Shinobu Takayasu

SOX6 attenuates glucose stimulated insulin secretion by repressing PDX1 transcriptional activity and is down-regulated in hyperinsulinemic obese mice

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing β-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca2+ mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in β-cells may contribute to the β-cell adaptation in obesity-related insulin resistance.

Mutations in NFKB2 and potential genetic heterogeneity in patients with DAVID syndrome, having variable endocrine and immune deficiencies

BackgroundDAVID syndrome is a rare condition combining anterior pituitary hormone deficiency with common variable immunodeficiency. NFKB2 mutations have recently been identified in patients with ACTH and variable immunodeficiency. A similar mutation was previously found in Nfkb2 in the immunodeficient Lym1 mouse strain, but the effect of the mutation on endocrine function was not evaluated.MethodsWe ascertained six unrelated DAVID syndrome families. We performed whole exome and traditional Sanger sequencing to search for causal genes. Lym1 mice were examined for endocrine developmental anomalies.ResultsMutations in the NFKB2 gene were identified in three of our families through whole exome sequencing, and in a fourth by direct Sanger sequencing. De novo origin of the mutations could be demonstrated in three of the families. All mutations lie near the C-terminus of the protein-coding region, near signals required for processing of NFΚB2 protein by the alternative pathway. Two of the probands had anatomical pituitary anomalies, and one had growth and thyroid hormone as well as ACTH deficiency; these findings have not been previously reported. Two children of one of the probands carried the mutation and have to date exhibited only an immune phenotype. No mutations were found near the C-terminus of NFKB2 in the remaining two probands; whole exome sequencing has been performed for one of these. Lym1 mice, carrying a similar Nfkb2 C-terminal mutation, showed normal pituitary anatomy and expression of proopiomelanocortin (POMC).ConclusionsWe confirm previous findings that mutations near the C-terminus of NFKB2 cause combined endocrine and immunodeficiencies. De novo status of the mutations was confirmed in all cases for which both parents were available. The mutations are consistent with a dominant gain-of-function effect, generating an unprocessed NFKB2 super-repressor protein. We expand the potential phenotype of such NFKB2 mutations to include additional pituitary hormone deficiencies as well as anatomical pituitary anomalies. The lack of an observable endocrine phenotype in Lym1 mice suggests that the endocrine component of DAVID syndrome is either not due to a direct role of NFKB pathways on pituitary development, or else that human and mouse pituitary development differ in its requirements for NFKB pathway function.

Ghrelin stimulates corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) play a central role in regulating the stress response. In response to stress, CRF and AVP neurons in the hypothalamic paraventricular nucleus secrete the peptides to stimulate the release of adrenocorticotropic hormone from the anterior pituitary. Ghrelin, an endogenous ligand of the growth hormone-releasing peptide receptors (GHSR), has been shown to stimulate the release of CRF and AVP by rat hypothalamic explants. However, little is known about the ability of the ghrelin signaling pathways to activate the CRF and AVP genes in the hypothalamus. In the present study, we examined the direct effect of ghrelin on CRF and AVP gene expression in hypothalamic 4B cells, which show the characteristics of the hypothalamic parvocellular paraventricular nucleus neurons. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. Ghrelin stimulated the promoter activities and mRNA levels for both CRF and AVP. The involvement of a protein kinase pathway was examined using inhibitors. Protein kinase A and phospholipase C pathways were shown to be involved in ghrelin-induced increases in both CRF and AVP promoter activities. GHSR type 1a (GHSR1a) mRNA levels were also increased by ghrelin, and these ghrelin-induced levels were suppressed by a GHSR1a antagonist. Thus, ghrelin-dependent pathways are involved in the regulation of CRF and AVP gene expression in the hypothalamus: ghrelin, an orexigenic hormone, stimulates CRF, an anorexigenic/anxiogenic factor in the hypothalamus, resulting in hypothalamic–pituitary–adrenal axis activation to stimulate the release of glucocorticoids.

Altered pain perception in schizophrenia

In June, 2009, a 51-year-old woman presented with a 4-day history of abdominal swelling and fever, and a 2-week history of loss of appetite. There was no history of abdominal pain or nausea. She was febrile (38·4°C) and her abdomen was swollen but soft without spontaneous pain or tenderness. She had a history of schizophrenia since the age of 40 years which was well controlled with perospirone 12 mg/day and quetiapine 150 mg/day. At the age of 45 years, she was diagnosed with diabetes mellitus and had achieved good glycaemic control (HbA1c 6·5%) with biphasic insulin aspart 30 (6 U/day). There was no sign of obvious peripheral neuropathy. Laboratory test results showed normocytic anaemia (haemoglobin 95 g/L), leucocytosis (13·3×109/L) with neutrophilia (91·5%), high concentration of C-reactive protein (129 mg/L), and normal liver and kidney function. Urinalysis showed negative protein and no leucocytosis. Chest radiography showed normal lungs with no cardiac enlargement. Abdominal CT showed a large volume of partly encapsulated ascites (fi gure). The peritoneum in her pelvic cavity was thickened, and ascites in the right inferior abdominal cavity contained calcifi cation and air (fi gure). The aspirated ascitic fl uid had an unpleasant smell and was suppurated. Culture of the fl uid was positive for pyogenic agents, including Peptostreptococcus spp and Bacillus subtilis. Blood culture was negative. We diagnosed severe bacterial pan-peritonitis. Surprisingly, her general condition was stable and she did not complain of any abdominal pain. Abdominal drainage was done and antibiotics started (imipenem/ cilastatin 1·5 g/day), but her symptoms did not improve. Therefore, laparotomy was done on the seventh day of admission. On laparotomy, a large amount of yellowish, turbid, gel-like material fi lled the abdominal cavity with retention of pus in the pouch of Douglas. The appendix was necrotic and contained faecolith. The bacterial peritonitis was suspected to be caused by perforation associated with acute appendicitis. Postoperative course was uneventful, and the patient recovered. When last seen in October, 2009, the patient was well. Even though our patient had a purulent pan-peritonitis caused by a perforated appendix, she did not present with abdominal pain, tenderness, or guarding. Diminished pain sensitivity or loss of pain sensation in people with schizophrenia has previously been reported in cases of acute myocardial infarction and perforated gastrointestinal tract. Clinically, diminished pain sensitivity in schizophrenia has been linked to key features of the disorder, such as positive symptoms, aff ective fl attening, or attention defi cits. Disturbances in dopamine, serotonin, glutamate, and opioids have been proposed to account for hypoalgesia in schizophrenia. Hypoalgesia in schizophrenia has been reported throughout the acute phase of illnesses and in medicated stable or drug-free patients; neuroleptic drugs have been suggested to have minor analgesic eff ects. Diabetes occurs in people with schizophrenia two to four times more often than in the general population. Compared with people without diabetes, appendicitis in people with diabetes is more likely to result in perforation or other complications. Decreased pain sensation may result in aggravation of the condition and in a delay of diagnosis and treatment. Our case is an important reminder that people with schizophrenia do not always present with typical clinical features of acute abdominal disease pathology.

The Cables1 Gene in Glucocorticoid Regulation of Pituitary Corticotrope Growth and Cushing Disease

CONTEXT Cushing disease (CD) is due to pituitary corticotrope adenomas that produce unrestrained ACTH secretion and have lost the negative feedback exerted by glucocorticoids (GCs). GCs also restrain corticotrope proliferation, and the mechanisms of this inhibition are poorly understood. OBJECTIVE The aim of the study was to identify cell cycle regulatory genes that are regulated by GCs and the glucocorticoid receptor and to assess regulatory genes that have a rate-limiting action on corticotrope proliferation and may be disregulated in CD. DESIGN The mouse corticotrope tumor cells AtT-20 were used to identify GC-regulated genes that contribute to control of cell cycle progression. Surgery sections from patients with CD were used to assess expression of CABLES1 in corticotrope adenomas. METHODS Gene expression profiling, small interfering RNA knockdowns, cell cycle analyses, and genetic manipulations were performed in AtT-20 cells. Sequencing of chromatin immunoprecipitation for pituitary-restricted transcription factors and RNA polymerase II were used to identify regulatory elements and genes that bind GR and are direct transcriptional targets. A panel of previously well-characterized corticotrope adenomas was used to correlate expression of CABLES1 with that of other markers. RESULTS GCs altered expression of 3 positive and 3 negative regulators of cell cycle progression. Two Myc genes (L-Myc and N-Myc) and E2F2 are repressed by GCs, whereas genes for the negative regulators of the cell cycle, Gadd45β, Gadd45γ, and Cables1 are activated by GCs. Cables1 small interfering RNA knockdown strongly stimulates AtT-20 cell proliferation and antagonizes the growth inhibition produced by GCs. The Gadd45 and Cables1 genes have the hallmarks of direct GC targets. CABLES1 is expressed in normal human pituitary cells, but expression is lost in ∼55% of corticotrope adenomas, and this is strongly correlated with the loss of p27(Kip1) expression. CONCLUSIONS CABLES1 is a critical regulator of corticotrope proliferation that defines a pathway often inactivated in CD and links proliferation to GC resistance.

Involvement of Nuclear Factor-ĸB and Nurr-1 in Cytokine-Induced Transcription of Proopiomelanocortin Gene in AtT20 Corticotroph Cells

Objective: The precise mechanism whereby proinflammatory cytokines activate the hypothalamo-pituitary-adrenal axis is still unclear. We examined whether transcription factors nuclear factor (NF)-ĸB and Nurr-1 are involved in the cytokine-induced proopiomelanocortin (POMC) gene expression. Methods: The mouse corticotropinoma cell line AtT20 was treated with tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). Real-time PCR, luciferase assay and Western blotting were conducted to assess the gene expression, promoter activity and protein expression in various conditions. Results: Intrinsic expression of NF-ĸB was confirmed by RT-PCR. An active component of NF-ĸB (p65) was upregulated in the nuclear fraction by both TNF-α and IL-1β treatment in a dose- and time-related manner. These cytokines potently stimulated the promoter activity of NF-ĸB and Nurr-1. We also found rapid upregulation of the Nurr-1 gene and protein after treatment with these cytokines. Cotreatment of the cells with either of the cytokines and corticotropin-releasing hormone resulted in additive effects. Cytokine-induced Nurr-1 transcription and Nurr-1 transcription induced by overexpression of NF-ĸB were both blunted by mutagenesis within the NF-ĸB responsive element, which implies that Nurr-1 upregulation specifically requires NF-ĸB binding to its own DNA-binding site. Proinflammatory cytokines exert positive effects on POMC gene expression, which were inhibited by pretreatment with a specific NF-ĸB inhibitor. Conclusion: These results together imply that Nurr-1 expression is a connecting point between neuroendocrine and immune systems in mediating cytokine-induced POMC gene expression.

Cytokines Induce NF-ĸB, Nurr1 and Corticotropin-Releasing Factor Gene Transcription in Hypothalamic 4B Cells

Objective: In the hypothalamus, corticotropin-releasing factor (CRF) plays a central role in regulating stress responses. Cytokines are important mediators of the interaction between the neuroendocrine and immune systems, and are implicated in the regulation of CRF expression. Following inflammatory challenges, interleukin (IL)-1 or IL-6 stimulates the hypothalamic-pituitary-adrenal axis. CRF promoter contains multiple nuclear factor (NF)-ĸB and Nurr1 binding sites. In the present study, we determined the ability of the signaling pathways to activate the CRF gene in the hypothalamic paraventricular nucleus following inflammatory challenge. Methods: Cytokine-induced changes in CRF gene expression were examined in the hypothalamic system. Luciferase assay and Western blotting were performed to assess transcriptional activity and the nuclear translocation of transcriptional factors. Results: IL-1β, IL-6 and tumor necrosis factor (TNF)-α stimulated the nuclear expression levels of NF-ĸB, NF-ĸB-dependent Nurr1 and c-Fos proteins. Direct stimulatory effects of TNF-α and IL-1β, in addition to IL-6, were found on the transcriptional activity of the CRF gene in hypothalamic 4B cells. Conclusion: These cytokines are involved in the regulation of CRF gene activity in hypothalamic cells.

Involvement of regulatory elements on corticotropin-releasing factor gene promoter in hypothalamic 4B cells.

Introduction: Corticotropin-releasing factor (CRF) plays a central role in controlling the hypothalamic-pituitary-adrenal (HPA) axis during stressful periods. CRF is synthesized and secreted in the hypothalamic paraventricular nucleus (PVN) in response to stress, and stimulates ACTH in the pituitary corticotrophs. ACTH stimulates the release of glucocorticoids from the adrenal glands, and glucocorticoids sequentially inhibit hypothalamic PVN production of CRF and pituitary production of ACTH. The effects of glucocorticoids on CRF gene regulation, however, are possibly tissue-specific since glucocorticoids stimulate CRF gene expression in the placenta and the bed nucleus of the stria terminalis, while they inhibit it in the hypothalamus. Methods and results: In a hypothalamic cell line, 4B, we found that forskolin-stimulated CRF gene transcription was mediated by a functional cAMP-response element (CRE), which included −220 to −233 bp on the CRF 5′-promoter region. Protein kinase A, protein kinase C, and p38 mitogen-activated protein kinase pathways contributed to forskolin-induced transcriptional activity of CRF in hypothalamic 4B cells. Glucocorticoid-dependent repression of cAMP-stimulated transcriptional activity of CRF was localized to promoter sequences between −278 and −233 bp, which included a glucocorticoid regulatory element and a serum response element. Conclusion: Taken together, these findings indicate that the regulatory elements, including CRE, negative glucocorticoid regulatory element, and a serum response element on the promoter, contribute to the regulation of CRF gene transcription in hypothalamic 4B cells.

Association between Higher Serum Cortisol Levels and Decreased Insulin Secretion in a General Population

Glucocorticoids (GCs) are well known to induce insulin resistance. However, the effect of GCs on insulin secretion has not been well characterized under physiological conditions in human. We here evaluated the effect of GCs on insulin secretion/ß-cell function precisely in a physiological condition. A population-based study of 1,071 Japanese individuals enrolled in the 2014 Iwaki study (390 men, 681 women; aged 54.1 ± 15.1 years), those excluded individuals taking medication for diabetes or steroid treatment, were enrolled in the present study. Association between serum cortisol levels and insulin resistance/secretion assessed by homeostasis model assessment using fasting blood glucose and insulin levels (HOMA-R and HOMA-ß, respectively) were examined. Univariate linear regression analyses showed correlation of serum cortisol levels with HOMA-ß (ß = -0.134, p <0.001) but not with HOMA-R (ß = 0.042, p = 0.172). Adjustments for age, gender, and the multiple clinical characteristics correlated with HOMA indices showed similar results (HOMA-ß: ß = -0.062, p = 0.025; HOMA-R: ß = -0.023, p = 0.394). The correlation between serum cortisol levels and HOMA-ß remained significant after adjustment for HOMA- R (ß = -0.057, p = 0.034). When subjects were tertiled based on serum cortisol levels, the highest tertile was at greater risk of decreased insulin secretion (defined as lower one third of HOMA-ß (≤70)) than the lowest tertile, after adjustment for multiple factors including HOMA- R (odds ratio 1.26, 95% confidence interval 1.03–1.54). In conclusion, higher serum cortisol levels are significantly associated with decreased insulin secretion in the physiological cortisol range in a Japanese population.

A Case of Adrenocortical Oncocytoma Occurring with Aldosteronoma

A 48-yr-old woman was referred to our hospital for evaluation of a left adrenal mass. An abdominal computed tomography scan demonstrated a 5.4 3.7-cm tumor in the left adrenal gland (Fig. 1, A and B). She had untreated hypertension on admission and no Cushingoid features. When in a seated position, the patient’s urinary aldosterone excretion rate was 19.4 g/d, plasma aldosterone concentration (PAC) was 20.8 ng/dl, and plasma renin activity (PRA) was 0.8 ng/ml h mid morning. Her plasma aldosterone to renin ratio was 26.0. She was diagnosed with normokalemic (serum potassium, 3.8 mmol/ liter) primary aldosteronism after captopril challenge (PAC, 15.2 ng/dl; PRA, 0.4 ng/ml h at 2 h) and furosemide-plus-upright tests (PAC, 65.4 ng/dl; PRA, 0.3 ng/ ml h at 2 h) (1). 18F-Fluorodeoxyglucose (FDG) positron emission tomography demonstrated a marked uptake of FDG in the left adrenal mass (Fig. 1, C and D). The mass was therefore clinically diagnosed as an aldosterone-producing adrenocortical carcinoma (2). After left adrenalectomy, the patient’s hypertension improved with normalizationofaldosterone to renin ratio.Lightmicroscopic examination revealed the cells within the main tumor body to have abundant eosinophilic cytoplasm and three positive scores in Weiss criteria, namely, nuclear atypia, architecture, and an eosinophilic cytoplasm (Fig. 2A). The eosinophilic tumor cells were positive for steroidogenic factor-1, but negative for steroidogenic enzymes [3 -hydroxysteroid dehydrogenase (3BHSD), P450c21, P450c17, and dehydroepiandrosterone sulfotransferase]. This main tumor body was therefore diagnosed as a true nonfunctioning adrenocortical tumor, an “oncocytoma” (3). A wellcircumscribed localized mass of clear cortical cells demonstrating marked P450 side-chain cleavage enzyme, 3BHSD and P450c21, and weak P450c17 immunoreactivity was detected around one margin of the oncocytoma (Fig. 2, B–E).Theattachedzonaglomerulosawashyperplasticbut negative for 3BHSD, evidence pointing toward a nonfunctioning albeit hyperplastic response in the neighboring normal glomerulosa. This part was therefore histopathologically diagnosed as an aldosteronoma. Although it could be one tumor with heterogeneity, this is the first