Hirotaka Shibata

Adrenocortical Zonation in Humans under Normal and Pathological Conditions

CONTEXT Aldosterone synthase (CYP11B2) and steroid 11 beta-hydroxylase (CYP11B1) catalyze the terminal steps for aldosterone and cortisol syntheses, respectively, thereby determining the functional differentiation of human adrenocortical cells. Little is known, however, about how the cells expressing the enzymes are actually distributed in the adrenals under normal and pathological conditions. OBJECTIVE The objective of the study was to determine the localization of CYP11B2 and -B1 in human adrenal specimens by using developed antibodies capable of distinguishing the two enzymes from each other. RESULTS Under normal conditions, CYP11B2 was sporadically detected in the zona glomerulosa, whereas CYP11B1 was entirely detected in the zonae fasciculata-reticularis. Adrenocortical cells lacking both enzymes were observed in the outer cortical regions. In addition to conventional zonation, we found a variegated zonation consisting of a subcapsular cell cluster expressing CYP11B2, which we termed aldosterone-producing cell cluster, and a CYP11B1-expressing area. Aldosterone-producing adenomas differed in cell populations expressing CYP11B2 from one another, whereas CYP11B1-expressing and double-negative cells were also intermingled. Adenomas from patients with Cushings syndrome expressed CYP11B1 entirely but not CYP11B2, resulting in atrophic nontumor glands. The nontumor portions of both types of adenomas bore frequently one or more aldosterone-producing cell clusters, which sustained CYP11B2 expression markedly under the conditions of the suppressed renin-angiotensin system. CONCLUSION Immunohistochemistry of the human normal adrenal cortex for CYP11B2 and CYP11B1 revealed a variegated zonation with cell clusters constitutively expressing CYP11B2. This technique may provide a pathological confirmatory diagnosis of adrenocortical adenomas.

Effect of KCNJ5 Mutations on Gene Expression in Aldosterone-Producing Adenomas and Adrenocortical Cells

CONTEXT Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA). OBJECTIVE The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells. METHODS A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA. RESULTS We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P < 0.05). APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P < 0.05). Real-time PCR confirmed increases in CYP11B2 and its transcriptional regulator, NR4A2. CONCLUSIONS KCNJ5 mutations are prevalent in APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production.

G-protein-coupled receptors in aldosterone-producing adenomas: a potential cause of hyperaldosteronism

The source of aldosterone in 30-40% of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined the expression of G-protein-coupled receptors (GPCRs) in APA and demonstrated that when compared with normal adrenals, there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs; n = 13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least 1 out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than threefold when compared with normal adrenals, suggesting a general increase in expression when compared with normal adrenal glands. Four GPCR transcripts exhibited a > 15-fold increase of expression in one or more of the APA samples when compared with normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be LH receptor, serotonin receptor 4, GnRH receptor, glutamate receptor metabotropic 3, endothelin receptor type B-like protein, and ACTH receptor. There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study.

Coactivation of the N-terminal Transactivation of Mineralocorticoid Receptor by Ubc9

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.

Fulminant type 1 diabetes mellitus with anti‐programmed cell death‐1 therapy

Anti‐programmed cell death‐1 (PD‐1) antibodies are regarded as a risk factor for insulin‐dependent diabetes mellitus as a side‐effect. While a small number of cases have been reported, evidence remains limited. This is the first report of an Asian patient developing insulin‐dependent diabetes during anti‐PD‐1 therapy. A 55‐year‐old euglycemic woman receiving nivolumab for malignant melanoma showed abrupt onset of ketonuria, and elevated levels of plasma glucose (580 mg/dL) and hemoglobin A1c (7.0%). Over the next 2 weeks, serum C‐peptide levels fell below the limit of detection. Islet autoantibodies were negative, and the patient showed a human leukocyte antigen haplotype associated with type 1 diabetes. Anti‐PD‐1 therapy can cause rapid onset of insulin‐dependent diabetes, possibly because of inappropriate activation of T cells. Human leukocyte antigen haplotypes might be related to the onset of this disease. Physicians should be aware of this serious adverse event and carry out routine blood glucose testing during anti‐PD‐1 therapy.

Increased expression of vascular angiotensin II type 1A receptor gene in glucocorticoid-induced hypertension.

Objectives We recently demonstrated that glucocorticoid increases the number of angiotensin II type 1 (AT1) receptors and their gene expression in cultured vascular smooth muscle cells. To clarify whether this mechanism participates in glucocorticoid-induced hypertension, we investigated the effect of dexamethasone on the modulation of vascular AT1 receptor messenger RNA (mRNA) expression in rats. Methods The effects were studied of administering dexamethasone orally to rats at a dose of 0.1 mg/day on the modulation of the expression of vascular AT1 receptor mRNA and the blood pressure. The effects of enalapril maleate, an angiotensin converting enzyme inhibitor, were also evaluated. Results The dexamethasone-treated rats showed a rise in systolic blood pressure beginning on day 3. On day 5 the blood pressure rose significantly. Before dexamethasone administration the vascular AT1 receptor mRNA was difficult to detect by Northern blot analysis. However, the level of vascular AT1A receptor mRNA began to increase on day 2, when the blood pressure was not yet elevated, and increased further on day 5. The concurrent administration of dexamethasone and enalapril maleate attenuated the elevation of blood pressure. However, as in the dexamethasone-treated rats, the level of AT1A receptor mRNA began to increase on day 2 and increased further on day 5. In rats treated with enalapril maleate alone, the AT1 receptor mRNA was difficult to detect during the experiment. Conclusion Glucocorticoid increased the amount of vascular A1 receptor mRNA and contributed to the elevation of blood pressure.

Effects of a nonnutritive sweetener on body adiposity and energy metabolism in mice with diet-induced obesity

OBJECTIVE Nonnutritive sweeteners (NNSs) have been studied in terms of their potential roles in type 2 diabetes, obesity, and related metabolic disorders. Several studies have suggested that NNSs have several specific effects on metabolism such as reduced postprandial hyperglycemia and insulin resistance. However, the detailed effects of NNSs on body adiposity and energy metabolism have not been fully elucidated. We investigated the effects of an NNS on energy metabolism in mice with diet-induced obesity (DIO). METHODS DIO mice were divided into NNS-administered (4% NNS in drinking water), sucrose-administered (33% sucrose in drinking water), and control (normal water) groups. After supplementation for 4 weeks, metabolic parameters, including uncoupling protein (UCP) levels and energy expenditure, were assessed. RESULTS Sucrose supplementation increased hyperglycemia, body adiposity, and body weight compared to the NNS-administered and control groups (P<0.05 for each). In addition, NNS supplementation decreased hyperglycemia compared to the sucrose-administered group (P<0.05). Interestingly, NNS supplementation increased body adiposity, which was accompanied by hyperinsulinemia, compared to controls (P<0.05 for each). NNS also increased leptin levels in white adipose tissue and triglyceride levels in tissues compared to controls (P<0.05 for each). Notably, compared to controls, NNS supplementation decreased the UCP1 level in brown adipose tissue and decreased O2 consumption in the dark phase. CONCLUSIONS NNSs may be good sugar substitutes for people with hyperglycemia, but appear to influence energy metabolism in DIO mice.

Chicken ovalbumin upstream promoter transcription factor II in human breast carcinoma: Possible regulator of lymphangiogenesis via vascular endothelial growth factor‐C expression

Chicken ovalbumin upstream promoter transcription factors (COUP‐TF) are orphan members of the nuclear receptor superfamily and consist of COUP‐TFI and COUP‐TFII. COUP‐TFI was reported to be overexpressed in human breast cancer and to promote estrogen‐independent transcriptional activity of estrogen receptor α. COUP‐TFII, however, has not been examined in the breast. Therefore, we carried out immunohistochemical analysis of COUP‐TFII in human breast cancer in order to clarify its biological and clinical significance. We immunolocalized COUP‐TFII in 119 human breast cancers and correlated the findings with various clinicopathological parameters. Fifty‐nine percent of the cases were immunohistochemically positive for COUP‐TFII. COUP‐TFII positivity was correlated with poor clinical outcome, and a statistically significant correlation was detected between COUP‐TFII and the following clinicopathological parameters: clinical stage, lymph node status, histological grade, and estrogen receptor α status. In addition, short interfering RNA‐mediated knockdown of COUP‐TFII in the breast carcinoma cell line MCF‐7 decreased the level of vascular endothelial growth factor‐C mRNA expression, which is a known inducer of lymphangiogenesis and lymph node metastasis. These results suggest that COUP‐TFII is involved in the development of advanced human breast cancer. (Cancer Sci 2009; 100: 639–645)

NF-YC Functions as a Corepressor of Agonist-bound Mineralocorticoid Receptor

The role of aldosterone has been implicated in the metabolic syndrome and cardiovascular diseases. The biological actions of aldosterone are mediated through mineralocorticoid receptor (MR). Nuclear receptor-mediated gene expression is regulated by dynamic and coordinated recruitment of coactivators and corepressors. To identify new coregulators of the MR, full-length MR was used as bait in yeast two-hybrid screening. We isolated NF-YC, one of the subunits of heterotrimeric transcription factor NF-Y. Specific interaction between MR and NF-YC was confirmed by yeast two-hybrid, mammalian two-hybrid, coimmunoprecipitation assays, and fluorescence subcellular imaging. Transient transfection experiments in COS-7 cells demonstrated that NF-YC repressed MR transactivation in a hormone-sensitive manner. Moreover, reduction of NF-YC protein levels by small interfering RNA potentiated hormonal activation of endogenous target genes in stably MR-expressing cells, indicating that NF-YC functions as an agonist-dependent MR corepressor. The corepressor function of NF-YC is selective for MR, because overexpression of NF-YC did not affect transcriptional activity mediated by androgen, progesterone, or glucocorticoid receptors. Chromatin immunoprecipitation experiments showed that endogenous MR and steroid receptor coactivator-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner, and endogenous NF-YC was sequentially recruited to the same element. Immunohistochemistry showed that endogenous MR and NF-YC were colocalized within the mouse kidney. Although aldosterone induces interaction of the N and C termini of MR, NF-YC inhibited the N/C interaction. These findings indicate that NF-YC functions as a new corepressor of agonist-bound MR via alteration of aldosterone-induced MR conformation.

Determinants for the development of hypertension in adolescents. A 6-year follow-up.

Objective This study was aimed to evaluate the determinants of elevated blood pressure (BP) in adolescents. Design and methods We retrospectively evaluated the BP and anthropometric data in 419 Japanese students (268 males and 151 females) during high school and university. Their annual health records were analysed for BP, heart rate, height and body weight between the ages of 15 and 21 years. Results The number of hypertensive students did not vary significantly during the 6 years. Concerning changes in BP categories according to the modified JNCVI classification between the ages of 15 to 21 years, 150 males kept a normal BP (keeping normal BP group); 39 males developed high BP (developing high BP group); 37 males kept high BP (keeping high BP group); and 42 males became normal BP (becoming normal BP group). The majority of females (n = 144, 95.4%) were included in the keeping normal BP group. In male students, both the keeping and becoming normal BP groups, especially the latter, showed a significant decrease in heart rate over the 6 years, while the other two groups showed no change. The height and body weight of each of the four groups showed a significant increase, but the body mass index (BMI) of the males in the becoming normal BP group did not increase over the 6 years. Body weight and BMI at the age of 15 years in the male keeping normal BP group were significantly below that of the other three groups; this difference persisted at the age of 21 years. Furthermore, male university students who showed a BP above ‘high–normal’ at the age of 21 years exhibited a significantly higher BP, heart rate, body weight and BMI than did the normotensives, when they were high school students. Stepwise regression analysis of the data showed that the best predictors of BP at the age of 21 years were the initial high school BP and BMI levels and changes in BMI and heart rate during the 6-year period for male students. Conclusion Results indicate that the BP and BMI during high school and the changes in BMI and heart rate from high school to university influenced the BP at the age of 21 years in male students. Data indicate that information on the prevention and management of hypertension including weight control should begin early, especially in male adolescents.