To elucidate the spatiotemporal profiles of final secretory stage, we have established two-photon extracellular polar tracer (TEP) imaging, with which we can quantify all exocytic events in the plane of focus within the intact tissues. With such technique, we can estimate the precise diameters of vesicles independently of the spatial resolution of optical microscope, and measure the fusion pore dynamics at nanometer resolution. At insulin exocytosis in the pancreatic islets, it took two seconds for the fusion pore to dilate from 1.4 nm in diameter to 6 nm in diameter, and such unusual stability of the pore may be due to the crystallization of the intragranular contents. Opening of the pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pores, supporting the idea that this structure was mainly composed of membrane lipids. TEP imaging has been also applied to other representative secretory glands, and has revealed hitherto unexpected diversity in spatial organizations of exocytosis and endocytosis, which are relevant for physiology and pathology of secretory tissues. In the pancreatic islet, compound exocytosis was characteristically inhibited (<5%), partly due to the rarity of SNAP25 redistribution into the exocytosed vesicle membrane. Such mechanisms necessitate transport of insulin granules to the cell surface for fusion, and possibly rendering exocytosis more sensitive to metabolic state. Two-photon imaging will be powerful tools to elucidate molecular and cellular mechanisms of exocytosis and related disease, and to develop new therapeutic agencies as well as diagnostic tools.

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The pathophysiological role of aldosterone in the development of cardiovascular disease has long been considered to be due its potent volume expansion/hypertensive effect mainly via mineralocorticoid receptor (MR) expressed in renal tubular epithelial cells. However, recent accumulating lines of evidence from clinical and experimental studies have suggested that direct cardiovascular effect of aldosterone contributes to the development of cardiovascular injury via MRs in non-epithelial tissue. A series of recent clinical studies have revealed that patients with primary aldosteronism have higher incidence of cardiovascular and renal complications than those with essential hypertension, and that aldosterone antagonism has cardiovascular protective effect in patients with heart failure independent from blood pressure. Numerous experimental studies have shown that both inflammation and oxidative stress play an initial and key role in the development of aldosterone-induced cardiovascular injury via non-epithelial MR activation. In this review, we discuss recent research progress in aldosterone and MR effects, with special emphasis on the pathophysiological role of aldosterone in cardiovascular diseases and the possible molecular mechanism(s) of cardiovascular injury by non-epithelial MR activation.

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TSH-secreting adenoma is a rare pituitary adenoma, and the expression levels of the specific subtypes of somatostatin receptors (sstr) mRNAs have remained obscure. To determine the quantitative expression of the sstr1-5 mRNAs in TSH-secreting adenomas that may be related to the efficacy of treatment with a somatostatin analogue, expression of the sstr1-5 mRNAs was examined and compared in TSH-secreting adenomas and other pituitary adenomas. The pituitary adenomas were obtained at transsphenoidal surgery from 4 cases of TSH-secreting adenoma, including 1 patient showing a significant shrinkage of the tumor size after only 10 days of octreotide treatment, 2 patients without tumor size reduction and 1 patient without treatment, and 5 GH-secreting adenomas, 6 prolactinomas, 5 nonfunctioning adenomas, 4 ACTH-secreting adenomas and normal pituitaries at autopsy from 4 normal subjects. In comparison to the normal pituitary, sstr2A>sstr1>sstr5>sstr3 mRNAs were expressed in the TSH-secreting adenomas examined. No expression of sstr2B or sstr4 mRNA was observed. The expression level of sstr2 mRNA was significantly higher than those in normal pituitary, prolactinomas, ACTH-secreting and nonfunctioning pituitary adenomas. The patient with marked shrinkage of the tumor showed the highest expression of both sstr2 and sstr5 mRNAs among all the cases of pituitary adenoma. A TSH-secreting tumor without shrinkage showed a similar expression level of sstr2 mRNA. These findings demonstrated that TSH-secreting adenomas express sstr1, 2A, 3 and 5 mRNAs, predominantly sstr2A, and in addition to the expression of sstr2 mRNA, the expression level of sstr5 mRNA may be a factor affecting the tumor shrinkage by somatostatin analogues against TSH-secreting adenomas.

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Psychosomatic symptoms in primary hyperparathyroidism (PHPT) are various and include such conditions as obsessive-compulsive disorder, depression, anxiety, and paranoia. In the elderly the clinical features of the disease are often non-specific and difficult to diagnose. To quantify subjective symptoms of patients with hyperparathyroidism in the elderly, we determined whether these clinical manifestations resolved after surgical parathyroidectomy (PTX) in three PHPT patients over eighty years old. They were diagnosed with hypercalcemia, hypophosphatemia, high PTH concentrations, and osteoporosis. A single parathyroid adenoma was confirmed in each patient by Tc-MIBI scintigram, neck ultrasonography and computed tomographic scanning. PTX was performed in these three patients. Assessments of psychologic symptoms, using the Hamilton Rating Scale for Depression (HAM-D), serum calcium, and intact PTH were obtained before and after PTX. Mean weight of the resected adenomas was 438 ± 138 mg (mean ± SD). After PTX, serum calcium decreased from 11.1 ± 0.5 to 9.2 ± 0.5 mg/dl and intact PTH from 160.0 ± 25.2 to 45.3 ± 22.2 pg/ml. Total HAM-D scores in each patient decreased from 45 to 9, 17 to 1 and 15 to 5, respectively. Especially, there were marked improvements in depressive mood, psychomotor inhibition, anxiety and somatic symptoms after PTX. The quality of life in those patients was also improved by PTX. We propose here that PTX in elderly PHPT patients with psychiatric symptoms should be considered instead of oral administration, such as anti-depressants or bisphosphonates.

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Controversies concerning the role of frozen section (FS) have been a matter of debate. The aim of this study was to identify the role of FS analysis in intraoperative decision making and analyze the effect of the cost in detecting thyroid malignancies in Turkey. Out of 214 consecutive patients who had been operated on for thyroid cancer between January 1996 and August 2004, 178 patients were evaluated retrospectively. All 178 patients were subjected to FS. Intraoperative FS correctly identified the pathology as malignant in 58.4% of patients. A true-positive FS result changed the surgical strategy in 30 (27.6%) cases False negative FS lesions were defined histologically as papillary microcarcinoma in 54%, follicular variant of papillary cancer in 18% and follicular cancer in 8% of cases. The sensitivities of FNAB and intraoperative FS in thyroid cancer patients were 22.5% and 58.4%, respectively. False negative FS results increased the cost for each informative FS from €25 to €42.7. Despite limitations, results of this study reject the idea that the role of FS is becoming limited. We recommend routine frozen section in the operative assessment of thyroid nodules. Omitting FS may be suggested only in cases with a FNAB revealing malignancy.

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A 66-year-old woman had massive bilateral adrenal macronodular hyperplasia, found incidentally on an abdominal ultrasonogram. Her plasma ACTH and serum cortisol levels were normal, but they were not suppressed by low-dose dexamethasone. The patient did not exhibit any typical signs or symptoms of Cushing's disease. MRI showed no evidence of a tumor in the pituitary gland. A diagnosis of preclinical Cushing's disease was made, and she was treated with 11-hydroxylase inhibitor metyrapone. As the dose of metyrapone was increased, plasma ACTH levels gradually increased. After three years of treatment, she developed moon-face. Her plasma ACTH and serum cortisol concentrations were at their highest levels. A pituitary microadenoma was detected by MRI, whose source of ACTH was demonstrated by the definite step-up of central/peripheral ratio of ACTH obtained by cavernous sinus sampling. Overt Cushing's disease was diagnosed, and a pituitary tumor was removed by transsphenoidal surgery. In conclusion, the clinically and endocrinologically overt Cushing's disease characterized by macronodular adrenal hyperplasia was converted from a preclinical form. This case offers some insight into the clinical and biological features of preclinical Cushing's disease.

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The activating point mutation of the BRAF gene, BRAF^T1799A, is the most common and specific genetic alteration in adult papillary thyroid carcinoma (PTC) and a possible marker of malignant potential of PTC. We have applied the PCR-RFLP method using fine-needle aspiration biopsy samples not only to our clinical practice but also to the international medical assistance effort around the Semipalatinsk Nuclear Testing Site in Kazakhstan. Seventy-seven cases (100 nodules) from Japan and 131 cases (137 nodules) from Kazakhstan were examined. There were 14 Japanese and 76 Kazakhstani cases of cytological malignant tumors from the examined samples. We detected 12 (85.7% of PTC) and 19 (25% of PTC) cases with BRAF^T1799A among the Japanese and Kazakhstani cases, respectively. Of these cases, we found mutations in one cytologically "suspicious" case and even in two pathologically "benign" cases (after surgery in Kazakhstan). All of the BRAF mutation-positive cases, including those three, were confirmed as PTC by careful pathological examination, including immunohistochemical analysis. In summary, our PCR-RFLP method for BRAF^T1799A detection using FNAB samples is useful not only for preoperative diagnosis of PTC but also as a complementary diagnostic tool for accurate pathological diagnosis, even after surgery.

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Cyclic Cushing's disease is a rare clinical entity that is defined as a periodic excessive production of adrenocorticotropic hormone (ACTH) and cortisol. Only 42 cases with cyclic Cushing's disease have been reported in the literature. The diagnosis is very difficult because of the fluctuating secretion of ACTH and cortisol. We report a 78-year-old woman with a pituitary adenoma presenting with cyclic Cushing's disease. In the present case, several interesting issues are pointed out: 1) MRI study detected the presence of an adenoma and selective venous sampling in the cavernous sinus disclosed the hypersecretion of ACTH from a pituitary adenoma. These neuroimaging and endocrinological studies were helpful for the diagnosis, even in the remission phase. 2) The disease was in the long-term remission phase after transsphenoidal surgery despite the high recurrence rate in this clinical entity, although it recurred four years later. Even in the remission phase of cyclic Cushing's disease, meticulous endocrinological and neuroimaging examinations can reveal the presence of a pituitary adenoma, which should be treated surgically.

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The basal promoter activity of the human AT₁ receptor gene was characterized using a human hepatoma cell line with a considerably high expression of AT₁, PLC-PRF-5. Four cis-acting, positively regulating elements termed AT₁PRE1 (−113 to −102 bp), AT₁PRE2 (−49 to −43 bp), AT₁PRE3 (−5 to −2 bp) and AT₁PRE4 (+44 to +50 bp) were identified. AT₁PRE2 contained a GC-box-like sequence and bound to Sp1. AT₁PRE1 contained two tandem GC-boxes and was bound to several nuclear proteins in addition to Sp1. Nuclear proteins that were bound sequence-specifically to AT₁PRE1, AT₁PRE2 and AT₁PRE4 were found in both PLC-PRF-5 cells and 8505C cells, while those bound to AT₁PRE3 were not found in 8505C cells, which showed no expression of AT₁ and almost no promoter activity for the AT₁ gene. Significant promoter activity was still observed even when AT₁PRE1, AT₁PRE2 and AT₁PRE4 were all mutated. Mutagenesis of AT₁PRE3, however, substantially inactivated promoter activity. AT₁PRE1, AT₁PRE2 and AT₁PRE4 synergistically enhanced AT₁ gene transcription promoted by AT₁PRE3. These results suggested that AT₁PRE3 is responsible for the tissue-specific expression of the human AT₁ gene, and that AT₁PRE1, AT₁PRE2 and AT₁PRE4 function as a general enhancer in liver-derived cells.

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We here report a 77-year-old Japanese male who suffered general fatigue with progressive thirst and polyuria. Central diabetes insipidus was diagnosed by depletion of vasopressin secretion in response to increases in serum osmolality. Secretory responses of anterior pituitary hormones including adrenocorticotropin, thyrotropin, gonadotropins and growth hormone were severely impaired. Diffuse swelling of the infundibulum as well as lack of T1-hyperintense signal in the posterior lobe was noted by pituitary magnetic resonance imaging. The presence of bilateral hilar lymphadenopathy and increased CD4/CD8 ratio in bronchoalveolar lavage fluid was diagnostic for lung sarcoidosis. Physiological doses of corticosteroid and thyroid hormone were administered in addition to desmopressin supplementation. Complete regression of the neurohypophysial swelling was notable two years after corticosteroid replacement. Diffuse damage of anterior pituitary combined with hypothalamic involvement leading to central diabetes insipidus is a rare manifestation in such elderly patients with neurosarcoidosis.

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Beneficial effects of peroxisome proliferator-activated receptor alpha (PPAR alpha) agonists have been reported in improving insulin sensitivity and raising serum total adiponectin. High molecular weight (HMW) adiponectin, which is secreted from adipocytes, and visfatin, which is also expressed in adipose tissue, is related to glucose metabolism. In view of the additive effects of PPAR alpha agonists on these adipocytokines and glucose metabolism, we investigated male hypertriglyceridemic subjects who were treated with fenofibrate. Eleven male subjects with hypertriglyceridemia were treated with fenofibrate and serum total cholesterol (T-cho), triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting glucose, fasting insulin, total and HMW adiponectin, and serum visfatin levels were determined before and 3 months after treatment. Fenofibrate treatment significantly lowered T-cho, triglyceride, and LDL-C levels. There was a statistically significant increase of HDL-C. No differences in insulin sensitivity indices (G/I ratio and HOMA-IR) were observed between before and after treatment with fenofibrate. The treatment did not alter the levels of serum total adiponectin and visfatin in the hypertriglyceridemic patients, while serum HMW adiponectin increased significantly. This study demonstrates that fenofibrate increases serum HMW adiponectin levels, whereas visfatin is not regulated by fenofibrate in hypertriglyceridemic subjects. Further investigations are warranted to determine whether the elevation of HMW adiponectin caused by fenofibrate might improve insulin sensitivity.

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We report the case of a 64-year-old woman who had a severe hypercalcemia. Serum calcium, intact parathyroid hormone (PTH), 1α, 25 (OH)₂ vitamin D₃ levels were all elevated, and serum phosphorus level was decreased, which were all consistent with primary hyperparathyroidism (PHPT). ²⁰¹Tl/^99mTc subtraction scintigraphy failed to detect any abnormal accumulation in the neck and chest, while ^99mTc-MIBI scintigraphy demonstrated the focal accumulation of increased radiotracer uptake in the mediastinum only on the early image, but not on the delayed image. Neck and chest computerized tomography scanning showed a small nodule at the retrosternal region, and a selective venous sampling study of the intact PTH suggested PTH production from the nodule. Together with the observation of the early image of ^99mTc-MIBI scintigraphy, it was diagnosed that the patient had an ectopic parathyroid adenoma. Video-assisted thoracic surgery was performed. A 15-mm diameter mass, visualized by an intravenous infusion of methylene blue, was excited. The histopathology was consistent with the parathyroid adenoma. The adenoma was composed of mainly chief cells and rarely oxyphil cells. The absence of oxyphil cells would explain the lack of ^99mTc-MIBI retention on late-phase imaging in our case. Even without uptake on the delayed image of ^99mTc-MIBI scintigram, the early image was available for the localization of an ectopic parathyroid adenoma.

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While a large numbers of clinical trials using various kinds of statins has been reported, a possible preventive effect on new onset of type 2 diabetes mellitus was shown only by the subanalysis of The West of Scotland Coronary Prevention Study (WOSCOPS) using pravastatin. The aim of this study was to investigate whether pravastatin has a preferable effect on glucose tolerance among statins. An open-label prospective cross-over trial was performed to compare the effect of pravastatin (10 mg/day) or atorvastatin (10 mg/day) in Japanese early-state type 2 diabetes mellitus with hypercholesterolemia. The analyzed study subjects were treated with pravastatin (10 mg/day, n = 12) or atorvastatin (10 mg/day, n = 12) for 12 weeks. After a 4-week-washout period, the drugs were switched and treatment was continued for another 12 weeks. Oral glucose tolerance test (OGTT) was performed to evaluate several parameters including the appropriateness of beta cell function for the individual insulin sensitivity (disposition index: product of a validated secretion parameter and sensitivity) at the end of each therapy. HbA_1c and 2 h-glucose levels during OGTT in the pravastatin treatment were significantly lower than atorvastatin treatment. Disposition index after pravastatin treatment was significantly higher than after atorvastatin treatment. In conclusion, our study suggests that pravastatin has a favorable effect on pancreatic beta cell function compared with atorvastatin.

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TGF-β-like activities of proteins unrelated to the cytokine could mimic its actions in fibrosis and cell proliferation. Thyroglobulin (Tg) has been identified as having a TGF-β receptor (TGFβR)-binding activity and is deposited in the glomerulus in certain immune-complex diseases. The aim of the present study is to determine whether Tg can reproduce the transcriptional activity of TGF-β1 in the mouse glomerular mesangial cell (MC), and to examine whether such activity is manifested through TGFβR. Real-time RT-PCR was employed to examine the effects of TGF-β1 and bovine Tg on the expression of three genes (TGF-β1, plasminogen activator inhibitor 1 [PAI-1], and Pax-8) regulated by TGF-β1 in other cell types. In addition, a pentacosapeptide TGF-β1 antagonist, β₁²⁵ (41-65) was employed to determine whether the transcriptional activity of Tg was mediated through the TGF-β binding site on the TGFβR. A 6h exposure to TGF-β1 resulted in increased TGF-β1 and PAI-1 transcript, and a decrease in Pax-8. Similarly, a 6h exposure to Tg resulted in increases of about 5-fold in TGF-β1 and PAI-1 mRNA and a decrease of 53% in Pax-8. In comparison with other proteins, Tg had the greatest positive effect on TGF-β1 transcript levels. β₁²⁵ (41-65) significantly reduced the TGF-β1-, but not the Tg-induced changes in TGF-β1, PAI-1 and Pax-8 transcript levels. We conclude from these studies that Tg possesses a TGF-β-mimetic transcriptional activity in the MC that is not mediated by its binding to TGFβR. These results suggest that Tg and other proteins could initiate glomerular injury by reproducing the actions of TGF-β1 in the mesangial cell.

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The objective of the present study was to investigate the effects of octreotide long acting release (S-LAR) preparation on GH and IGF-1 serum concentrations and pituitary tumor size in patients with persistent and difficultly controlled acromegaly even after adjuvant irradiation and/or dopamine agonists. Thirty-three patients with active acromegaly (26 female and 7 male, mean age; 43.94 ± 14.01 SD years) were included in this study. Patients were evaluated at baseline and at 6, 12, 30 and 40 months for GH, IGF-1, and GH response to OGTT and biliary ultrasonography. Sella MRI was performed at initial and at 40 months. All patients received 20 mg S-LAR. Afterwards, the dosage was titrated to improve individual GH response and reduction of IGF-1 into normal ranges. Basal serum IGF-1 levels decreased from median: 530 μg/l [IQR: 420–600] to 340 μg/l [IQR: 230–460] at 6 months (p = 0.01), to 400 μg/l [IQR: 222.4–600] at 12 months (p = 0.48), to 396 μg/l [IQR: 318–468] at 30 months (p = 0.49), to 482 μg/l [308–580] at 40 months (p = 0.47). Nadir GH levels in OGTT fell from 2.70 ng/ml [IQR: 1.35–6.90] to 1.60 ng/ml [IQR: 0.36–4.10] at 6 months (p = 0.03), to 0.31 ng/ml [IQR: 0.18–0.65] at 12 months (p<0.0001), to 1.50 ng/ml [IQR: 0.83–4.00] at 30 months (p = 0.398) and to 0.89 ng/ml [IQR: 0.58–1.35] at 40 months (p<0.0001). Initially, pituitary adenoma volume was median: 1.18 ml [IQR: 0.08–3.50] and it shrank to 0.21 ml [IQR: 0–2.1] at 40 months (p = 0.08). Gallstones were detected in 12 patients and six of them underwent cholecystectomy. S-LAR is an effective treatment regimen in reducing GH and IGF-1 concentrations and as well as in shrinking tumor volume in persistent and difficultly controlled acromegalic patients.

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Hypoparathyroidism is a complication of thyroidectomy that causes hyperphosphatemia primarily due to enhanced reabsorption of phosphate in the kidney resulting from decreased parathyroid hormone (PTH) secretion. Fibroblast growth factor-23 (FGF23) is a hormone-like factor that is thought to play an important role in phosphate homeostasis. However, the changes and role of FGF23 in transient hypoparathyroidism after thyroidectomy are not clear. We examined changes in serum levels of calcium, phosphate, intact PTH, 1,25-dihydroxyvitamin D, and FGF23 in 12 patients (10 women, 2 men; mean age, 51 yr) who developed transient hypoparathyroidism after thyroidectomy. Serum phosphate reached its peak level (5.9 ± 0.5 mg/dl) approximately 4 days after development of hypoparathyroidism, and this was followed by a peak in the serum FGF23 level (71 ± 28 ng/l). Serum levels of calcium, phosphate, and FGF23 normalized after recovery of parathyroid function. There was a significant positive correlation between serum phosphate and FGF23 levels (P<0.05). Serum FGF23 was elevated in patients with hypoparathyroidism and hyperphosphatemia and normalized along with normalized phosphate levels after recovery of parathyroid function. The peak level of phosphate always preceded that of FGF23 by several days, suggesting that elevated phosphate is a primary stimulus for release of FGF23. This homeostatic regulation of phosphate differs considerably from that of serum calcium whose change is rapidly corrected within minutes.

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Hypothyroidism may result in accelerated atherosclerosis. Hyperhomocysteinaemia is an independent risk factor for premature atherosclerotic vascular disease. The aim of the present study was to assess plasma total homocysteine (tHcy), folate and cobalamin concentrations in hypothyroid patients before and after treatment. Thirty-one hypothyroid and thirty health young women were studied. The hypothyroid patients were investigated in the untreated state and again after restoration of euthyroidism. The levels of homocysteine, folate, cobalamin and thyroid stimulating hormone (TSH), free thyroxine (fT₄), free triiodothyronine (fT₃) and renal function were measured before and after treatment. In hypothyroidism tHcy was higher but not statistically significant than in control group. Serum level of folate was higher and serum cobalamin was lower in the hypothyroid state. Following L-thyroxine therapy tHcy significantly decreased as well as the concentration of cobalamin. Level of folate remained unchanged. Univariate analysis in hypothyroid group indicated that tHcy negative correlated with creatinine clearance, fT₃, fT₄, cobalamin and positive with TSH. In multivariate analysis tHcy correlated with creatinine clearance, cobalamin and fT₄. Thyroid status influences the plasma tHcy. Free triiodothyronine and next free thyroxine have the greatest negative influence. This would account for hyperhomocysteinemia in the hypothyroid state and premature atherogenesis.

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Insulin-induced localized lipoatrophy is a well-known localized side effect. Although an immune process has been suggested as its etiology, no definitive evidence has been reported to show that insulin is involved. Here, we report the first evidence for the phagocytosis of insulin by histiocytes as a very early stage of lipoatrophy, which was reproducible in two different lobular panniculitis tissues from a 71 year-old male patient. He had taken a subcutaneous insulin injection in his arms because of a sight disturbance. Since these subcutaneous tumors were likely due to inflammation by insulin, a biopsy sample was taken from the subcutaneous tumor of his right arm with his consent. The primary antibodies for insulin (1 : 200) and CD68 (1 : 50) were obtained from DAKO (guinea pig anti-insulin and mouse anti-human CD68). HE staining revealed the infiltration of mononuclear cells and histiocytes into the subcutaneous fat tissue, and some parts of this tissue had fibrosis with rich collagen fibers. These findings are consistent with a lobular panniculitis. Some histiocytes contained intracellular substances with a positive immunoreactivity to insulin. This activity was reduced when the anti-insulin antibody was preincubated with an excess amount of insulin antigen. The same substances were also positive to CD68. Thus, the phagocytosis of insulin by histiocytes appears to occur in this region. Therefore, the activation of subcutaneous macrophages by the complex of insulin and insulin antibodies may account for the initial autoimmune process.

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Fibroblast growth factor (FGF)23 is a hormone that regulates serum phosphate and 1,25-dihydroxyvitamin D levels. Hyperphosphatemia is sometimes observed in patients with acromegaly while the detailed mechanism of this abnormal phosphate metabolism remains to be elucidated. We have measured FGF23 levels in 18 patients before and after the surgery for acromegaly. Serum GH, IGF-I and phosphate significantly decreased after the surgery. In addition, FGF23 also reduced by the surgery. These results indicate that deficient action of FGF23 is not the cause of deranged phosphate metabolism in patients with acromegaly.

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