Search

Ad4BP, also known as SF-1, is a steroidogenic tissue-specific transcription factor that is also essential for adrenal and gonadal development. Two mechanisms for the transcriptional regulation of the mammalian FTZ-F1 gene encoding Ad4BP in adrenocortical cells have been proposed in the previous studies: the crucial role of a cis-element, an E box for the steroidogenic cell-specific expression of mouse and rat FTZ-F1 genes, and a possible autoregulatory mechanism of the rFTZ-F1 gene by Ad4BP itself through binding to the Ad4 (or SF-1) site in the first intron. In the present study, the transcriptional regulation of the human FTZ-F1 gene in adrenocortical cells was investigated from several angles, including the above two mechanisms. Using a series of deletion analyses of the 5'-flanking region of the hFTZ-F1 gene and site-directed mutagenesis for transient transfection studies, an E box element, CACGTG at -87/-82 from the transcriptional start site, was also found to be essential for the transcription of the hFTZ-F1 gene in mouse or human adrenocortical cell lines as well as in non-steroidogenic CV-1 cells. Despite the presence of a corresponding Ad4 site, CCAAGGCC at +163/+156 in the first intron of the hFTZ-F1 gene, an autoregulatory mechanism through the Ad4 site was found to be unlikely in the hFTZ-F1 gene mainly due to site-directed mutagenesis. In addition, the forced expression of Ad4BP had little effect on hFTZ-F1 gene transcription in non-steroidogenic CV-1 cells. Such Ad4BP-independent regulation of the hFTZ-F1 gene was in striking contrast to the regulation of steroidogenic CYP genes, such as the human CYP11A gene, in which the proximal promoter activity is Ad4BP-dependent and the transactivation by Ad4BP is silenced by DAX-1. Even though the Ad4BP-dependent transcriptional regulation of the DAX-1 gene has been reported, DAX-1 did not affect the transcriptional activity of the hFTZ-F1 gene in our study. Taken together, these observations suggest that the E box is indeed required for the expression of the FTZ-F1 gene, at least in mammalian species, but may not determine the tissue-specific expression of the hFTZ-F1 gene, and that, unlike the steroidogenic CYP gene, the regulation of the hFTZ-F1 gene appears to be independent of both Ad4BP and DAX-1.

Estrogen is the most important endocrine hormone that stimulates the growth of hormone-dependent breast cancer. The biosynthesis of estrogens in breast tissue is catalyzed by cytochrome P450 aromatase (P450arom). The expression of P450arom is controlled by the tissue- or cell-specific promoters of CYP 19 gene. The roles of nuclear receptor systems for the aromatase activity in breast cancer cells have not yet been fully investigated. In the present study, we investigated the effects of a nuclear receptor system constituted by retinoid X receptor (RXR) and its heterodimer partner on the aromatase activity in a cultured MCF-7 human breast cancer cell line, using each selective ligand for retinoic acid receptor (RAR) (TTNPB), RXR (LG100268), PPARgamma (troglitazone), and vitamin D(3) receptor (vitamin D(3)). The treatment of the cells with TTNPB or LG100268 alone for 2 days increased slightly the aromatase activity, but the increases were not statistically significant in comparison to the control. However, the combined treatment with TTNPB (10(-7) M) and LG100268 (10(-7) M) caused a dramatic stimulation of the aromatase activity. The treatment with other ligands had little or no effect on the aromatase activity. The stimulation of the aromatase activity by TTNPB plus LG100268 was dose-dependent, and a maximum stimulation was observed at 10(-7) M in both compounds. In addition, the increase in the aromatase activity was accompanied by an increase in the P450arom mRNA levels determined by RT-PCR in MCF-7 cells. The increase in the P450arom transcript was also found to be related to the specific usage of promoter 1a of the CYP 19 gene based on the analysis using RT-PCR. This is the first demonstration that a nuclear receptor system constituted by a RAR:RXR heterodimer is involved in the regulation of aromatase activity in MCF-7 breast cancer cells.

Ovarian granulosa cells synthesize estrogens from androgens, which are catalyzed by aromatase cytochrome P450 (P450arom). Troglitazone (Tro), one of the insulin-sensitizing compounds, thiazolidinediones (TZDs), is a ligand for peroxisome proliferator activated receptor gamma (PPARgamma) and is effective in the treatment of both non-insulin-dependent diabetes mellitus (NIDDM) as well as polycystic ovary syndrome (PCOS). PPARgamma exerts a transcriptional activity as a PPARgamma:RXR heterodimer. In this study, we investigated the effects of Tro and/or RXR ligand, LG100268 (LG) on the aromatase activity in cultured human ovarian granulosa cells obtained from patients who underwent in vitro fertilization. Human ovarian granulosa cells expressed PPARgamma mRNA assessed by RT-PCR. The treatment of the granulosa cells with Tro for 24 h resulted in a dramatic inhibition of the aromatase activity in a dose-dependent manner. While the treatment with LG alone also inhibited the aromatase activity, the combined treatment with both Tro and LG caused a much more reduction in the aromatase activity. The changes in the aromatase activity by Tro and/or LG were associated with comparable changes in P450arom mRNA assessed by RT-PCR. These results suggest that Tro directly inhibit the aromatase activity in human granulosa cells probably via nuclear receptor system PPARgamma:RXR heterodimer. The findings may provide a biochemical basis for the decrease in the blood concentrations of estrogens which is observed after the in vivo administration of Tro and may also possibly be useful as a novel therapy for estrogen-dependent diseases.

The activation of PPARgamma:RXR nuclear system induces monocytic differentiation of some myelogeneous leukemia cell lines. The present study was undertaken to examine the effect of PPARgamma ligand, TZD (troglitazone or pioglitazone) and/or RXR selective ligand, LG100268 on the erythroleukaemia cell line K562 which has both an erythroid character and a potential for differentiation into megakaryocytes. TZD suppressed cell proliferation and the erythroid phenotype of K562 cells. The suppression of erythroid phenotype of K562 cells by TZD was synergistically enhanced by the combined treatment with LG100268. Moreover, the marked suppression of erythroid phenotype in K562 cells was also accompanied by the downregulation of the erythroid lineage-transcription factor, GATA-1. These novel actions of troglitazone may provide a biochemical basis for anemia occasionally which is observed after the in vivo administration of TZD.

The DAX-1 (also known as AHC) gene encodes an unusual member of the nuclear hormone receptor superfamily. DAX-1 plays a critical role during gonadal and adrenal differentiation since mutations of the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. In recent studies, DAX-1 was reported to function as a transcriptional suppressor of Ad4BP/SF-1, a critical transcription factor in gonadal and adrenal differentiation. With respect to implication of Ad4BP/SF-1 in the transcriptional regulation of the DAX-1 gene, inconsistent findings have been previously reported. We investigated the upstream region of the mouse Dax-1 (also known as Ahch) gene and identified a novel Ad4/SF-1 site by transient transfection and electrophoretic mobility shift assays. In addition, immunohistochemical analyses with a specific antibody to Dax-1 indicated the presence of immunoreactive cells in steroidogenic tissues, pituitary gland, and hypothalamus. Although the distributions of Dax-1 and Ad4BP/SF-1 were very similar, they were not completely identical. The expression of Dax-1 was significantly impaired in knock-out mice of the Ftz-f1 gene, which encodes Ad4BP/ SF-1. Taken together, our findings indicate that Ad4BP/SF-1 controls the transcription of the Dax-1 gene.

Thiazolidinedione (TZD) is known to be a potent activator of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor that constitutes a heterodimer with retinoid X receptor (RXR). Since a considerable amount of PPARγ is expressed in various hematopoietic cells, the present study was undertaken to examine the effect of TZD in the absence or presence of LG100268, an RXR-selective ligand, on a cultured promyelocytic leukemia cell line, HL60. Treatment with TZD (25–50 µM troglitazone or pioglitazone) markedly suppressed cell proliferation of HL60. A cell cycle analysis revealed that the suppressive effect of troglitazone on HL60 cell proliferations was caused by G0/G1 cell cycle arrest as well as by an apoptotic effect. Treatment with the same concentration of troglitazone also induced the monocytic differentiation of HL60 cells. The apoptotic or the differentiating effect of TZD on HL60 cells was synergistically enhanced by the combined treatment with 1 µM LG100268, while LG100268 alone neither had an apoptotic nor a differentiating effect on HL60 cells. These results suggest that these actions are mediated through the nuclear receptor system constituted by the PPARγ: RXR heterodimer.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroid glands, the pancreatic islet cells, and the anterior pituitary. Germline mutations of the MEN1 gene in three independent Japanese cases with MEN1 were analyzed. Case 1 has revealed a 2-bp (TA) insertion at nucleotide position 341 (341insTA) in exon 2, which shifts the reading frame such that the mutant protein has a completely different amino acid sequence from codon 78 to the premature stop codon at 119. In case 2, a nucleotide substitution, i.e., TAG in place of TGG, which encodes tryptophan at codon 198 was identified (nonsense mutation). These mutations were heterozygously present and have not been reported previously. Case 3 showed no mutations in the protein-coding exons and exon-intron junctions of the MEN1 gene by single-strand conformation polymorphism or direct sequencing of the polymerase chain reaction (PCR) fragments. We confirmed the finding that patients with MEN1 carry heterozygous germline mutations in the MEN1 gene, which is compatible with the idea that the MEN1 gene is a tumor suppressor gene. The reason why mutations in the coding region of the MEN1 gene could not be detected by PCR-based analysis in some of the MEN1 patients, e.g. case 3, needs to be clarified further.

In 2005, the International Diabetes Federation (IDF) and the Japanese Committee for the Diagnostic Criteria of Metabolic Syndrome (Japanese definition) proposed metabolic syndrome definitions for the Japanese population (1,2). Both definitions included waist circumference as an essential component and adopted cut points of 85 cm for men and 90 cm for women based on the relationship between waist circumference and visceral fat area by computed tomography (3). However, those cut points have been in dispute because of their suboptimal sensitivity and specificity (4–6). Recently, the IDF proposed common cut points for the whole Asian population including the Japanese: 90 cm for men and 80 cm for women (7). In this study, we investigated optimal waist cut points to identify subjects with multiple risk factors in the Japanese population and verified the cut points to predict carotid intima-media thickening in metabolic syndrome subjects as a surrogate marker of early atherosclerosis and cardiovascular disease (8–10). We reviewed cross-sectional data from 1,658 men and 1,116 women (aged 48.8 ± 9.8 and 46.8 ± 10.4 years, respectively) who had annual medical checkup services provided by their employers. The medical checkup took place from May 2005 to November 2006 at the Human Dry Dock Center Wellness in Fukuoka, Japan. Subjects on antihypertensive and/or antidiabetic medications were included as individuals with hypertension and/or diabetes, respectively. We excluded 158 subjects on medications for dyslipidemia because we were unable to determine …

We cloned 537 basepairs (bp) of rat partial peroxisome proliferator-activated receptor gamma2 (PPARgamma2) cDNA and examined the effect of fasting or obesity on the expression of two isoforms of rat PPARgamma, gamma1 and gamma2, in either subcutaneous or mesenteric adipose tissue specimens using an RNase A protection assay. In Wistar rats, expression of both isoforms was dramatically reduced after 48 hours of fasting in the two fat tissue specimens. In comparing genetically obese (fa/fa) Zucker rats and lean control rats, no significant difference was observed in expression of the two isoforms in either type of adipose tissue. From these findings, we conclude that the adipose tissue level of rat PPARgamma depends on nutritional deprivation but is not closely associated with either obesity or insulin resistance in obese Zucker rats.

OBJECTIVES The overnight 8-mg dexamethasone suppression test is often used to differentiate Cushing's disease, due to an oversecretion of ACTH from the pituitary gland, from other kinds of Cushing's syndrome. However, a few patients with ACTH-producing pituitary adenoma show no suppression of plasma cortisol after the administration of 8 mg of dexamethasone. To clarify the relationship between the level of glucocorticoid receptor (GR) in the pituitary adenoma and the sensitivity to dexamethasone in Cushing's disease, we thus examined the levels of GRα and GRβ mRNAs in the pituitary adenomas in six patients who were proven at surgery to have pituitary ACTH-producing adenomas. MATERIALS Total RNA was extracted from six pituitary adenomas and pituitary tissue adjacent to one of the adenomas, and the mRNA levels of GRα, GRβ, pro-opiomelanocortin (POMC) and β-actin in these samples were sampled by quantitative RT–PCR. RESULTS The GRα mRNA levels in the adenomas from the two patients who showed no response to the 8-mg dexamethasone suppression test were significantly lower than those in the adenomas of four patients who showed suppression. The GRβ mRNA level was much lower than that of GRα mRNA but not significantly different among the six adenomas. CONCLUSIONS These results suggest strongly that decreased expression of GRα in pituitary adenomas may be the major reason for the marked insensitivity to the 8-mg dexamethasone suppression test observed in two patients with Cushing's disease.

X-linked adrenal hypoplasia congenita (AHC) is characterized by primary adrenal insufficiency and is frequently associated with hypogonadotropic hypogonadism (HHG). Mutations of the DAX1 gene have been reported in patients with AHC and HHG. We found a novel DAX1 mutation in our patient. Sequence analysis of the patient's DAX1 demonstrated a 1-bp (G) deletion at codon 49 in exon 1. The mutation shifts the reading frame, resulting in completely different amino acid sequences from codon 49 to the premature stop codon at 84. The G was present at this position in the sequences of the father and 2 younger brothers. Direct sequence and single-strand conformation polymorphism analyses of polymerase chain reaction fragments revealed that the mutation at codon 49 was heterozygously present in the mother's DAX1 gene. The codon 84 is located in the first half of the DNA binding domain, and the mutation site is closer to the N-terminus than those in previously reported cases. The onset of adrenal insufficiency in the neonatal period as seen in our patient has also been reported in other patients with different DAX1 mutations, especially in a patient with DAX1 protein lacking 11 amino acids at the C-terminus. Thereore, it is less likely that position of termination codons correlate to clinical manifestations. Am. J. Med. Genet. 76:62–66, 1998. © 1998 Wiley-Liss, Inc.

Cytochrome b5, a component of the electron transfer system increases the relative activity of 17,20-lyase to 17alpha-hydroxylase of P450c17 in vitro. In the present study, immunohistochemical analysis of cytochrome b5 was performed in the human adrenal gland and in adrenocortical adenomas from patients with Cushing's syndrome. In the human adrenal gland, cytochrome b5 was stained in all three adrenocortical layers but the staining was most remarkable in the zona reticularis. All of the adenomas were composed mainly of compact cells, which exhibited immunoreactive staining for cytochrome b5 as well as for P450c17 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The distribution of b5 in the adenomas was correlated with that of P450c17 rather than with that of 3beta-HSD. The immunoreactive staining for cytochrome b5 appeared to be more prominent in the two adenomas that produced relatively high concentrations of adrenal androgens than in adenomas that produced low concentrations of adrenal androgens. These results immunohistochemically support the functional association of b5 with androgen production through interaction with P450c17 and the previous finding that higher concentrations of cytochrome b5 are associated with greater production of adrenal androgens in adrenocortical adenomas from patients with Cushing's syndrome.

The sex-determining region Y chromosome (SRY) triggers testis determination. We report a 46,XX true hermaphrodite who had ambiguous genitalia at birth. A laparotomy at one year of age revealed this patient to have a testis on the right side and an ovotestis on the left side. By polymerase chain reaction analysis no SRY was detected in the DNA from the leukocytes but it was found in the DNA from the ovotestis. The hidden mosaicism with the Y-bearing cells in the gonads is likely the cause of the dual gonads in this patient.

A case of acinar-islet cell carcinoma presenting as insulinoma is reported. The patient was a 28-year-old man who presented with two convulsive episodes. Fajans' index [immunoreactive insulin (IRI; microU/ml/ glucose mg/dl)] and Turner's [IRI (microU/ml) x 100/glucose (mg/dl) - 30] index were high (2.8 and 308, respectively), as were serum proinsulin levels (550 pg/ml). Abdominal computed tomography and angiography revealed a highly vascular tumor in the pancreatic tail and several similar tumors in the liver. Histologic features of a biopsy specimen from a hepatic tumor were those of a malignant pancreatic endocrine tumor. Insulin secretion by the liver metastases was confirmed by venous sampling after arterial stimulation with calcium. These findings led us to diagnose malignant insulinoma with liver metastases. Serum levels of alpha-fetoprotein and trypsin were markedly elevated, to 2234 ng/ml (normal10) and 22,000 ng/ml (normal460) respectively, and these levels continued to rise with further growth of the liver metastases. Immunohistochemically, the metastatic liver tumor specimen was positive for alpha-fetoprotein, alpha 1-antichymotrypsin, chromogranin A, and neuron-specific enolase. These findings of amphicrine features in the tumor were indicative of acinar-islet cell carcinoma that produced alpha-fetoprotein and trypsin in addition to insulin.

Patients with systemic lupus erythematosus (SLE) are at risk of atherosclerosis. An increased carotid intima–media thickness (IMT) is considered to be a marker of early atherosclerosis. To determine influential factors for increased carotid IMT in SLE patients. We evaluated the impact of conventional risk factors for atherosclerosis on carotid IMT in 427 healthy controls and of clinical factors on carotid IMT in 94 SLE patients. Carotid IMT was measured by using a newly developed computer-automated system. Unconditional logistic regression was used to assess the adjusted odds ratios (ORs) and 95 % confidence intervals (95 % CI). Multivariate-adjusted mean carotid IMT (mm) was significantly reduced in SLE patients (0.51, 95 % CI = 0.36–0.66) compared to healthy controls (0.55, 95 % CI = 0.40–0.70) (P = 0.003). The SLE Disease Activity Index (SLEDAI) was associated with carotid IMT in a dose-dependent manner (P trend = 0.041). The current use of cyclosporine A (adjusted OR = 0.02, 95 % CI = 0.01–0.40, P = 0.011) and a history of steroid pulse therapy (adjusted OR = 0.01, 95 % CI = 0.01–0.25, P = 0.006) were significantly associated with a decreased risk of increased carotid IMT. Our findings suggest that the current use of cyclosporine A can protect against increased carotid IMT, leading to a decreased risk of arteriosclerosis. Future studies with a larger sample size need to confirm that this association holds longitudinally.

The Research Committee of Disorders of Adrenal Hormones, Japan, undertook a nationwide epidemiological study of primary aldosteronism (PA). The present study was undertaken as a part of this study to reveal the relationship between type of treatment and the prognosis of PA. In the primary survey, 4161 patients with PA during the period January 1, 2003-December 31, 2007 were reported from 3252 departments of internal medicine, pediatrics and urology. In the secondary survey, a questionnaire that requested detailed clinical information on individual patients was sent to those departments reporting patients in the primary survey. In total, data on 1706 patients with PA were available in the present study. Among patients with bilateral or unilateral aldosterone-producing adenoma, after adjustment for age at which prognosis was examined, sex, surgical treatment and medical treatment, surgical treatment was significantly associated with amelioration of hypertension (adjusted odds ratio [OR]: 0.47 [95% confidence interval (CI): 0.29-0.77]) and hypokalemia (adjusted OR: 0.17 [95% CI: 0.11-0.29]). No significant relationship was observed between medical treatment and such prognosis in this group of patients. Among patients with bilateral or unilateral adrenal hyperplasia, surgical, but not medical, treatment was significantly associated with amelioration of hypokalemia (adjusted OR: 0.23 [95% CI: 0.06-0.74]), while there was no relationship between surgical or medical treatment and the prognosis of hypertension. In conclusion, surgery offered a better prognosis of PA than medication with regards to hypertension and hypokalemia, with the limitation that a new anti-aldosterone drug, eplerenone, was not available during the study period.

The research to develop a drug, so called selective androgen receptor modulator (SARM) , which shows beneficial androgenic action on bone and muscle, but hardly possesses the stimulatory action on prostate has been making a progress. However, no drug is available in the market at present. Most of such drugs are developed, aiming at the application to age-related muscle reduction (sarcopenia) and osteoporosis. We are now trying to develop a SARM which may have beneficial effect on metabolic syndrome.

The prevalence of primary aldosteronism (PA) is around 3-15% in patients with hypertension. Hypertension is a frequent complication of type 2 diabetes mellitus (DM) because of the close etiological relationship between these two diseases. However, the possibility of PA in patients with DM and hypertension is often overlooked and the prevalence of PA in patients with DM and hypertension in Japan is unknown. We enrolled 124 hospitalized patients with both DM and hypertension. PA was diagnosed according to the modified criteria for Japanese patients. We examined the prevalence of PA and compared clinical characteristics between patients with and without PA. In another analysis of 43 patients with a confirmed diagnosis of PA, we compared the characteristics of patients with and without DM. Overall, 14/124 patients with DM and hypertension (11.3%) were diagnosed with PA. Multivariate logistic regression showed that the duration of DM was significantly shorter in the PA group. Fisher's direct probability test revealed that history of hypertension before the diagnosis of DM was a significant factor in patients with PA. Treatment with an angiotensin II receptor blocker (ARB) did not affect the diagnosis of PA in these patients. Among 43 patients with PA, those with DM were significantly older and the delay to the diagnosis of PA was significantly longer compared with patients without DM. In conclusion, almost 10% of patients with DM and hypertension actually have PA. More extensive screening for PA is recommended in patients with DM and hypertension, regardless of the use of ARBs.

Subclinical Cushing's syndrome (SCS) associated with adrenal incidentaloma is usually characterized by autonomous cortisol secretion without overt symptoms of Cushing's syndrome (CS). Although the diagnostic criteria for SCS differ among countries, the 1 mg dexamethasone suppression test (DST) is essential to confirm the presence and the extent of cortisol overproduction. Since 1995, SCS has been diagnosed in Japan based on serum cortisol levels ≥3 μg/dL (measured by radioimmunoassay [RIA]) after a 1 mg DST. However, the increasing use of enzyme immunoassays (EIA) instead of RIA has hindered the diagnosis of SCS because of the differing sensitivities of commercially available assays, particularly for serum cortisol levels of around 3 μg/dL. One way to overcome this problem is to lower the cortisol threshold level after a 1 mg DST. In the present study, we examined the clinical applicability of lowering the cortisol threshold to 1.8 μg/dL, similar to the American Endocrine Society's guidelines for CS, by reanalyzing 119 patients with adrenal incidentaloma. Our findings indicate that serum cortisol levels ≥1.8 μg/dL after 1 mg DST are useful to confirm the diagnosis of SCS if both of the following criteria are met: (1) basal ACTH level

A 52-year-old man was referred to our clinic. One week before his visit, he had complained of left eye pain and double vision. His clinical features were exacerbated. Despite the acute onset, which is atypical of thyroid eye disease (TED), TED was suspected due to the patient's history of Graves' disease (GD). After conducting clinical examinations and orbital magnetic resonance imaging, the patient was diagnosed with idiopathic orbital myositis (IOM), and intravenous glucocorticoid therapy was administered. After treatment, the patient's clinical manifestations dramatically improved. This is a rare case in that the history of GD made it difficult to differentiate IOM from TED.

We herein report a rare case of surgically proven normocalcemic primary hyperparathyroidism (NCHPT). A premenopausal 51-year-old woman was referred to our clinic because parathyroid adenoma was detected on neck ultrasonography (US). The patient's serum calcium concentration was 9.3 mg/dL and the intact parathyroid hormone (PTH) level was 128.8 pg/mL. The findings of almost all other examinations were also compatible with a diagnosis of NCHPT. Then, parathyroidectomy was performed. The serum calcium and PTH concentrations reduced significantly but remained within the normal ranges. A histological examination demonstrated parathyroid adenoma. A review of this case and the associated literature suggests that estrogen plays a significant role in the etiology of NCHPT in premenopausal women.

A 17-yr-old female Japanese patient, who was reported in 1968 as having glucocorticoid-responsive hyperaldosteronism but was presumed to have a defect of 17 alpha-hydroxylation mainly in the adrenal glands as the etiology of her illness, was followed. The relationship between clinical manifestations and molecular abnormalities in cytochrome P-45017 alpha gene (CYP17) was also reviewed based on the literature on Japanese patients with 17 alpha-hydroxylase deficiency. She has been treated with dexamethasone, resulting in normal blood pressure and normokalemia for 28 yr. She had almost normal gonadal function with regular menstruation on her first admission. Because of sustained genital bleeding, however, she underwent total hysterectomy with an ovarian biopsy at the age of 42 yr. No follicles or corpus luteum were detected in the ovarian specimen. At the age of 45 yr, the basal levels of sex steroids were decreased, while those of gonadotropins were increased. A genetic study on CYP17 revealed a homozygous deletion of phenylalanine (Phe) codon (TTC) at either amino acid position 53 or 54 in exon 1. A review of the literature revealed 4 patients with this type of CYP17 mutation, including the present patient, out of a total of 11 young adult Japanese patients. The clinical manifestations caused by congenitally deficient gonadal function were not marked in any of these 4 patients, but were marked in 5 of the 7 patients with different mutations of CYP17. The remaining 2 female patients had irregular menstruation. The pretreatment urine/plasma values of aldosterone were variable, normal to high, in individual patients, regardless of the structural abnormalities of CYP17. The following conclusions were suggested: 1) this type of CYP17 mutation is associated with well preserved gonadal function in young adult patients, but it likely causes early reduction of gonadal function with increasing age in these patients; 2) the prevalence of this type of CYP17 mutation is quite high in Japanese patients; and 3) the pretreatment hyperaldosteronism observed in the present patient seems not to be related to the mutation of CYP17.

To clarify the antiatherogenic mechanism of action of dehydroepiandrosterone (DHEA), we investigated the effects of DHEA on the accumulation of cholesteryl ester (CE) in cultured mouse macrophage J774-1 cells. The accumulation of CE in J774-1 cells in the presence of acetyl low density lipoprotein (AcLDL) and 10(-5) mol/l DHEA was significantly reduced to 30% of the control values for 24 h. The marked effect of DHEA was observed as early as 6 h and continued at least for 48 h. This reduction by DHEA was dose-dependent and occurred starting at a DHEA dose of 5 x 10(-7) mol/1 for 24 h. DHEA treatment did not induced any changes in the cell surface binding, cell-association, or degradation of AcLDL. In comparison, the DHEA analogues, 8354 and 8356, which are known to be much stronger inhibitors of glucose 6-phosphate dehydrogenase than DHEA, did not show as marked an effect as DHEA on the accumulation of CE during the first 6 h. However, after 24-48 h of incubation, both 8354 and 8356 caused a marked reduction in the accumulation of CE similar to that observed with DHEA. A quantitative analysis of the cellular cholesterol content revealed that DHEA caused a marked reduction in CE with a concomitant continuous increase in free cholesterol (FC), while the DHEA analogues caused a marked reduction in CE with no change in FC. DHEA demonstrated little inhibitory effect on 25-hydroxycholesterol-driven esterification. Moreover, 10(-5) mol/1 DHEA induced a CE reduction in the foam cells induced by AcLDL. The CE-reducing capacity was also observed in the DHEA analogues. This CE-reducing capacity disappeared, however, when acyl CoA:cholesterol acyltransferase inhibitor, 58-035, was also present. Based on these findings, it can be concluded that the inhibitory effect of DHEA on the CE storage in response to AcLDL can be explained, at least in part, by two mechanisms. First, a recently published mechanism, namely, the inhibitory action of DHEA on lysosomal cholesterol transport, correlates well with the inhibition against foam cell transformation by DHEA in the early phase (at 6 h) observed in our study. With regard to the second mechanism, the CE-reducing capacity of DHEA from CE-laden foam cells, which appears to be related to a decreased cholesteryl ester cycle, may contribute to the inhibitory effect on the CE storage in the late phase (at 24 h and 48 h). These phase-specific inhibitory mechanisms of DHEA on the CE-storage may therefore partly explain the antiatherogenic action of DHEA.

Yanase T, Hara T, Sakai Y, Takayanagi R, Nawata H. Expression of sterol carrier protein 2 (SCP2) in human adrenocortical tissue. Eur J Endocrinol 1996;134:501–7. ISSN 0804–4643 Sterol carrier protein 2 (SCP2) has been implicated in adrenal steroidogenesis by in vitro studies. In order to clarify the clinical significance of SCP2 in human steroidogenesis, we investigated the expression of SCP2 mRNA in various types of adrenocortical tissue and one testis and examined the correlation between the amounts of SCP2 and other values such as the free cholesterol content and the cholesterol side-chain cleavage (SCC) activity in the tissue mitochondria, The types of adrenocortical tissue examined included adrenocortical carcinomas (N = 3), adrenocortical adenomas from patients with Conn's syndrome (N = 3) and from patients with Cushing's syndrome (N = 3), non-functioning adrenocortical adenomas (N = 2) and normal adrenal glands (N = 2). Northern blot hybridization predominantly revealed a 1.8-kb SCP2 mRNA in all tissue specimens examined. The mRNA concentrations of SCP2 in two out of three adrenocortical carcinomas were relatively lower than those in other types of tissue. No other special tendency was observed regarding the mRNA expression levels in various tissue specimens. The mRNA concentrations of SCP2 correlated significantly with mitochondrial contents of free cholesterol (r = 0.67, p < 0.01), but was not correlated with the SCC activities in mitochondria measured by an in vitro enzyme assay. The mitochondrial SCC activities, however, were correlated significantly with the protein levels of mitochondrial P-450 scc determined by a Western blot analysis (r = 0.79, p < 0.01). The significant positive correlation between mRNA concentrations of SCP2 and the mitochondrial content of free cholesterol suggests that the central role of SCP2 in human steroidogenic tissues may be in part a translocation of cytoplasmic free cholesterol to the mitochondria, as demonstrated previously by in vitro studies. Toshihiko Yanase, Third Department of Internal Medicine, Faculty of Medicine, Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Japan

Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for the mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients' genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene.

A decreased concentration of dehydroepiandrosterone sulfate (DHEA-S) in patients with Alzheimer's disease (AD) has been reported but is still controversial. In the present study, serum concentrations of DHEA and DHEA-S were determined in 19 patients with AD, 21 patients with cerebrovascular dementia (CVD) and 45 age- and gender matched elderly control individuals from the Japanese community at large. Serum concentration of DHEA among controls, patients with AD and patients with CVD did not significantly differ from one another. However, patients with AD and patients with CVD were found to have lower concentration of serum DHEA-S and a lower DHEA-S/DHEA ration compared to normal control individuals. No significant difference was observed in the concentration of serum DHEA-S or the DHEA-S/DHEA ratio between patients with AD and those with CVD. These results suggest that reduced concentrations of serum DHEA-S may not be unique to AD, but instead reflect a common phenomenon in dementing diseases. However, since serum concentration of DHEA in these patients remained unchanged, the significance of DHEA in dementia remains unclear.

Although antiobesity effect of dehydroepiandrosterone (DHEA) has been reported in rats, it remains unclear whether the effect is brought about by itself or mediated by sex steroids converted from DHEA in gonads. In the present study, to clarify this point, the effect of DHEA on growth in obese Zucker male rats was reevaluated under two conditions: with or without castration. Castration did not affect the pattern of growth curve of obese Zucker male rats. Three-months treatment of castrated Zucker rats with 0.3% DHEA in the diet resulted in dramatic decrease of body weight gain in comparison to DHEA-untreated and castrated rats. The degree of antiobesity effect of DHEA in castrated rats was almost same as that observed in non-castrated rats. These results suggest that DHEA exerted its antiobesity effect by itself rather than through conversion to testosterone in testis.

Mutation of the insulin receptor gene can compromise the ability of the receptor to mediate insulin action. A homozygous point mutation that results in the substitution of histidine for arginine 252 in the insulin receptor alpha-subunit has now been identified by polymerase chain reaction and single stranded conformational polymorphism analysis in a 20-yr-old Japanese woman with type A syndrome and severe insulin resistance. The proband's consanguineous parents (diabetic mother and normal father) and her sister (impaired glucose tolerance), each of whom showed an exaggerated insulin response to an oral glucose load, were heterozygous for this mutation. Her brother showed a normal insulin response and lacked the mutation, as did 50 healthy Japanese control subjects. The chronic sc administration of insulin-like growth factor I (IGF-I) improved the patient's hyperglycemia and corrected certain metabolic abnormalities over a 9-month period, even though the binding of 125I-labeled IGF-I to her cultured fibroblasts was decreased by 40% relative to that to cells from healthy controls. Studies of the binding of 125I-labeled insulin to the proband's cultured fibroblasts, to COS-I cells transfected with complementary DNA encoding the mutant insulin receptor, and to partially purified mutant receptors revealed that the Arg252--His mutation decreased both cell surface expression and the affinity for insulin for the receptor. These observations suggest that the homozygous Arg252--His mutation is responsible for the type A insulin resistance of the proband, whereas in the heterozygous state, the mutation results in mild insulin resistance indistinguishable from that observed in noninsulin-dependent diabetes mellitus.

We investigated the genetic defects in two patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Their clinical manifestations including corneal opacities, anemia, proteinuria, and hypoalphalipoproteinemia were identical for familial LCAT deficiency. Their LCAT activities and the cholesterol esterification rate (CER) were nearly zero, and their LCAT masses were below 10% of normal control values. Sequence analysis of the amplified DNA of case 1 revealed one base deletion of G at base 873 (first position of Val264) in exon 6, leading to a premature termination by frameshift. Sequence analysis of amplified DNA of case 2 revealed a single G to A converting Gly (GGT) to Ser (AGT) substitution at residue 344. When COS-1 cells were transfected with these mutants, LCAT activity in the medium was nearly zero, and the LCAT mass was undetectable (< 0.01 microgram/ml). In contrast, LCAT activity in the medium of COS-1 cells, transfected with wild-type LCAT, was 1.7 nmol/h per ml and the LCAT mass was 0.09 micrograms/ml. The LCAT mass in the cell lysates of the mutants was less than 12% of control for case 1 and 18% of control for case 2. Northern blot analysis of the mRNA of COS-1 cells transfected with the mutants showed the same amounts of LCAT mRNA as compared with wild-type LCAT. Biosynthesis of mutant LCATs was analyzed by pulse-chase and immunocytochemistry in transfected baby hamster kidney cells. SDS-PAGE/fluorography demonstrated that wild-type LCAT was synthesized as a high-mannose type of 56 kDa, which was very slowly converted to a mature form of 67 kDa and was secreted into the media. In contrast to the wild-type LCAT, the mutant precursors were not processed into the mature form but slowly degraded along with chase times. On steady and continuous labeling in the case of wild-type LCAT, the mature 67 kDa form was observed in both the cell lysate and media, whereas no mature form was detected in the cell lysates and media which were transfected mutant LCATs. These data suggest that the mutant LCATs are actually synthesized in an amount comparable to that of wild-type, but they are slowly degraded without being processed into the mature form. The immunocytochemistry revealed that mutant LCATs were mainly retained in the endoplasmic reticulum. These data suggest that these two mutations may disrupt the mutant LCATs' transport from the endoplasmic reticulum into Golgi apparatus, resulting in LCAT deficiency.

Aryl hydrocarbon receptor interacting protein (AIP) is thought to be a tumor suppressor gene, as indicated by a mutational analysis of pituitary somatotroph adenomas. However, the physiological significance of AIP inactivation in somatotroph cells remains unclear. Using CRISPR/Cas9, we identified a GH3 cell clone (termed GH3-FTY) in which Aip was genetically disrupted, and subsequently investigated its character with respect to growth hormone (Gh) synthesis and proliferation. Compared with GH3, GH3-FTY cells showed remarkably increased Gh production and a slight increase in cell proliferation. Gh-induced Stat3 phosphorylation is known to be a mechanism of Gh oversecretion in GH3. Interestingly, phosphorylated-Stat3 expression in GH3-FTY cells was increased more compared with GH3 cells, suggesting a stronger drive for this mechanism in GH3-FTY. The phenotypes of GH3-FTY concerning Gh overproduction, cell proliferation, and increased Stat3 phosphorylation were significantly reversed by the exogenous expression of Aip. GH3-FTY cells were less sensitive to somatostatin than GH3 cells in the suppression of cell proliferation, which might be associated with the reduced expression of somatostatin receptor type 2. GH3-FTY xenografts in BALB/c nude mice (GH3-FTY mice) formed more mitotic somatotroph tumors than GH3 xenografts (GH3 mice), as also evidenced by increased Ki67 scores. GH3-FTY mice were also much larger and had significantly higher plasma Gh levels than GH3 mice. Furthermore, GH3-FTY mice showed relative insulin resistance compared with GH3 mice. In conclusion, we established a somatotroph cell line, GH3-FTY, which possessed prominent Gh secretion and mitotic features associated with the disruption of Aip.

To clarify the possible action of adrenal androgen on bone cell, the existence, characteristics and regulation of aromatase in human osteoblast-like osteosarcoma cells (HOS) and primary cultured osteoblast-like cells from normal human bones (HO) were examined in this study. Significant positive correlation between bone mineral density (BMD) and serum dehydroepiandrosterone sulfate (DHEA-S) was found in 120 postmenopausal women (51-99 years old) but no correlation was seen between BMD and serum estradiol (E2). In subset analysis, strongly positive correlation of serum DHEA-S and estrone (E1) with BMD was observed in postmenopausal women aged less than 69 years old. Administration of DHEA to ovariectomized rat significantly increased BMD and decreased relative osteoid volume in femur. These in vivo findings strongly suggested that serum adrenal androgen may be converted to estrogen in peripheral organ, especially, osteoblast and be important steroids to maintain BMD. [3H]DHEA was converted to [3H]androstenedione and [3H]androstenedione to [3H]estrone in primary cultured human osteoblast. Osteoblast-like cells showed aromatase activity, and an apparent Km and the Vmax were 4.74 +/- 0.78 nM (mean +/- SD, n = 3) and 0.83 +/- 0.79 fmol/mg protein/h for HOS, and 4.6 +/- 2.9 nM and 279 +/- 299 fmol/mg protein/h (mean +/- SD, n = 19) for HO, respectively. The aromatase activity was significantly increased by dexamethasone in a dose-dependent manner. Reverse transcription-polymerase chain reaction analysis revealed that dexamethasone increased the transcript of P450AROM gene. Osteoblast-specific promoters were also determined. Dexamethasone and 1 alpha,25-dihydroxyvitamin D3 synergistically enhanced aromatase activity and P450AROM mRNA expression. These results demonstrate that adrenal androgen, DHEA, is converted to E1 in osteoblast by P450AROM which is positively regulated by glucocorticoid and 1 alpha,25-dihydroxyvitamin D3 and important to maintain BMD in the 6 to 7th decade, after menopause.

Nishi Y, Haji M, Takayanagi R, Yanase T, Ikuyama S, Nawata H. In vivo and in vitro evidence for the production of inhibin-like immunoreactivity in human adrenocortical adenomas and normal adrenal glands: relatively high secretion from adenomas manifesting Cushing's syndrome. Eur J Endocrinol 1995;132:292–9. ISSN 0804–4643 To clarify whether adrenal gland secretes inhibin in vivo in physiological or pathological conditions, we measured the levels of inhibin-like immunoreactivity (inhibin-LI) in adrenal veins (A-vein) and compared them with those in inferior vena cava (IVC) using blood samples obtained at catheterization of adrenal vein in the patients with adrenal adenoma manifesting Cushing's syndrome (Cs), aldosterone-producing adenoma, clinically non-functioning adenoma and normal adrenal gland. The tumor sides of A-veins in the patients with adenomas and also both sides of A-veins in subjects with normal adrenal glands showed significantly higher contents of inhibin-LI than their IVC. When the inhibin-LI secretion rate from adrenal gland was estimated by the difference between the levels of A-vein (tumor side) and IVC, Cs adenomas showed the highest secretion rate. Similarly, the tissue inhibin-LI content and the basal secretion rate of inhibit-LI from primary cultured cells were the highest in Cs adenomas. These findings indicated that normal adrenal glands and adrenocortical adenomas produced and secreted inhibin-LI into the general circulation in vivo and Cs adenomas have relatively high capacity for secreting inhibin-LI, and the present study provided the first in vivo evidence for adrenal inhibin-LI production in pathological conditions Yoshihiro Nishi, Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812, Japan

We present a 32-year-old-woman with Cushing's disease and sarcoidosis. She had been diagnosed as having Cushing's disease in 1985. She had transsphenoidal surgery followed by pituitary radiation. Subsequently her plasma cortisol level gradually decreased to normal range. In 1991, she manifested bilateral hilar lymphadenopathy on chest X-ray, erythema nodosum, subcutaneous nodules and granulomatous uveitis. From the histological findings of the skin lesions, the patient was diagnosed as having sarcoidosis. Interestingly, an inverse relationship between the disease activity of sarcoidosis and the level of serum cortisol in Cushing's disease was observed in the clinical course of this patient. Co-existence of Cushing's disease and sarcoidosis is very rare. Since the therapeutic significance of steroids in sarcoidosis has been well established, this case provides insight to the relationship between sarcoidosis and steroids.

The present study was designed to clarify the transcriptional regulation of the human type I angiotensin II receptor (ATI) gene and its pathophysiological roles in steroidogenesis by adrenal tumors. A cDNA encoding type I angiotensin II receptor (ATI) was isolated from a human liver cDNA library encoding a protein of 359 amino acids with seven transmembrane segments. It is very likely that human has only one type of ATI receptor, in contrast with rodents. A genomic clone containing 217 bp of exon 1 and 2558 bp of the 5′-flanking region of human ATI receptor gene was isolated. Its proximal promoter region contained putative TATA and GC boxes, CRE and API sites. Aldosterone-producing adenoma (APA) contained significantly higher levels of mRNA for ATI and ACTH receptors than normal tissues adjacent to APA. There were no mutations within the cytoplasmic third loops of ATI and ACTH receptors in APAs examined. APA showed increased expression of the mRNA for P450c11 and decreased expression of the mRNA for P450c17. These results suggest that renin-independent overproduction and clinically observed ACTH-dependent production of aldosterone in APAs may result from the enhanced transcription of P450c11 and ACTH receptor genes. The mechanism of the discordantly increased expression of ATI receptor in APA remains to be clarified.

Background Although incretin therapy is clinically available in patients with type 2 diabetes undergoing hemodialysis, no study has yet examined whether incretin therapy is capable of maintaining glycemic control in this group of patients when switched from insulin therapy. In this study, we examined the efficacy of incretin therapy in patients with insulin-treated type 2 diabetes undergoing hemodialysis. Methods Ten type 2 diabetic patients undergoing hemodialysis received daily 0.3 mg liraglutide, 50 mg vildagliptin, and 6.25 mg alogliptin switched from insulin therapy on both the day of hemodialysis and the non-hemodialysis day. Blood glucose level was monitored by continuous glucose monitoring. After blood glucose control by insulin, patients were treated with three types of incretin therapy in a randomized crossover manner, with continuous glucose monitoring performed for each treatment. Results During treatment with incretin therapies, severe hyperglycemia and ketosis were not observed in any patients. Maximum blood glucose and mean blood glucose on the day of hemodialysis were significantly lower after treatment with liraglutide compared with treatment with alogliptin (p < 0.05), but not with vildagliptin. The standard deviation value, a marker of glucose fluctuation, on the non-hemodialysis day was significantly lower after treatment with liraglutide compared with treatment with insulin and alogliptin (p < 0.05), but not with vildagliptin. Furthermore, the duration of hyperglycemia was significantly shorter after treatment with liraglutide on both the hemodialysis and non-hemodialysis days compared with treatment with alogliptin (p < 0.05), but not with vildagliptin. Conclusions The data presented here suggest that patients with type 2 diabetes undergoing hemodialysis and insulin therapy could be treated with incretin therapy in some cases.

The first framework is for analysis phase, in which we find out how to utilize business data through trial and error. The proposed framework stores tree-structured data using vertical partitioning technique, and uses Hadoop MapReduce for distributed computing. These methods enable to reduce disk I/O load, and to avoid computationally-intensive processing, such as grouping and combining of records.

We herein report a case of severe postsurgical hypocalcemia associated with primary hyperparathyroidism (pHPT), Graves' disease (GD) and acromegaly (AC). A 54-year-old woman was referred to our clinic for treatment of pHPT and GD. She also had active AC and was clinically diagnosed as multiple endocrine neoplasm type 1 because of pHPT and AC. Two enlarged parathyroid glands were detected by preoperative examinations. We performed total parathyroidectomy and thyroidectomy. After the operation, she showed severe hypocalcemia induced by postsurgical hypoparathyroidism and hungry bone syndrome. This is a rare case of postsurgical severe hypocalcemia associated with pHPT, GD and AC.