We had a patient with asymptomatic hyper-immunoreactive glucagonemia and with no evidence of pancreatic tumor detected by radiological examinations. The glucagon level was not decreased by the administration of glucose or somatostatin analogue (SMS 201-995). Gel filtration studies revealed that most glucagon immunoreactivity was eluted at the position of 150, 000 daltons [big plasma glucagon (BPG)]. Binding studies with ¹²⁵I-glucagon showed that glucagon autoantibody wasnegative. Acid treatment of plasma and reduction of immunoglobulin G (IgG) did not result in a shift of BPG to normal glucagon (3485 daltons). Glucagon immunoreactivity determined with anti-glucagon antiserum OAL 123 (C-terminal specific antiserum used in the present radioimmunoassay kit) did not dilute out in parallel to normal glucagon (3485 daltons), and the plasma glucagon level was normal with Unger's 30K (another C-terminal specific antiserum) and OAL 196 (N-terminal specific antiserum). The patient's IgG dose-dependently reduced the binding of ¹²⁵I-glucagon to anti-glucagon antiserum OAL-123. Glucagon degrading activity (GDA) was negative in the patient's plasma. These results suggest that the patient's IgG cross-reacted with the present anti-glucagon antiserum OAL 123, and caused a spuriously high plasma Immunoreactive glucagon level.

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We investigated the in vitro effect of glycation on LDL oxidation. Native LDL (nLDL) was glycated in 0, 5, 10, or 20mM glucose. This glycated LDL (gLDL) was oxidized by 1μM copper ion. Compared to nLDL and gLDL, oxidized gLDL (ogLDL) has a greater negative charge. The thiobarbituric acid reactive substance (TBARS) value of ogLDL increased with the glucose concentration tested during glycation in a dose-dependent manner. OgLDL glycated in 20mM glucose had a significantly higher TBARS level than did oxidized LDL incubated without glucose. In conclusion, LDL glycated in vitro is prone to oxidation. Thus, glycated LDL, which increases in the diabetic state, may contribute to the pathogenesis of atherosclerosis in diabetic patients.

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The localization of epidermal growth factor (EGF) in the ovary and its effect on proliferation of granulosa cells were investigated in gonadotrophin-primed immature female rats. Immunoreactions with anti-rat EGF monoclonal antibody were observed sparsely in the granulosa layer and antrum of follicles, but not in the theca layer or stromal tissue. The EGF-positive cells were round or oval shaped and often larger than granulosa cells. The localization and morphological appearances of these cells in the follicles were in good agreement with those of macrophages. Although EGF alone did not promote granulosa cell growth in vitro, the labelling index with [³H]thymidine of granulosa cells cultured with 0.1ng/ml EGF and 0.1ng/ml basic fibroblast growth factor was significantly greater than that without the growth factors (18.4% vs. 15.8%, P<0.01). These results suggest that macrophages in follicles may modulate follicular development through a paracrine mechanism by secreting EGF and other growth factors.

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Regulation of the ACTH receptor (R) in the adrenal gland by its own ligand “ACTH” has beena matter of controversy. In the present study, whether ACTH regulates the expression of mRNA for its own receptor in the adrenal gland was studied in human subjects and in rats in vivo. In the human study, adrenal adenoma tissues as well as adjacent normal tissues were obtained at surgery from two patients with typical Cushing's syndrome. Northern blot analysis revealed two ACTH-R mRNA species with 4.0kb and 2.0kb. ACTH-R mRNAs in the adenoma tissues were much more abundant than those in the normal tissues from the two patients, suggesting that the mRNA in normal adrenal tissue is either suppressed by cortisol excess or the absence of ACTH. To examine the mechanism involved in ACTH-R mRNA regulation, the changes in the receptor mRNA caused by ACTH were studied in dexamethasone-treated rats. Administration of dexamethasone for 5 days resulted in a marked decrease in ACTH-R mRNA to an undetectable level. A bolus administration of ACTH_1-24 intravenously or ACTH-Z_1-24 intramuscularly to the dexamethasone-treated rat did not cause any significant change in ACTH-R mRNA from 0.5 to 12h after the administration. However, a significant increase in the receptor mRNA was observed at 24h after the ACTH-Z_1-24 and the level was further increased until 48h followed by a sustained increase at 72h when it was given once every 24h. These data suggest that the ACTH-receptor is increased by ACTH at a pretranslational level. Although it remains to be studied whether the increased receptor mRNA level in the adenoma tissues of the patients with Cushing's syndrome is a general phenomenon, the results suggest that the regulatory mechanism of the receptor in the adenoma is different from that in normal tissue and this could contribute to the pathogenesis of autonomous production of cortisol.

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A small subgroup of primary aldosteronism due to aldosteronoma, named aldosterone-producing renin-responsive adenoma (AP-RA), has been reported to masquerade as idiopathic hyperaldosteronism (IHA) because of the responsiveness of the plasma aldosterone concentration (PAC) to upright posture (UP). We found two patients with AP-RA in 19 patients with aldosteronoma who were examined by UP stimulation and were treated surgically. In 17 patients with typical aldosterone-producing adenoma (APA), PAC decreased or increased only slightly (less than 200% of the basal level); in contrast, it increased to over 300% of the basal level in two patients with AP-RA. The two groups were comparatively studied as to their hormonal levels, adrenal computed tomography (CT) scan and histological findings in order to clarify the characteristics of AP-RA. Basal PAC was within the normal range (11.1 and 13.0ng/dl) in AP-RA but in APA it ranged from 14.8 to 58.1ng/dl with a mean of 32.3± 2.7ng/dl. The diameters of the adenoma in AP-RA were apparently smaller (6 and 9mm) than those in APA ranged from 10 to 25mm with a mean of 15.5±1.1mm. After a contrast medium was injected at CT scan, the density of the normal adrenal gland adjacent to the adenoma increased but that of the adenoma did not in APA, making a clear distinction between the adenoma and the gland. On the other hand, the density of the adenoma and gland increased to almost the same degree in AP-RA. Thus, in AP-RA it was difficult to detect adrenal tumor by CT scan because of its size and because of the response to the contrast medium. Adenomas in both groups were mainly composed of clear cells, and no histological difference was found between the two groups. In summary, AP-RA was rarely present in primary aldosteronism and should be carefully diagnosed as primary aldosteronism because of normal PAC. Its PAC increased over to 300% of the basal level. It was showed that AP-RA are difficult to distinguish from IHA not only because of the similar responsiveness of PAC to UP but also because of difficulties in detecting adrenal tumor by adrenal CT scanning.

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The inhibitory effect of timoprazole on iodide uptake by cultured thyrocytes was observed using FRTL-5 cells. The inhibition was noticed to be dose dependent and was eliminated completely by rinsing. Timoprazole pretreated with acid had inhibitory effects, but [³H] leucine incorporation experiments indicated that acidified timoprazole inhibited protein synthesis to some degree. In addition, TSH-induced cAMP production was also inhibited by acidified timoprazole. Timoprazole pretreated in a neutral solution did not have any cytotoxic effects on TSH-induced cAMP production or protein synthesis at concentrations less than 1000μM, suggesting that timoprazole by itself has specific effects on iodide uptake. The absence of any direct interaction of the drug with iodide was confirmed by separation of timoprazole and iodide on a C₁₈ Sep-Pak column. It is concluded that timoprazole by itself could inhibit iodide transport through the thyroid cell membrane. This may provide a useful system in further investigating the mechanism of iodide transport.

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With the widening use of computerized tomography, the incidentaloma, an adenoma found incidentally in the adrenal, in computerized tomograms obtained for problems not necessarily related to the adrenal, has emerged as a recent clinical entity. Nine cases with such tumors are presented, here, along with a brief review of the related medical literature. Endocrine and other studies have shown that two of these nine patients had hormone secreting adrenal tumors, two pheochromocytomas. Surgical resection of the tumor was performed in six of the cases and aspiration biopsy was done in four with three completely benign cytological examination results (Class I or II) and one Class III result. The tumor with the class III result turned out to be a benign pheochromocytoma. CT estimates of the tumor size were 25mm to 80mm in the whole group and 30 to 80mm in the patients who were operated on. Operation and histopathologic examination revealed three cortical adenomas, two pheochromocytomas, and one myelolipoma. Although no malignant tumors were found, the percentage of functioning adrenal neoplasms is rather high (22.2%) in this group of nine incidentalomas. Cases of adrenal incidentaloma therefore require a thorough endocrine evaluation along with other examinations which allow the clinician to follow tumor size.

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We report two cases of congenital adrenal hyperplasia (CAH) in which administration of chlormadinone acetate (CMA), a substituted progestational agent for prostatic disease, suppressed ACTH hypersecretion and lowered plasma testosterone levels. Case 1 was 83-year-old male with advanced prostatic carcinoma and CAH due to 21-hydroxylase deficiency. His plasma testosterone did not decrease in spite of a bilateral orchiectomy. Case 2 was 40-year-old female with CAH due to 21-hydroxylase deficiency suffering from virilization after the cessation of cortisol supplement therapy because of her breast carcinoma. In these two cases, oral administration of CMA at a daily dose of 75-100mg suppressed ACTH and cortisol to subnormal levels and reduced testosterone levels. With the suppressive effect on ACTH excess and antiandrogenic action, CMA may be suitable for patients with CAH suffering from symptoms due to overproduced ACTH or adrenal androgen.

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We demonstrated the functional evaluation of adrenal incidentaloma in 8 patients who had no abnormal finding associated with Cushing's syndrome. Adrenal tumors were incidentally discovered by abdominal echogram in 5 patients and by computed tomography (CT) in 3 patients. Serum cortisol levels and urinary excretion of 17-hydroxycorticosteroids (17-OHCS) were within normal limits in four of 8 patients. Urinary excretion of free cortisol was also within normal limits except for patient 8. Urinary excretion of 17-OHCS, however, was not suppressed by dexamethasone administration in five of 8 patients. Excretion of urinary 17-OHCS did not increase in response to metyrapone in 3 of 4 dexamethasone-insuppressible patients, but increased in 3 dexamethasone-suppressible ones. Serum cortisol increased in response to exogenous ACTH in all 6 patients examined. ¹³¹I-Adosterol accumulation was found in only the tumor side in 7 patients and bilaterally in one patient. Adrenalectomy was done in 7 patients, and microscopic findings showed adrenocortical adenoma. Serum cortisol was significantly suppressed in response to dexamethasone in the post-operative stage in all 7 patients examined. These results indicate that these adrenal incidentalomas seem to have a cortisol over-production which is dexamethasone-insuppressible and ACTH-dependent, and that they can be classified as “Pre-Cushing's Syndrome”.

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Medullary thyroid carcinoma (MTC) and pheochromocytoma appear in either a sporadic or a hereditary form as components of multiple endocrine neoplasia (MEN). Many germline mutations of the RET proto-oncogene have been reported in patients with MEN 2A and 2B, and familial MTC(FMTC). To elucidate the etiological roles in tumorigenesis of sporadic MTCs and pheochromocytomas, mutations in the cysteine-rich region of the RET proto-oncogene were analyzed by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Exons 10 and 11 were studied in genomic DNAs from 3 clinically apparent sporadic MTCs, MTCs and pheochromocytomas from 2 patients with MEN 2A, 1 with FMTC, 4 with MEN 2B, 3 with neurofibromatosis type 1(NF1), 12 sporadic pheochromocytomas and an MTC cell line, TT. All tumors from two patients with MEN 2A and one patient with FMTC had mutations at codon 618 and 634 as well as their leukocytes, reflecting their germline mutations. In this region, no mutations were detected in any tumors from patients with MEN 2B and NF1, and sporadic pheochromocytomas. But mutations were detected and identified in 3 clinically apparent sporadic MTCs and TT cells. A 6 base pair (bp) deletion causing the loss of a cysteine residue at codon 634 and a mutation causing substitution from cysteine to tyrosine at codon 634 were detected in 2 sporadic MTCs as somatic events. In a female patient diagnosed as having sporadic MTC, a mutation at codon 618 was detected not only in tumor tissues, but also in her leukocytes, suggesting a germline mutation of the RET protooncogene. In TT cells a heterozygous mutation at codon 634 was detected. These results suggest that RET mutations within a cysteine-rich region may also play an important role in the tumorigenesis of sporadic MTCs, and mutations of RET protooncogene should be screened in clinically sporadic cases to exclude hereditary MTCs.

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Genetic linkage analyses have traced the loci for multiple endocrine neoplasia type 2A (MEN 2A) to an interval on chromosome 10q11.2. This region encompasses the RET proto-oncogene, a receptor tyrosine kinase gene expressed in medullary thyroid carcinoma (MTC) and pheochromocytoma. By means of genomic polymerase chain reaction (PCR) amplification and DNA sequencing, we have analysed 19 individuals from two Japanese MEN 2A families for mutations of the RET proto-oncogene in exons 10 and 11. We conducted single-strand conformational polymorphism (SSCP) analysis of the RET proto-oncogene amplified from affected and unaffected family members. The DNA alterations in the RET proto-oncogene caused substitution of a cysteine for a serine at codon 620 in the exon 10 in three patients in one MEN 2A family, 1, and of a cysteine for a tyrosine at codon 634 in the exon 11 in six patients in the MEN 2A family, 2. We could find two asymptomatic MEN2A gene carriers who had no symptoms or signs of MEN 2A by DNA analysis of the RET proto-oncogene. No mutations in these exons were detected in any unaffected normal members of MEN 2A. A DNA alteration in the RET proto-oncogene coding sequence in exon 10 caused a shift on SSCP gels that was characteristic of the disease chromosome in the MEN 2A family, present only in affected members of the family. The DNA change could also be detected by restriction enzyme digestion with RsaI in family 2. Two MEN 2A patients with a cysteine for a tyrosine substitution at codon 634 in the exon 11 had parathyroid hyperplasia. We conclude that the identification of a DNA alteration in the MEN2A gene will permitpredictive molecular testing of individuals at risk in these MEN 2A families and the PCR-restriction enzyme system will be useful for genetic diagnosis of members of these MEN 2A families. This information, by providing diagnostic certainty, should improve medical care for affected family members.

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The prolactin (PRL) gene is known to be expressed not only in the anterior pituitary but also in the decidualized human endometrium. This study was designed to detect the site of synthesis of PRL during pregnancy by in situ hybridization histochemistry. Decidual and trophoblast tissues from early pregnancy were obtained from patients undergoing therapeutic abortion at 8-10 weeks of gestation. Term placentae were obtained from patients with uncomplicated deliveries at 38-40 weeks. Sections of these tissues were hybridized with ³⁵S-labeled RNA probe complementary to human PRL mRNA. Specific hybridization signals were distributed over the decidual cells in early and term pregnancy. In the decidua capsularis of early pregnancy, labeled cells were concentrated close to the amniotic cavity, although decidual cells were distributed evenly. In the decidua parietalis, almost all decidual cells werelabeled, but no specific labeling was seen in the endometrial glands or capillary endothelium. In the decidua basalis, greater signals were always detected over the decidual cells in early pregnancy than in term pregnancy, when sections, which were hybridized with the same probe and exposed simultaneously, were compared. No specific hybridization was detected in the trophoblast cells. These results not only confirm that PRL is specifically synthesized in the decidual cells but also indicate that there are regional and periodical differences in PRL gene expression in the decidual cells during pregnancy.

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Body composition was assessed by bioelectrical impedance analysis (BIA) in 11 female patients with Graves' disease and in 49 age-matched healthy Japanese women. Patients with Graves' disease were examined in the hyperthyroid state before treatment and in the stable euthyroid state after treatment with antithyroid drugs for 6 to 18 months. Body weight (BW), percent body fat (BF/BW), percent lean body mass (LBM/BW) and percent total body water (TBW/BW) were not statistically different between hyperthyroid Graves' patients and healthy subjects. Percent body cell mass (BCM/BW) was much lower in hyperthyroid Graves' patients than in healthy subjects (mean±SEM; 33.9± 2.4% vs. 41.5±0.5%, P<0.001). Percent ratio of extracellular water to total body water (ECW/TBW) was much greater in hyperthyroid Graves' patients than in healthy subjects (53.9±3.0% vs. 41.8±0.5%, P<0.001). These abnormal ratios, BCM/BW and ECW/TBW, were normalized after treatment. Serum free T₄ levels showed a positive correlation with ECW/TBW (r=0.779) and a reverse correlation with BCM/BW (r=-0.760) in all of the patients with Graves' disease examined. These findings indicate that body composition is affected by thyroid hormones and that body composition in hyperthyroid Graves' disease is characterized by decreased BCM associated with increased ECW.

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Systemic administration of human interferon-α stimulates the pituitary-adrenal axis in men, but the exact mechanism still remains to be established. The present study was undertaken to examine the hypothesis that interferon-α may alter the circulating concentrations of the cytokines which involve the activation of the pituitary-adrenal axis. Eleven patients with chronically active hepatitis C were treated with human lymphoblastoid interferon-α (IFN: 6×10⁶IU/day) and changes in plasma adrenocorticotropin (ACTH), serum cortisol and cytokine concentrations were observed on both the first and second days of the treatment. Subcutaneous administration of IFN significantly increased plasma ACTH and serum cortisol concentrations by 3h after the injection. Serum interleukin-6 (IL-6) increased with the increase in circulating ACTH and cortisol. There was a significant correlation between serum cortisol and IL-6 concentrations at 3h. In contrast, an increase in serum interleukin-1β was only observed in one case. On the second day of IFN treatment, simultaneous administration of 25mg diclofenac sodium eliminated the IFN effects on circulating ACTH, cortisol and IL-6 concentrations. The present studies demonstrated that circulating IL-6 increases after systemic IFN administration, resulting in activation of the pituitary-adrenal axis.

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Several lines of evidence have suggested that insulin resistance may play an important role in the pathogenesis of hypertension. CS-045 is a new hypoglycemic drug by which improves insulin sensitivity in peripheral tissues. We assessed the effect of CS-045 on hypertension in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) as a control. At 20 weeks of age, the treatment with CS-045 was started by being mixed with chow pellets in the proportion of 0.2% (w/w) and continued for 6 weeks. Nonfasting plasma glucose levels were not changed in either strain of rats treated with CS-045, but plasma insulin levels decreased in SHR 4 and 5 weeks after the start of CS-045 therapy. Blood pressure increased with age in SHR without treatment, but CS-045 decreased blood pressure only 4 and 6 weeks after the treatment. These findings suggest that insulin resistance is not strongly associated with the pathogenesis of hypertension in SHR.

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To evaluate the sequential changes in secretion of insulin and glucagon by the pancreas of Zucker fa/fa rats, we examined the secretion of these peptides by the perfused pancreas of the rats and by that of their lean littermates aged 9, 18, and 54 wks. Obese rats weighed significantly more than lean rats at all ages and had increased plasma insulin except for those at 54 wks of age. Isolated pancreas of obese rats at 9 wks showed greater insulin secretion response to glucose and arginine than that of lean rats at the same age. Insulin secretion response to arginine from pancreas of obese rats at 18 wks was also greater than that from pancreas of lean rats at 18wks. Increased glucose concentration in the perfusion medium caused less suppression in obese rats than in lean rats. 10mM arginine stimulation resulted in a higher secretion response in lean rats than in obese rats at 18 and 54wks. Impairment of glucagon secretion was observed even at 54wks of age, when the obese rats no longer had significantly increased plasma insulin. This suggested that abnormal glucagon secretion was due not only to hyperinsulinemia, but also to a possible defect in the secretory mechanism.

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Insulin-like growth factors (IGFs) have 6 types of binding proteins (IGFBPs), and IGFBP-3 is the major IGFBP in human sera. A proteolytic enzyme for IGFBP-3 has recently been reported to be present in human and animal pregnant sera. Although the physiological significance of a pregnancy-associated IGFBP-3 protease remains to be established, the proteolysis could result in lowering the affinity for IGFs, thereby enhancing their delivery to target tissues by increasing free IGFs in the circulation. The methods for detection of IGFBP-3 protease which have been widely used so far are a method reported by Lamson et al. which used affinity crosslinking or western ligand blotting. These methods need radioactive materials (iodinated IGFs and IGFBP-3) and it takes at least a few days to get the results. We have now developed a simple assay for the proteolysis of IGFBP-3. The method is western immunoblotting without radioactive materials. The results can be obtained in a day. With this method, we proved the absence of significant proteolytic activity in sera from rapidly growing children (early stage of puberty or precocious puberty), and sera from a severe type of growth hormone deficiency. Significant proteolytic activity, as in pregnant women, was detected in 6 out of 11 patients with acute disorders such as measles, Kawasaki disease, bacterial meningitis and mycoplasma pneumonia, some of whom were probably in a catabolic condition. These data suggests that the proteolysis of IGFBP-3 might also be important in modulating IGF action in some acute diseases during childhood. The increased bioavailability of IGFs by IGFBP-3 proteolysis may play a role in overcoming catabolic conditions.

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The blood FSH level is often high in patients with severe testicular disorders including cryptorchids. To examine whether inhibin is involved in the increase in FSH we measured immunoreactive inhibin, FSH, LH, and testosterone in 17 patients after orchidopexy. FSH was extremely high (20mIU/ml or above) in 3 patients. The inhibin level was significantly lower (P<0.01) in these 3 patients (6.47±2.19IU/ml; mean±SEM) than in the other 14 patients (14.31±3.96 IU/ml). All 3 high-FSH patients had azoospermia. Testosterone and LH were normal in one of them. Even considering problems involved in the inhibin assay, the high FSH levels are considered to reflect reductions in the blood inhibin level due to Sertoli cell dysfunction. These findings suggest that inhibin plays an important role in the suppression of FSH at least in some patients after orchidopexy.

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The cases of two boys, a 14 years 10 months old and an 18-year-old, with delayed puberty are presented. The first patient also had a short stature. Both patients had a pituitary adenoma, as shown by computed tomography, with high prolactin levels. After bromocriptine therapy was started, there was a spontaneous progression of normal puberty. The first patient used a synthetic growth hormone together with bromocriptine, however, even after the growth hormone was stopped progression in puberty and gain in height continued. The favorable response obtained in these patients implies that bromocriptine can be an effective therapy for adolescent patients with prolactinoma.

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