To determine if subacute thyroiditis (SAT) is associated with changes in the regional perfusion of the thyroid gland, we performed Tc-99 m sestamibi scans on eleven patients with SAT who had painful goiter and clinical thyrotoxicosis. Eleven patients had Tc-99 m pertechnetate and Tc-99 m sestamibi scintigraphy during the acute stage of SAT. The thyroid uptake ratio of sestamibi was compared with the laboratory data and color Doppler ultrasonography. Tc-99 m pertechnetate scintigraphy in the thyroid was markedly reduced during the acute stage of SAT. Conversely, Tc-99 m sestamibi showed diffuse increased uptake in the thyroid region, suggesting increased perfusion. On the other hand, there was near absence of vascularization in the acute phase and slight increase in the recovery phase by color Doppler ultrasonography. The clearance rate of Tc-99 m sestamibi during the early phase (from 10 min to 1 h) was decreased in the acute stage of SAT. The sestamibi uptake ratio correlated with serum immunosuppressive acidic protein (IAP) in the acute stage of SAT and the sestamibi uptake ratio in the recovery stage of SAT was correlated with serum thyrotropin levels. Tc-99 m sestamibi uptake in the early phase in the acute stage of SAT may reflect the inflammatory process associated with SAT.

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hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common α-subunit, but differ in their hormone-specific β-subunits. The α-subunit has two N-glycosylation sites at Asn⁵² and Asn⁷⁸. To obtain more information on the relationship between the structure and function of the α-subunit, we introduced a novel N-glycosylation site in the N-terminal region by mutating Asp³ and Gln⁵ into Asn and Thr, respectively. Glycosylation mutants were expressed alone or with hCGβ-subunit in Chinese hamster ovary cells. New N-linked oligosaccharides were efficiently added to the wild-type and mutant α-subunits lacking N-glycan at Asn⁵²(αΔAsn1), Asn⁷⁸(αΔAsn2), and both (αΔAsn(1 + 2)). The new sugar chain did not affect secretion and assembly except that 1) it increased the intracellular degradation of αΔAsn(1 + 2), and 2) it augmented the assembly of αΔAsn1 with hCGβ-subunit. Amino acid changes generated the attachment of O-glycosylation in free α-subunit but not in assembled form. These data indicate that the newly introduced N-glycosylation consensus sequence is functional, and that the N-terminal region of the α-subunit is flexible and can be modified without affecting the intracellular function. Furthermore, amino acid sequences in the N-terminus are involved in the O-glycosylation in free α-subunit.

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We previously reported that the daily urinary unidentified ketosteroid glucuronide(US-G) level in patients with Cushing’s syndrome was much higher than that in the healthy subjects. Furthermore, urine samples from patients with Cushing’s syndrome, including those with pituitary adenoma and adrenal adenoma, yielded almost the same high excretion levels, despite the different sites of the adenomas. We extracted US obtained by hydrolysis of US-G in urine of patients with Cushing’s syndrome, purified it, and analyzed its chemical structure. Molecular weight and molecular formula were analyzed by MS spectrometry, and the chemical structure was analyzed by NMR spectrometry, utilizing small quantities of refined US. The substance has a molecular weight of 304Da, a molecular formula of C₁₉H₂₈O₃, and its chemical structure is 3α, 11β-dihydroxyandrost-4-en-17-one.

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To analyze the effects of treatments with GH and cyclic estrogen/progesterone (E/P) replacement on bone mineralization in patients with Turner’s syndrome (TS), bone mineral density (BMD) was measured longitudinally. BMDs of the whole body and the lumbar spine in 16 adult female patients with TS (17-38 year old; 0-20 years by length of E/P treatment) were assessed using dual energy X-ray absorptiometry one to 5 times over a treatment period of up to 7 years maximum. GH treatment was performed in 9 cases (GH group), but not in the remaining 7 (non-GH group). E/P replacement therapy was initiated in all patients after they finished GH administration. The BMDs of both the whole body and the lumbar spine in the patients with TS were significantly less than those in age-matched normal subjects, and did not improve with E/P treatment. Although there were no differences in final body height and age at the beginning of E/P administration between the GH and non-GH groups, whole body BMD in the GH group was significantly lower than that in the non-GH group. These results indicate that GH administration in childhood and adolescence and E/P treatment in adulthood did not increase bone mineralization in the TS patients. Therefore, we can conclude that the optimal protocol of hormonal replacement therapy with GH and E/P during childhood and adolescence should be established as soon as possible.

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The small intestine plays an important role in the digestion and absorption of many nutrients. To investigate the contribution of carbohydrate digestion to diabetes mellitus, we examined the morphological changes of the small intestine, and the expression of sucrase-isomaltase, which is one of the intestinal disaccharidases, in diabetic model rat, that is the streptozotocin-induced (STZ) diabetic rat (insulin-deficient model), and the Otsuka Long-Evans Tokushima Fatty (OLETF) rats and the Goto-Kakizaki (GK) rats (type 2 diabetic models). Intestinal hyperplasia was observed in STZ, OLETF, and GK rats. Moreover, in the small intestine of each diabetic strain, the proliferating cell nuclear antigen (PCNA)-labeling index, which is a marker of proliferation, was higher than in the respective control. Cdx1 and Cdx2, known to be transcriptional factors related to intestinal proliferation and differentiation, were more highly expressed in STZ, OLETF and GK rats than in the respective controls. These findings indicate that small intestinal hyperplasia, and thereby the resultant increase of total activity of disaccharidases such as sucrase and isomaltase in the entire small intestine, might be one of the reasons for postprandial hyperglycemia in diabetes mellitus.

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Although ERβ is known to be expressed at high levels in the rat prostate gland, its regulation is not well understood. Here we examined ER mRNA expression and the effects of testosterone administration in male rats at 1, 4 and 9 weeks of age who were castrated and/or treated with testosterone for a week, and then sacrificed. ERα was the major type of ER expressed in 2 week-old animals while dominant expression of ERβ mRNA was apparent in older age groups. Interestingly while ERβ expression was diminished and ERα mRNA increased in the castrated group, testosterone administration reversed this effect. A time-course study indicated that induction of ERβ mRNA increased within 9 hr and ERα decreased in 2 days after an injection (i.p.) of testosterone. Our results suggested that 1) testosterone up-regulates ER β mRNA expression while ER α is down-regulated; and that 2) great changes in ERα and β expression in the prostate gland during development from the newborn to adult may be due to the influence of testosterone.

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To clarify the direct effects of ghrelin on growth hormone (GH) release from anterior pituitary (AP) cells in cattle, GH-releasing effects of human ghrelin (hGhrelin) and rat ghrelin (rGhrelin) on bovine AP cells were compared with those of GH-releasing hormone (GHRH) in vitro. The AP cells were obtained from Holstein steers and were incubated for 2 h with the peptides after incubating in DMEM for 3 days. hGhrelin and rGhrelin significantly stimulated GH release from the cultured cells at doses from 10^-10 to 10^-7 M and from 10^-9 to 10^-7 M, respectively (P<0.05). The rates of increase in GH at 10^-10, 10^-9, 10^-8 and 10^-7 M hGhrelin were 26, 26, 59 and 100% compared with controls, respectively, and those of increase in GH at 10^-9, 10^-8 and 10^-7 M rGhrelin were 58, 74 and 106%, respectively. GHRH significantly increased GH concentrations in cultured media at a dose as low as 10^-13 M compared with the control (P<0.05). When hGhrelin (10^-8 M) and GHRH (10^-8 M) were added together, the release of GH induced by both peptides was significantly greater than that by hGhrelin alone (P<0.05), and tended to be greater than that by GHRH alone. Somatostatin (SS, 10^-7 M) significantly blunted GH release induced by hGhrelin (10^-8 M) and GHRH (10^-8 M)(P<0.05). In the presence of SS, the percent increase in GH released with hGhrelin plus GHRH was 42% and 14% greater than that by either hGhrelin or GHRH alone, respectively (P<0.05). These results show that ghrelin directly stimulates the release of GH from anterior pituitary cells, and that SS modifies ghrelin-stimulated GH release in cattle.

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The aim of the present study was to determine the clinical and hormonal characteristics with Sheehan’s syndrome in 28 cases that we had diagnosed and followed in the last 20 years. Twenty-eight patients with Sheehan’s syndrome, diagnosed and followed at our University Endocrinology Clinic in the last 20 years were reported in the study. Medical history, physical examination, routine laboratory examinations, pituitary hormone analysis, CT and/or MRI scan of the sella of the patients were reviewed. All patients had a history of massive hemorrhage at delivery and physical signs of Sheehan’s syndrome. Twenty-six of them lacked postpartum milk production, followed by failure of resumption of menses. There were 9 subjects with disturbances in consciousness associated with hyponatremia on admittance. All 28 patients had secondary hypothyroidism, adrenal cortex failure, hypogonadotrophic hypogonadism and growth hormone deficiency. Diabetes insipidus has not been found in any patient. Empty sellae were revealed in 8 patients by CT and/or MRI scan. Sheehan’s syndrome is still encountered in clinical practice occasionally. If not diagnosed early, it could cause increased morbidity and mortality. The most important clues for diagnosis of Sheehan’s syndrome are lack of lactation and failure of menstrual resumption after a delivery complicated with severe hemorrhage.

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We report on a 2 years and 9 months old Japanese boy with adrenal hypoplasia and mental retardation (MR) (developmental quotient ∼60) which occurred in the absence of severe adrenal crisis and resultant brain damage. Cytogenetic and molecular studies were performed in this boy and his parents with normal phenotype, showing that the boy had a maternally derived ∼2 Mb interstitial Xp deletion involving DAX1 (DSS-AHC critical region on the X chromosome, gene 1) for adrenal hypoplasia congenita and disrupting IL1RAPL (interleukin-1 receptor accessory protein-like) for non-specific MR. The results explain the development of MR in this boy in terms of contiguous gene syndrome, and suggest the importance of IL1RAPL analysis in patients with adrenal hypoplasia and MR.

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Components of cyclinD1/cyclin-dependent kinase 4 (CDK4)/p16^INK4a/pRb pathway are the frequent target of many tumor types. We examined the role of retinoblastoma susceptibility gene (RB1) and the CDK4 gene in human pituitary tumorigenesis. For the RB1 gene, pRb expression and loss of heterozygosity (LOH) on 13q in pituitary adenomas were analysed. Immunostaining of pRb revealed lack of expression in 1 of 29 pituitary adenomas. In 4 of 31 pituitary adenomas, allelic imbalances including LOH of RB1 on 13q14 were detected. Three of 4 pituitary adenomas, in which one adenoma lacked pRb expression, had a common LOH region at least from D13S219 on 13q12.3-q13 to D13S265 on 13q31-32. Interphase fluorescence in situ hybridization with a probe of RB1 showed 2 copies of RB1 gene suggesting that mitotic recombination events, not deletion or chromosome loss, led to LOH in the 3 pituitary adenomas analyzed. All 27 exons, intron-exon boundaries, and essential promoter region of RB1 gene were then sequenced in genomic DNA from 4 pituitary adenomas with allelic imbalance on 13q14 including one adenoma without pRb expression and 3 adenomas with pRb expression. Any somatic mutations, insertions, or microdeletions in the RB1 gene were not detected in 4 pituitary adenomas. Methylation sensitive (MS)-polymerase chain reaction (PCR) and bisulfite sequencing analysis revealed hypomethylated status of CpG islands in the promoter region of the RB1 genes of 4 pituitary adenomas. In addition, activating mutations of CDK4 gene, which is a component of cyclinD1/CDK4/p16^INK4a/pRb pathway, were not detected in 31 pituitary adenomas. Based on these results, it is concluded that somatic mutations of the RB1 gene or CDK4 gene do not appear to play a major role in pituitary tumorigenesis. This supports the presence of potential tumor suppressor gene(s) on 13q12.3-q13 to 13q31-32 in pituitary adenomas.

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We report on a Japanese boy with Noonan syndrome who had short stature, bilateral cryptorchidism, poor pubertal development, mild mental retardation, complex cardiac lesions consisting of hypertrophic cardiomyopathy, mitral valve stenosis and insufficiency, subvalvular aortic stenosis, and single coronary artery, and various dysmorphic features including hypertelorism, epicanthic folds, low set malrotated ears, high arched palate, micrognathia, webbed neck, low posterior hairline, shield chest, pectus excavatum, cubitus valgus, borderline short metatarsals, lymphedema, redundant skin, and nail dysplasia. Because of marked lymphedema in the bilateral lower legs, lymphatic scintigraphy was carried out at 13.3 years of age, indicating extreme lymphstasis in the lower extremities, severe lymphstasis in the forearm, the elbow, and the axillary regions, moderate lymphstasis around the ascending aorta, and mild lymphstasis in the bilateral lungs. The results, in conjunction with those suggested in Turner syndrome, imply that lymphatic hypoplasia/dysplasia and resultant distended lymphatics and lymphedema are relevant to the development of not only soft tissue and visceral anomalies but also skeletal anomalies in Noonan syndrome.

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A 75-year-old woman had tumors in her pituitary, thyroid and left adrenal gland. Plasma ACTH and cortisol levels were both mildly elevated. Both plasma ACTH and cortisol concentrations were partially suppressed by 1 mg of overnight dexamethasone suppression test, while both were inhibited with a dosage of 8 mg dexamethasone. Plasma ACTH and cortisol levels were increased in response to human CRH and desmopressin. Together with the observation of pituitary microadenoma, the patient had a pituitary ACTH-producing tumor. The patient, however, had no typical Cushingoid features, hypertension, or impaired glucose tolerance, suggesting that the tumor had an autonomic ACTH secretion that was insufficient for expressing clinical symptoms, the so-called preclinical Cushing’s disease. A case of preclinical Cushing’s disease is extremely rare. Further, the patient had thyroid papillary carcinoma and non-functioning adrenal tumor. Molecular genetic analysis demonstrated a polymorphism of the menin gene in the patient. Even without Cushingoid features in pituitary incidentaloma, we concluded that the elevated ACTH and cortisol levels should be followed up by CRH, desmopressin and dexamethasone suppression tests. This patient with preclinical Cushing’s disease would be observed whether the physical conditions in the patient develop to overt Cushing’s disease.

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ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a rare cause of Cushing’s syndrome. Recently, aberrant expression of adrenal receptors for various hormones and/or cytokines has been identified in several cases with AIMAH, which may act as a pathogenetic factor for the disorder. We report here an AIMAH patient with a Rathke’s cleft cyst. Endocrinological examinations revealed that the pituitary cyst had no hormonal secretion. Administrations of either AVP or isoproterenol provoked cortisol production in the patient, whereas DDAVP, mosapride or endogenous LH induced by GnRH did not. Reverse transcriptional-PCR analysis of total RNA obtained from the patient’s adrenal tissue revealed the expression of mRNA of receptors for V1a, V1b, V2, and LH/hCG. Three of these receptors except for V1a receptor were not expressed in normal adrenal tissue. Hyperosmolar saline infusion promoted the patient’s cortisol secretion through the increase in endogenous AVP (peak plasma AVP level reached 90.4 pg/ml during the test). These results suggest that endogenous AVP and catecholamines are involved in the pathophysiology of the patient. Further study will be necessary to clarify the molecular mechanisms that regulate tissue-specific expression of these receptors and their role in the overgrowth of adrenal in AIMAH.

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Luteinizing hormone (LH) consists of α- and β-subunits, and synthesis and secretion of LH are regulated by gonadotropin-releasing hormone (GnRH). In order to examine the molecular mechanisms by which GnRH regulates LH secretion, we transfected αT3-1 cells with rat LHβ-subunit cDNA under the control of a constitutive promoter and established a stable cell line of LH2 cells which secreted LH in response to GnRH. Pulsatile and continuous GnRH pretreatments increased gene expression of the α-subunit and synthesis of LH, and enhanced the LH secretion by brief treatments with GnRH and 56 mM KCl. The LH secretions were partially blocked by elimination of extracellular Ca²⁺. GnRH-induced LH secretion was completely inhibited by calphostin C (a protein kinase C inhibitor) and 1 μM wortmannin. In contrast to the GnRH induction, high K⁺-induced LH secretion was inhibited by KN93, a Ca²⁺/calmodulin-dependent protein kinase II inhibitor, as well as by 1 μM wortmannin. We also confirmed that activation of cAMP-pathway induced LH secretion, but activation of mitogen-activated protein (MAP) kinase pathway was not involved in LH secretion. These results suggest that GnRH directly regulates LH secretion as well as LHβ-subunit synthesis, and that LH2 cells are a useful model for the study of LH secretion induced by several secretagogues.

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The purpose of the present study was to investigate changes in serum leptin levels during GnRH agonist therapy. Twenty regularly menstruating women with uterine leiomyomas were enrolled. These subjects were given GnRH agonist (leuprorelin acetate, 3.75 mg) monthly for 4 months. Serum leptin and estradiol (E2) levels were measured at the two time points of day 1 or 2 of the menstrual cycle and the end of GnRH agonist therapy. Weight, total body fat mass, percentage of body fat, and total body lean mass were measured by whole body scanning with dual-energy X-ray absorptiometry. The ratio of serum leptin levels to total body fat mass (leptin-fat mass ratio), and the ratio of serum leptin levels to total body lean mass (leptin-lean mass ratio) were calculated. All subjects became amenorrheic after the initial administration of GnRH agonist. Baseline E2 levels were 45.4 ± 21.0 pg/mL, which significantly decreased after GnRH agonist therapy (13.3 ± 4.2 pg/mL, p<0.01). Baseline leptin levels were 8.7 ± 8.1 ng/mL, which did not differ from the values after 4 months of GnRH agonist administration (8.9 ± 6.8 ng/mL). Total body fat mass significantly increased from 20.0 ± 10.4 to 21.0 ± 9.4 kg (p<0.05), while total body lean mass significantly decreased (34.5 ± 4.2 kg to 33.3 ± 3.9 kg, p<0.01). However, leptin-fat mass ratio after GnRH agonist therapy did not differ from the baseline values (0.39 ± 0.16 ng/mL/kg vs 0.38 ± 0.16 ng/mL/kg). Hypogonadism does not have a major impact on circulating leptin levels.

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