona glomerulosa consistent with paradoxical hyperplasia associated with primary aldosteronism. Postoperatively, PRA was normalized and his high blood pressure was well controlled with lower doses of antihypertensive drugs than those used before surgery. The clinicopathological features of our case are consistent with the diagnosis of unilateral multiple adrenocortical micronodules (UMN), a new subset of primary aldosteronism, in which SAVS proved to be a useful diagnostic tool for its localization.

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To explore the relationship between pituitary morphology and function, we performed mid-sagittal MRI and endocrinological evaluation in 38 patients with asthenia. Six patients were diagnosed as having complete empty sella (ES) and 16 patients partial empty sella (PES). BMI, blood pressure, serum Na, ACTH, cortisol, TSH and T₄ were lower in ES group and PES group than in the group with normal pituitary size. Age in the patients with ES was oldest. Multiple regression analysis revealed that serum cortisol level was independently correlated with the size of the pituitary (β = 0.586, p = 0.0069). Other variables, including age, BMI, blood pressure, serum Na, ACTH, TSH and T₄, were not correlated with the pituitary size when multivariate analysis was employed. In conclusion, there is a close relationship between the reduction of size of pituitary gland and the degree of adrenocortical dysfunction in asthenic patients. It is suggested that the pituitary-adrenal axis is especially vulnerable in empty sella syndrome, and therefore, meticulous evaluation of the hypophysial adrenal axis is recommended in subjects with reduced pituitary size even in elderly population.

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Steroidogenic factor-1 (SF-1) regulates the transcription of multiple genes involved in the androgen biosynthesis, and SF-1 Gly146Ala polymorphism is known to reduce the transactivation function by ~20%. To examine whether the Gly146Ala polymorphism constitutes a susceptibility factor for the development of micropenis (MP), we analyzed this polymorphism in a total of 52 patients with micropenis (T-MP) consisting of 30 patients with severe MP below –2.5 SD (S-MP) and 22 patients with mild MP from –2.1 SD to –2.5 SD (M-MP), together with 115 control males. The Ala allele, the Ala/Gly genotype, and the Ala/Ala plus Ala/Gly genotype frequencies were significantly higher in the S-MP patients than in the control males, whereas the allele and the genotype frequencies were comparable between the M-MP patients and the control males. The results suggest that the SF-1 Gly146Ala polymorphism may constitute a susceptibility factor for the development of S-MP, and that M-MP can be regarded as a normal variation in terms of the polymorphism effect.

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The aim of this study was to investigate whether administration of exogenous estrogen affects the changes of leptin and GnRH levels in women with normal menstrual cycle. A total of 18 women received a bolus intravenous injection of 20 mg conjugated estrogen (premarin group) at 0800 during the fifth day of menstrual cycle, while another 18 women were administered 20 mL of normal saline as the control group. Fasting blood samples were collected at 0, 4, 8, 24, 28, 32, 48, 56, 72 and 96 hours after injection for analyses of leptin, GnRH, estrone (E₁), estradiol (E₂), LH and FSH. Both the mean plasma levels of E₁ and E₂ were significantly increased from 4 hours and significantly sustained elevated levels up to 72 hours after injection of premarin. Simultaneous significant increases of leptin and GnRH levels were observed at 28, 32 and 48 hours after injection, while the controls remained constant. The mean LH and FSH levels were initially suppressed and then significantly increased at 56 and 72 hours after premarin administration. Leptin appears to be involved in the regulation of positive feedback mechanism of estrogen by conveyance of metabolic signal to affect the release of GnRH in hypothalamus, while its participation in the modulation of negative feedback remains unknown.

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This study was designed to clarify the long-term efficacy and safety of percutaneous ethanol injection (PEI) therapy in benign nodular and cystic thyroid diseases, and to evaluate response by criteria defined as disappearance of hot nodule. Solid nodule and complex cyst were classified into three groups in accordance with volume reduction. In autonomously functioning thyroid nodule (AFTN), disappearance of hot nodule with normalization of thyroid hormone level and restored extra-nodular uptake was defined to be curative. In solid nodule (n = 198) and complex cyst (n = 432), initial volume was significantly reduced to post-PEI and final volumes, and volume reduction persisted during follow-up period. Complete response, partial response and no response were as follows: 17.2%, 71.7%, 11.1% in solid nodule; 19.0%, 60.4%, 20.6% in complex cyst, respectively. Differences of volume reduction according to initial volume (≥10 mL vs. <10 mL) were significant. Correlations between initial and final volumes, and between initial volume and volume reduction were also significant. In 24 patients with AFTN, when effectiveness was assessed by disappearance of hot nodule, only 1 case was curative. Reexpansion or recurrence was observed in 5 cases. Complications developed in 9.0% but there was no permanent or serious complication in this study. In conclusion, our data suggest that PEI therapy could be an effective and safe therapeutic modality for benign nodular and cystic thyroid diseases especially when initial volume is more than 10 mL, but may not induce disappearance of hot nodule itself in AFTN.

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According to the diagnostic criteria for adrenal preclinical Cushing's syndrome (PreCS) established by a group headed by the Ministry of Health, Labor and Welfare (MHLW), low- and high-dose dexamethasone suppression tests (DSTs) must be performed to prove autonomous cortisol secretion, i.e., ≥3 μg/dL serum cortisol following 1-mg DST administration, and ≥1 μg/dL serum cortisol following 8-mg DST administration. However, discrepancies have been documented in the results of low-and high-dose DSTs. We therefore investigated the validity of the DST for diagnosing PreCS by performing 1-mg and 8-mg DSTs in 39 patients with adrenal incidentaloma, but no characteristic Cushingoid symptoms. In about half of these patients (20/39, 51.3%), high-dose DST was positive but low-dose was negative, and one or more of the other abnormalities of hypothalamus-pituitary-adrenal axis dysfunction was seen in 75% of these patients. Furthermore, no significant difference in incidence of glucose intolerance and hypertension was noted in patients with positive high-dose DST and negative low-dose DST compared with patients with positive low- and high-dose DST. Under the current MHLW diagnostic criteria, patients with positive high-dose DST and negative low-dose DST are not diagnosed with PreCS, but some of these patients should be. Discrepancies in the results of low- and high-dose DSTs appear attributable to the current cutoff values, and further investigations are necessary to resolve these discrepancies.

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Graves' disease (GD) is an autoimmune disorder with genetic predisposition. CD40, which stimulates lymphocyte proliferation and differentiation, is an important immunomodulator and is expressed in the thyroid follicular cells as well as antigen-presenting cells. A single nucleotide polymorphism (SNP) at position –1 of the Kozak sequence of the CD40 gene has been reported to be associated with the development of GD. The aim of the present study was to investigate whether CD40 gene polymorphism confers susceptibility to GD in Japanese. CD40 gene polymorphisms were studied in Japanese GD patients (n = 324) and healthy control subjects without anti-thyroid autoantibodies or a family history of autoimmune disorders (n = 229). A C/T polymorphism at position –1 of the CD40 gene was measured using the polymerase chain reaction restriction fragment length polymorphism. There was no significant difference in allele or genotype frequency of the CD40 SNP between GD and control subjects. There was a significant decrease in the TT genotype frequency in the GD patients, who developed GD after 40 years old, than those under 40 year of age. These data suggest that the SNP of CD40 gene is associated with susceptibility to later onset of GD in Japanese.

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The treatment policy for patients with well-differentiated thyroid cancer varies among institutions. Although surgery has been the mainstay of treatment for this cancer, there is no consensus concerning the optimal extent of thyroid resection or the extent of lymph node dissection. Furthermore, controversy remains with regard to the indications for radiation therapy and hormonal therapy in surgical or non-surgical cases and the treatment modalities for cancer recurrence. To determine the actual status of treatment policies for thyroid cancer in Japan, a questionnaire was distributed to all physicians who participated in the 37th meeting of the Japanese Society of Thyroid Surgery, to inquire into treatment options for well-differentiated thyroid cancer in relation to clinical stages in actual situations. The replies to this questionnaire were analyzed in this study.

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After antithyroid drug (ATD) treatment for Graves' disease, either a relapse of Graves' thyrotoxicosis or painless thyroiditis can develop. It is important to differentiate these two types of thyrotoxicosis because of the difference in required therapy. However, differentiation of thyrotoxicosis is usually difficult without radioactive iodine uptake (RAIU) which is not available in general practice. We investigated the clinical usefulness of the 2nd generation assay for anti-TSH receptor antibodies (TRAb) to differentiate these two types of thyrotoxicosis after ATD treatment for Graves' disease. We recruited 26 patients who developed thyrotoxicosis after ATD treatment for Graves' disease. These patients once became negative for TRAb and seemed to be in remission after ATD treatment. Upon development of thyrotoxicosis after ATD treatment, TSH, free T4, free T3 and TRAb were measured. TRAb were measured by the 2nd generation assay using recombinant human TSH receptors instead of porcine TSH receptors. Fourteen patients relapsed into Graves' thyrotoxicosis and 12 patients developed painless thyroiditis. Twelve (85.7%) of 14 patients with relapse of Graves' thyrotoxicosis were positive for TRAb. Eleven (91.7%) of 12 patients with development of painless thyroiditis after ATD treatment for Graves' disease were negative for TRAb. Levels of TRAb were significantly different between patients with relapse of Graves' thyrotoxicosis (4.86 ± 6.45 IU/L) and those with painless thyroiditis (0.62 ± 0.61 IU/L) (P<0.001). The 2nd generation assay for TRAb was useful to differentiate relapse of Graves' thyrotoxicosis from development of painless thyroiditis in patients who seemed to be in remission after ATD treatment for Graves' disease.

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Defects in insulin receptor function have been associated with insulin resistant states such as obesity and type 2 diabetes mellitus. Several types of mutations in the insulin receptor gene have been identified in patients with genetic syndromes of extreme insulin resistance. We have studied a 10-year-old Japanese girl with type A insulin resistance with hirsutism and hyperinsulinemia but without the dysmorphic features characteristic of leprechaunism or Rabson-Mendenhall syndrome. Despite the presence of severe insulin resistance, the patient did not develop overt diabetes mellitus at the time of investigation. Using direct sequencing, we identified a nonsense mutation causing premature termination after amino acid 345 in the α subunit of the insulin receptor.

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