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Supplementary Data from Transcriptional Activation of the Mixed Lineage Leukemia–p27Kip1 Pathway by a Somatostatin Analogue

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of hepatic triglycerides (TG) and hyperglycemia arising due to persistent insulin resistance, and is profoundly linked to obesity. However, there is currently no established treatment for NAFLD in obese human subjects. We previously isolated Helz2, the expression of which was upregulated in human and mouse NAFLD, and its deletion activated the hepatic expression of functional leptin receptor long form (Leprb) and suppressed NAFLD development and body weight (BW) gain in obese mice. A high-throughput assay of small-molecule drugs revealed that guanabenz acetate (Ga), originally used to treat hypertension, possesses a high affinity constant against HELZ2, and its administration activates LEPRB expression in HepG2 cells in vitro. The chronic oral administration of Ga shows the selective leptin sensitization in the liver via upregulation of hepatic Leprb expression, which affects expression of genes involved in lipogenesis and fatty acid β-oxidation and diminishes hepatocyte hypertrophy with droplets enriched in TG in high-fat diet-induced obese mice. This activity significantly improves insulin resistance to decrease hyperglycemia and hepatocyte and adipocyte weights, resulting in BW reduction without reducing food intake. Regarding drug repositioning, Ga has the potential to effectively treat NAFLD and hyperglycemia in obese patients.

A 39-year-old woman was admitted to our hospital with symptoms of general fatigue, nausea, and vomiting that appeared three months after she stopped seven years of medroxyprogesterone acetate (MPA) medication for endometrial stromal sarcoma. Laboratory tests demonstrated moderate hypercalcemia. Several tests demonstrated that she was suffering from adrenal insufficiency. Glucocorticoid supplementation decreased her calcium level to a normal range, indicating that hypercalcemia was induced by adrenal insufficiency. It was suggested that she was suffering from MPA-induced adrenal insufficiency, but hypocortisolemia was being compensated by a high dose of MPA; hypocortisolemia and hypercalcemia then became evident after MPA treatment was discontinued.

Ewing's sarcoma usually arises in the bones of children and adolescents. We herein report a 74-year-old man with Ewing's sarcoma in the adrenal gland. The diagnosis was confirmed by a genetic test, pathological studies, and several imaging studies. He already had multiple liver metastases when he was transferred to our hospital and died on the 37th day. The diagnosis was further confirmed by autopsy studies. Adrenal Ewing's sarcoma is very rare, and our patient was older than other reported cases. Ewing's sarcoma should be considered even in elderly patients with adrenal tumors.

Somatic mutations in KCNJ5 gene have been identified in patients with adrenal aldosterone-producing adenomas (APAs). We previously reported that Japanese patients with APAs had distinct characteristics from patients in Western countries; i.e. they had a high frequency of KCNJ5 mutations and exhibited a frequent association with cortisol co-secretion. Therefore, APAs among Japanese patients may have different features from those in Western countries. We added recent cases, examined 47 cases (43% male) of APAs, including clinicopathological features, KCNJ5 mutations, and the mRNA levels of several steroidogenic enzymes, and compared the results obtained to those reported in other countries. While the prevalence of KCNJ5 mutations is approximately 40% in Western countries, 37 APA cases (78.7%) showed mutations: 26 with p.G151R and 11 with p.L168R. Although a significant gender difference has been reported in the frequency of KCNJ5 mutations in Europe, we did not find any gender difference. However, the phenotypes of Japanese patients with mutations were similar to those of patients in Western countries; patients were younger and had higher plasma aldosterone levels, lower potassium levels, and higher diastolic blood pressure. Reflecting these phenotypes, APAs with mutations had higher CYP11B2 mRNA levels. However, in contrast to APAs in Western countries, Japanese APAs with mutations showed lower CYP11B1, CYP17A1, and CYP11A1 mRNA levels. These findings demonstrated that Japanese APA patients may have distinct features including a higher prevalence of KCNJ5 mutations, no gender difference in the frequency of these mutations, and characteristics similar to the zona glomerulosa.

Context Congenital isolated TSH deficiency (i-TSHD) is a rare form of congenital hypothyroidism. Five genes (IGSF1, IRS4, TBL1X, TRHR, and TSHB) responsible for the disease have been identified, although their relative frequencies and hypothalamic/pituitary unit phenotypes have remained to be clarified. Objectives To define the relative frequencies and hypothalamic/pituitary unit phenotypes of congenital i-TSHD resulting from single gene mutations. Patients and Methods Thirteen Japanese patients (11 boys and 2 girls) with congenital i-TSHD were enrolled. IGSF1, IRS4, TBL1X, TRHR, and TSHB were sequenced. For a TBL1X mutation (p.Asn382del), its pathogenicity was verified in vitro. For a literature review, published clinical data derived from 74 patients with congenital i-TSHD resulting from single-gene mutations were retrieved and analyzed. Results Genetic screening of the 13 study subjects revealed six mutation-carrying patients (46%), including five hemizygous IGSF1 mutation carriers and one hemizygous TBL1X mutation carrier. Among the six mutation carriers, one had intellectual disability and the other one had obesity, but the remaining four did not show nonendocrine phenotypes. Loss of function of the TBL1X mutation (p.Asn382del) was confirmed in vitro. The literature review demonstrated etiology-specific relationship between serum prolactin (PRL) levels and TRH-stimulated TSH levels with some degree of overlap. Conclusions The mutation screening study covering the five causative genes of congenital i-TSHD was performed, showing that the IGSF1 defect was the leading genetic cause of the disease. Assessing relationships between serum PRL levels and TRH-stimulated TSH levels would contribute to predict the etiologies of congenital i-TSHD.

Intravascular large B-cell lymphoma (IVLBCL) is a rare subtype of diffuse large B-cell lymphoma. There have been only a limited number of reports regarding pituitary dysfunction associated with IVLBCL. We present a 71-year-old woman with hypopituitarism without any hypothalamic/pituitary abnormalities as assessed by magnetic resonance imaging. She presented with edema, abducens palsy, and elevated levels of lactate dehydrogenase and soluble interleukin-2 receptor. Provocative testing showed that the peaks of luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone and adrenocorticotropic hormone were evoked to normal levels by simultaneous administration of luteinizing hormone-releasing hormone, thyrotropin-releasing hormone and corticotropin-releasing hormone, but the responses of these four pituitary hormones showed a delayed pattern. She was diagnosed with IVLBCL with cerebrospinal invasion by pathological findings of the bone marrow, skin, and cerebrospinal fluid. She achieved hematological remission after immunochemotherapy. Pituitary function was also restored without hormonal replacement, and the improvement of the pituitary function was confirmed by dynamic testing. We reviewed the literature with respect to hypopituitarism associated with IVLBCL. There were less than 20 case reports and most of the patients died. Endocrinological course was described in only two cases, and both of them required hormonal supplementation. To our knowledge, this is the first case of hypopituitarism induced by IVLBCL that was successfully managed by immunochemotherapy alone. This case suggests that early diagnosis and treatment of IVLBCL might improve anterior pituitary function and enable patients to avoid hormone replacement therapy.

Mutations in GNAS, which encodes Gsα, have been documented in detail, particularly in human pituitary GH-secreting adenomas. Mutations have also recently been reported in adrenal cortisol-producing adenomas (CPAs), in addition to those in the PRKACA gene. However, mutations have not yet been examined in aldosterone-producing adenomas (APAs). Therefore, we herein investigated mutations in the GNAS gene in APAs. Two of the 15 (13%) CPAs with overt Cushing's syndrome and one of the 9 (11%) CPAs with subclinical Cushing's syndrome examined had the somatic mutations, p.R201S and p.R201C in the GNAS gene. We identified mutations in the GNAS gene (p.R201C) in 2 out of the 33 (6%) APAs tested, both of which showed autonomous cortisol secretion, while 24 APAs had mutations in the KCNJ5 gene (18 with p.G151R and 6 with p.L168R). These GNAS and KCNJ5 mutations were mutually exclusive in these adenomas. We herein demonstrated for the first time the presence of GNAS mutations in APAs, as well as in some cortisol-secreting adenomas. Our results suggest that these mutations, in addition to mutations in the KCNJ5 gene and other genes such as ATP1A1, ATP2B3 and CACNA1D, may be responsible for the tumorigenesis of APAs and CPAs with subclinical Cushing's syndrome.

The anti-programmed cell death-1 monoclonal antibody (mab), nivolumab has recently been approved for the treatment of unresectable or metastatic malignant melanoma and non-small-cell lung cancers in Japan. Ipilimumab, an anti-cytotoxic T lymphocyte antigen-4 mab for malignant melanoma that was approved earlier than nivolumab in Western countries, is known to frequently cause endocrine immune-related adverse events such as hypophysitis and thyroid dysfunction. We herein report a patient with advanced melanoma who appeared to develop hypophysitis as a consequence of the inhibition of PD-1 by nivolumab. One week after the 6th administration of nivolumab, the patient developed progressive fatigue and appetite loss. Laboratory data on admission for the 7th administration of nivolumab showed eosinophilia and hyponatremia. Since ACTH and cortisol levels were low, nivolumab was discontinued and a large dose of hydrocortisone (100 mg/d) was promptly administered intravenously. A magnetic resonance imaging scan revealed the mild enlargement of the anterior pituitary gland and thickening of the stalk with homogenous contrast. A detailed assessment of anterior pituitary functions with hypothalamic hormone challenges showed that hormonal secretions other than ACTH and TSH were normal. With a replacement dose of hydrocortisone (20 mg/d), the 7th administration of nivolumab was completed without exacerbating the patient's general condition. The present report provides the first detailed endocrinological presentation of nivolumab-induced hypophysitis showing the enlargement of the pituitary gland and stalk in a malignant melanoma patient in Japan. Oncologists and endocrinologists need to be familiar with potentially life-threatening hypophysitis induced by immune-checkpoint inhibitors.

Thyroid storm is an endocrine emergency which is characterized by multiple organ failure due to severe thyrotoxicosis, often associated with triggering illnesses. Early suspicion, prompt diagnosis and intensive treatment will improve survival in thyroid storm patients. Because of its rarity and high mortality, prospective intervention studies for the treatment of thyroid storm are difficult to carry out. We, the Japan Thyroid Association and Japan Endocrine Society taskforce committee, previously developed new diagnostic criteria and conducted nationwide surveys for thyroid storm in Japan. Detailed analyses of clinical data from 356 patients revealed that the mortality in Japan was still high (∼11%) and that multiple organ failure and acute heart failure were common causes of death. In addition, multimodal treatment with antithyroid drugs, inorganic iodide, corticosteroids and beta-adrenergic antagonists has been suggested to improve mortality of these patients. Based on the evidence obtained by nationwide surveys and additional literature searches, we herein established clinical guidelines for the management of thyroid storm. The present guideline includes 15 recommendations for the treatment of thyrotoxicosis and organ failure in the central nervous system, cardiovascular system, and hepato-gastrointestinal tract, admission criteria for the intensive care unit, and prognostic evaluation. We also proposed preventive approaches to thyroid storm, roles of definitive therapy, and future prospective trial plans for the treatment of thyroid storm. We hope that this guideline will be useful for many physicians all over the world as well as in Japan in the management of thyroid storm and the improvement of its outcome.

Brief refeeding times (~60 min) enhanced hepatic Angptl8 expression in fasted mice. We cloned the mouse Angptl8 promoter region to characterise this rapid refeeding-induced increase in hepatic Angptl8 expression. Deletion of the −309/−60 promoter region significantly attenuated basal promoter activity in hepatocytes. A computational motif search revealed a potential binding motif for hepatocyte nuclear factor 1α/1β (HNF-1α/β) at −84/−68 bp of the promoter. Mutation of the HNF-1 binding site significantly decreased the promoter activity in hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfection of HNF-1 in a non-hepatic cell line. Silencing Hnf-1 in hepatoma cells and mouse primary hepatocytes reduced Angptl8 protein levels. Electrophoretic mobility-shift assays confirmed direct binding of Hnf-1 to its Angptl8 promoter binding motif. Hnf-1α expression levels increased after short-term refeeding, paralleling the enhanced in vivo expression of the Angptl8 protein. Chromatin immunoprecipitation (ChIP) confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region. Insulin-treated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement. HNF-1 appears to play essential roles in the rapid refeeding-induced increases in Angptl8 expression. HNF-1α may therefore represent a primary medical target for ANGPTL8-related metabolic abnormalities. The study revealed the transcriptional regulation of the mouse hepatic Angptl8 gene by HNF-1.

Mutations in TBL1X, a component of the nuclear receptor co-repressor (N-CoR) and silencing mediator of retinoic acid and thyroid hormone receptor co-repressor complexes, have recently been implicated in isolated central hypothyroidism (CeH). However, the mechanisms by which TBL1X mutations affect negative feedback regulation in the hypothalamus-pituitary-thyroid axis remain unclear. N-CoR was previously reported to paradoxically enhance the ligand-independent stimulation of TRH and TSHβ gene promoters by thyroid hormone receptors (TR) in cell culture systems. We herein investigated whether TBL1X affects the unliganded TR-mediated stimulation of the promoter activities of genes negatively regulated by T3 in cooperation with N-CoR. In a hypothalamic neuronal cell line, the unliganded TR-mediated stimulation of the TRH gene promoter was significantly enhanced by co-transfected TBL1X, and the co-transfection of TBL1X with N-CoR further enhanced promoter activity. In contrast, the knockdown of endogenous Tbl1x using short interfering RNA significantly attenuated the N-CoR-mediated enhancement of promoter activity in the presence of unliganded TR. The co-transfection of N365Y or Y458C, TBL1X mutants identified in CeH patients, showed impaired co-activation with N-CoR for the ligand-independent stimulation of the TRH promoter by TR. In the absence of T3, similar or impaired enhancement of the TSHβ gene promoter by the wild type or TBL1X mutants, respectively, was observed in the presence of co-transfected TR and N-CoR in CV-1 cells. These results suggest that TBL1X is needed for the full activation of TRH and TSHβ gene promoters by unliganded TR. Mutations in TBL1X may cause CeH due to the impaired up-regulation of TRH and/or TSHβ gene transcription despite low T3 levels.

Patients with adrenal insufficiency require appropriate glucocorticoid replacement therapy; however, reliable biological parameters for optimizing glucocorticoid supplementation are limited. The physician has to rely primarily on clinical judgment, carefully taking into account signs and symptoms potentially suggestive of over- or under-replacement. We have found that some patients who are viewed as receiving sufficient doses of glucocorticoids occasionally exhibit morning headache or morning discomfort, which may be caused by unrecognized nocturnal hypoglycemia. Our aim in this study was to evaluate the usefulness of continuous glucose monitoring (CGM) for detecting unrecognized hypoglycemia and optimizing glucocorticoid replacement therapy in adult patients with central hypoadrenalism. Six patients with central hypoadrenalism of various etiologies were included in this study. All patients exhibited occasional morning headache or discomfort. We performed CGM to measure plasma glucose levels in all patients, and CGM identified unrecognized hypoglycemia episodes at midnight and early in the morning in five patients (83%). The CGM findings were used to fine-tune the dosing and regimens of glucocorticoid replacement and to re-evaluate glucose levels to avoid further unrecognized hypoglycemic events. This optimization of hydrocortisone supplementation prevented additional nocturnal hypoglycemia incidences in all cases. The addition of L-thyroxine with hydrocortisone continued to provide favorable glycemic control. Occasional symptoms also improved after maintenance in all patients. These findings demonstrated that CGM may represent a powerful tool for identifying unrecognized hypoglycemia and for optimizing supplementary hormones in patients with central hypoadrenalism, thereby improving their quality of life.

SummaryObjective Thyroid storm (TS) is a life-threatening endocrine emergency. This study aimed to achieve a better understanding of the management of TS by analyzing therapeutic modalities and prognoses reported by nationwide surveys performed in Japan. Design, patients and measurements Retrospective analyses were performed on clinical parameters, outcomes, and treatments in 356 TS patients. Results Patient disease severities assessed via Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores significantly correlated with mortality. Free triiodothyronine (FT3) and the FT3/free thyroxine (FT4) ratio inversely correlated with disease severity. Methimazole (MMI) was used in the majority of patients (78·1%), and there were no significant differences in mortality or disease severity between those treated with MMI and those receiving propylthiouracil (PTU). Patients who received inorganic iodide (KI) demonstrated higher disease severity but no change in mortality compared to those who did not. Patients treated with corticosteroids (CSs) demonstrated significantly higher disease severity and mortality than those who were not. Disease severity in patients treated with intravenous administration of beta-adrenergic antagonists (AAs) was significantly higher than those treated with oral preparations, although no significant difference in mortality was observed between these groups. In addition, mortality was significantly higher in patients treated with non-selective beta-AAs as compared with other types of beta-AAs. Conclusion In Japan, MMI was preferentially used in TS and showed no disadvantages compared to PTU. In severe TS, multimodal treatment, including administration of antithyroid drugs, KI, CSs and selective beta1-AAs may be preferable to improve outcomes.

Fibroblast growth factor 21 (FGF21), liver-derived hormone, exerts diverse metabolic effects, being considered for clinical application to treat obesity and diabetes. However, its anorexigenic effect is debatable and whether it involves the central mechanism remains unclarified. Moreover, the neuron mediating FGF21’s anorexigenic effect and the systemic energy state supporting it are unclear. We explored the target neuron and fed/fasted state dependence of FGF21’s anorexigenic action. Intracerebroventricular (ICV) injection of FGF21 markedly suppressed food intake in fed mice with elevated blood glucose. FGF21 induced c-Fos expression preferentially in hypothalamic paraventricular nucleus (PVN), and increased mRNA expression selectively for nucleobindin 2/nesfatin-1 (NUCB2/Nesf-1). FGF21 at elevated glucose increased [Ca2+]i in PVN NUCB2/Nesf-1 neurons. FGF21 failed to suppress food intake in PVN-preferential Sim1-Nucb2-KO mice. These findings reveal that FGF21, assisted by elevated glucose, activates PVN NUCB2/Nesf-1 neurons to suppress feeding under fed states, serving as the glycemia-monitoring messenger of liver-hypothalamic network for integrative regulation of energy and glucose metabolism.

Somatic mutations of the catalytic subunit of the cyclic AMP-dependent protein kinase (PRKACA) gene have recently been identified in about 35% of cortisol-producing adenomas (CPAs), with the affected patients showing overt Cushing’s syndrome. Since we recently reported higher prevalence of mutations of the KCNJ5 gene and associations with autonomous cortisol secretion in Japanese aldosterone-producing adenomas than in Western countries, there might be different features of CPAs between Japan and the West. We therefore investigated mutations of the PRKACA gene in Japanese patients with several adrenal tumors secreting cortisol, including overt Cushing’s syndrome, subclinical Cushing’s syndrome, and aldosterone-producing adenomas (APAs) co-secreting cortisol operated on at Gunma University Hospital. Of the 13 patients with CPA who showed overt Cushing’s syndrome, 3 (23%) had recurrent somatic mutations of the PRKACA gene, p.L206R (c.617 T>G), and there were no mutations in subclinical Cushing’s syndrome. Among 33 APAs, 24 had somatic mutations of the KCNJ5 gene, either G151R or L168R, 11 (33%) had autonomous cortisol secretion, but there were no mutations of the PRKACA gene. We established a PCR-restriction fragment length polymorphism assay and revealed that the mutated allele was expressed at a similar level to the wild-type allele. These findings demonstrated that 1) the prevalence of Japanese patients with CPA who showed overt Cushing’s syndrome and whose somatic mutations in the PRKACA gene was similar to that in Western countries, 2) the mutation might be specific for CPAs causing overt Cushing’s syndrome, and 3) the mutant PRKACA allele was expressed appropriately in CPAs.

Previously we reported that the phosphorylation of Synip on serine 99 is required for Synip dissociation from Syntaxin4 and insulin-stimulated Glut4 translocation in cultured 3T3-L1 adipocytes. We also reported that the dissociated Synip remains anchored to the plasma membrane by binding to Phosphatidylinositol (3,4,5)-triphosphate. Recently Synip was reported to arrest SNARE-dependent membrane fusion as a selective t-SNARE binding inhibitor. In this study, we have found that Synip is expressed in podocytes although at a somewhat lower level than in adipocytes. To determine whether phosphorylation of Synip on serine 99 is required for insulin-stimulated Glut4 translocation and glucose uptake in podocytes we expressed a phosphorylation deficient Synip mutant (S99A-Synip) that inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake in adipocytes. We conclude that serine 99 phosphorylation of Synip is required for Glut4 translocation and glucose uptake in both adipocytes and podocytes, suggesting that defects in Synip phosphorylation may underlie insulin resistance and associated diabetic nephropathy.

The physiology of insulin signaling under normal and disease conditions is well studied in classical insulin target tissues, but not in podocytes. To examine insulin stimulation of podocyte GLUT4 translocation, we established a protocol involving treatment with the PPARα agonist fenofibrate to induce E11 podocyte differentiation within 48 hours rather than 7-10 days, which is required for differentiation under the reported protocol. This allowed us to transiently introduce GLUT4 reporter cDNA and RNAi and thereby to examine the regulatory pathway involved. Here we demonstrate that treatment with 200 μM fenofibrate for 36 hours following transfection had a dramatic effect on podocyte morphology, induced several podocyte specific protein expression markers (G protein-coupled receptor 137B, chloride intracellular channel 5, and nephrin) and resulted in insulin-stimulated GLUT4 translocation. In addition, Nucleobindin-2 was found to constitutively associate with Septin 7 (the repressor of GLUT4 translocation), and knockdown of Nucleobindin-2 was found to completely abrogate insulin-stimulated GLUT4 translocation. Together, these data suggest that Nucleobindin-2 may repress Septin7-induced inhibition of insulin-stimulated GLUT4 translocation in podocytes.

Context: Subclinical hypothyroidism (SCH) and metabolic syndrome (MetS) increase with age; however, their relationship remains unclear. Objective: Our objective was to investigate the relationship between SCH and indices of metabolic syndrome and follow up subjects for 1 year. Design: Cross-sectional and longitudinal follow-up studies of cases were collected from Takasaki Hidaka Hospital between 2003 and 2007. Participants: Overall, 11 498 participants of health checkups were analyzed. The mean age was 48 ± 9 years. Main Outcome Measures: The relationship between SCH and indices of MetS were examined. Results: Serum free T4 levels were lower in women than men in most of the age groups, and the prevalence of SCH, 6.3% in women vs 3.4% in men, increased with age, reaching 14.6% in 70-year-old women. Multivariate logistic-regression analyses revealed that waist circumference and the serum triglyceride and low-density lipoprotein-cholesterol levels were significantly higher in subjects with SCH than without among women. Reflecting these findings, the adjusted odds ratio of MetS in patients with SCH was higher than in the euthyroid subjects in women with an odds ratio of 2.7 (95% confidence interval 1.1–5.6; P = .017) but not in men. Furthermore, progression from euthyroid into SCH resulted in a significant increase in the serum triglyceride levels but not low-density lipoprotein-cholesterol in women. Conclusion: Japanese women exhibited a high prevalence of SCH associated with low free T4 levels. There was a strong association between SCH and several indices of metabolic syndrome in women. SCH may affect serum triglyceride levels and be a risk factor for metabolic syndrome.

Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral response is mediated by HELZ2 transcriptional upregulation, enrichment of HELZ2 protein levels in the nucleus, and activation of a transcriptional program that appears to modulate intracellular lipid state. IEGs identified in this study may serve as both (1) potential targets for host directed antiviral design, downstream of the common flaviviral subversion point, as well as (2) possible biomarkers, whose variation, natural, or iatrogenic, could affect host response to viral infections.

Mutations in GNAS, which encodes Gsα, have been documented in detail, particularly in human pituitary GH-secreting adenomas. Mutations have also recently been reported in adrenal cortisol-producing adenomas (CPAs), in addition to those in the PRKACA gene. However, mutations have not yet been examined in aldosterone-producing adenomas (APAs). Therefore, we herein investigated mutations in the GNAS gene in APAs. Two of the 15 (13%) CPAs with overt Cushing's syndrome and one of the 9 (11%) CPAs with subclinical Cushing's syndrome examined had the somatic mutations, p.R201S and p.R201C in the GNAS gene. We identified mutations in the GNAS gene (p.R201C) in 2 out of the 33 (6%) APAs tested, both of which showed autonomous cortisol secretion, while 24 APAs had mutations in the KCNJ5 gene (18 with p.G151R and 6 with p.L168R). These GNAS and KCNJ5 mutations were mutually exclusive in these adenomas. We herein demonstrated for the first time the presence of GNAS mutations in APAs, as well as in some cortisol-secreting adenomas. Our results suggest that these mutations, in addition to mutations in the KCNJ5 gene and other genes such as ATP1A1, ATP2B3 and CACNA1D, may be responsible for the tumorigenesis of APAs and CPAs with subclinical Cushing's syndrome.

Nucleobindin-2 is a 420 amino acid EF-hand Ca²⁺ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.

Mutations of the KCNJ5 gene have recently been identified in patients with aldosterone-producing adenomas (APA).Our objective was to investigate the expression and mutations of the KCNJ5 gene in Japanese patients with APA.We sequenced KCNJ5 cDNA and measured KCNJ5 mRNA levels in 23 patients with APA operated on at Gunma University Hospital.Mutations and mRNA levels of the KCNJ5 gene were examined and compared to those in cortisol-producing adenomas (Cushing's syndrome) and pheochromocytomas.Of the 23 patients with APA, 15 (65.2%) had two somatic mutations of the KCNJ5 gene: 12 cases of p.G151R (eight with c.451GA, and four with c.451GC) and three cases of p.L168R (c.503TG). Levels of KCNJ5 mRNA were significantly higher in the APA with mutations than those without. Immunohistochemistry also showed a stronger staining of KCNJ5 on the cell membrane in the tumor with a mutation. Furthermore, a PCR-restriction fragment length polymorphism assay with c.503TG revealed the mutant mRNA to be expressed at a similar level to the wild type. The level of KCNJ5 mRNA in cortisol-producing adenomas was approximately 30% of that in APA, and almost no expression was observed in pheochromocytomas.We found that: 1) a significant number of patients with APA had somatic mutations of the KCNJ5 gene; 2) KCNJ5 mRNA levels were higher in the APA with KCNJ5 mutations; and 3) the expression of KCNJ5 mRNA was significantly higher in APA than cortisol-producing adenomas and pheochromocytomas.

Adrenal aldosterone-producing adenomas (APA) are rarely associated with the clear co-secretion of cortisol. Somatic mutations of the potassium channel KCNJ5 gene, with the hotspots G151R and L168R, have been recently identified in patients with APA. However, whether APAs that secrete cortisol have these mutations remains unclear. We examined three patients with APAs showing clear autonomous secretion of cortisol who possessed a 1 mg dexamethasone suppression test (DST) with a failure of the serum cortisol level to drop below 3.0 μg/dL, a morning plasma ACTH level of less than 10 pg/mL, and suppressed accumulation in the intact adrenal on (131)I- adosterol scintigraphy, or postoperative adrenal insufficiency. Laparoscopic adrenectomy revealed all tumors to be golden yellow, and histological examination confirmed them to be adrenocortical adenomas. All these patients required replacement therapy with hydrocortisone after surgery. Sequencing demonstrated that 2 of three cases showed a mutation of the KCNJ5 gene, one with c.451G>A, p.G151R and one with c.503T>G, p.L168R. Furthermore, the mRNA levels of steroidogenic enzymes including CYP11B1, CYP11B2, HSD3B2, CYP17A1, CYP11A1 and KCNJ5 in the 3 cases did not differ from those in 8 pure APAs not showing any of the above conditions for autonomous cortisol secretion. In addition, all 8 pure APAs harbored mutations of the KCNJ5 gene. These findings suggested that at least some aldosterone- and cortisol-co-secreting adrenal tumors have mutations of the KCNJ5 gene, suggesting the origin to be APA, and pure APAs may show a high incidence of KCNJ5 mutations.

We recently identified a novel satiety peptide, nesfatin-1, containing 82 amino acids derived from the precursor peptide, nucleobindin 2 (NUCB2), from a troglitazone (TZ)-induced cDNA library. We examined the molecular mechanism underlying TZ-induced NUCB2 mRNA expression. Although TZ induced the mRNA expression in HTB185 cells, a nuclear run-on assay revealed no significant change in the transcription of the gene. Surprisingly, HTB185 cells possessed no functional peroxisome proliferator-activated receptor-γ. We therefore examined the effect of TZ on the mRNA’s stability. The half-life of NUCB2 mRNA was approximately 6 h, and incubation with TZ increased this to 27 h. Furthermore, this increase was completely inhibited by an ERK inhibitor, PD98059, and phosphorylated ERK1/2 was significantly increased after 30 min incubation with TZ. In addition, we cloned the entire NUCB2 gene and identified four adenylate/uridylate-rich elements (AREs) in the 3′ untranslated region (UTR), to which several proteins of HTB185 extracts treated with TZ bound. The reporter assay fused with 3′UTR showed that the second and third AREs were crucial. Furthermore, the human NUCB2 gene spanned 55 kb and contained 14 exons and 13 introns. The transcriptional start site formed clusters around 246 bp upstream from the translational start site. We confirmed that a construct containing 5889 bp of the promoter region was very active in neuron-derived cell lines but not stimulated by TZ. These findings demonstrated a novel action of derivatives of thiazolidinediones, oral insulin-sensitizing antidiabetic agents, to stabilize the mRNA of NUCB2 through AREs in the 3′UTR by activating the ERK1/2 pathway independently of peroxisome proliferator-activated receptor-γ.

Nesfatin-1 is a novel satiety molecule in the hypothalamus and is also present in peripheral tissues. Here we sought to identify the active segment of nesfatin-1 and to determine the mechanisms of its action after peripheral administration in mice. Intraperitoneal injection of nesfatin-1 suppressed food intake in a dose-dependent manner. Nesfatin-1 has three distinct segments; we tested the effect of each segment on food intake. Injection of the midsegment decreased food intake under leptin-resistant conditions such as db/db mice and mice fed a high-fat diet. After injection of the midsegment, expression of c-Fos was significantly activated in the brainstem nucleus tractus solitarius (NTS) but not in the hypothalamic arcuate nucleus; the nicotinic cholinergic pathway to the NTS contributed to midsegment-induced anorexia. Midsegment injection significantly increased expression of proopiomelanocortin and cocaine- and amphetamine-regulated transcript genes in the NTS but not in the arcuate nucleus. Investigation of mutant midsegments demonstrated that a region with amino acid sequence similarity to the active site of agouti-related peptide was indispensable for anorexigenic induction. Our findings indicate that the midsegment of nesfatin-1 causes anorexia, possibly by activating POMC and CART neurons in the NTS via a leptin-independent mechanism after peripheral stimulation. Peripherally administered nesfatin-1 and its mid-segment suppress food intake in mice. The nicotinic cholinergic pathway to the nucleus tractus solitarius contributes to the anorexigenic action of the mid-segment.

The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour. Here we show that nesfatin, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains nesfatin-1, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of nesfatin-1 decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing nesfatin-1 stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to nesfatin-1 is necessary to induce feeding suppression. Chronic i.c.v. injection of nesfatin-1 reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. Nesfatin-1-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. In contrast, central injection of alpha-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by nesfatin-1 is abolished by an antagonist of the melanocortin-3/4 receptor. We identify nesfatin-1 as a satiety molecule that is associated with melanocortin signalling in the hypothalamus.

The physiology of insulin signaling under normal and disease conditions is well studied in classical insulin target tissues, but not in podocytes. To examine insulin stimulation of podocyte GLUT4 translocation, we established a protocol involving treatment with the PPARα agonist fenofibrate to induce E11 podocyte differentiation within 48 hours rather than 7-10 days, which is required for differentiation under the reported protocol. This allowed us to transiently introduce GLUT4 reporter cDNA and RNAi and thereby to examine the regulatory pathway involved. Here we demonstrate that treatment with 200 μM fenofibrate for 36 hours following transfection had a dramatic effect on podocyte morphology, induced several podocyte specific protein expression markers (G protein-coupled receptor 137B, chloride intracellular channel 5, and nephrin) and resulted in insulin-stimulated GLUT4 translocation. In addition, Nucleobindin-2 was found to constitutively associate with Septin 7 (the repressor of GLUT4 translocation), and knockdown of Nucleobindin-2 was found to completely abrogate insulin-stimulated GLUT4 translocation. Together, these data suggest that Nucleobindin-2 may repress Septin7-induced inhibition of insulin-stimulated GLUT4 translocation in podocytes.

Obesity arises from impaired energy balance, which is centrally coordinated by leptin through activation of the long form of leptin receptor (Leprb). Obesity causes central leptin resistance. However, whether enhanced peripheral leptin sensitivity could overcome central leptin resistance remains obscure. A peripheral metabolic organ targeted by leptin is the liver, with low Leprb expression. We here show that mice fed a high-fat diet (HFD) and obese patients with hepatosteatosis exhibit increased expression of hepatic helicase with zinc finger 2, a transcriptional coactivator (Helz2), which functions as a transcriptional coregulator of several nuclear receptors, including peroxisome proliferator-activated receptor γ in vitro. To explore the physiological importance of Helz2, we generated Helz2-deficient mice and analyzed their metabolic phenotypes. Helz2-deficient mice showing hyperleptinemia associated with central leptin resistance were protected against HFD-induced obesity and had significantly up-regulated hepatic Leprb expression. Helz2 deficiency and adenovirus-mediated liver-specific exogenous Leprb overexpression in wild-type mice significantly stimulated hepatic AMP-activated protein kinase on HFD, whereas Helz2-deficient db/db mice lacking functional Leprb did not. Fatty acid-β oxidation was increased in Helz2-deficeint hepatocytes, and Helz2-deficient mice revealed increased oxygen consumption and decreased respiratory quotient in calorimetry analyses. The enhanced hepatic AMP-activated protein kinase energy-sensing pathway in Helz2-deficient mice ameliorated hyperlipidemia, hepatosteatosis, and insulin resistance by reducing lipogenic gene expression and stimulating lipid-burning gene expression in the liver. These findings together demonstrate that Helz2 deficiency ameliorates HFD-induced metabolic abnormalities by stimulating endogenous hepatic Leprb expression, despite central leptin resistance. Hepatic HELZ2 might be a novel target molecule for the treatment of obesity with hepatosteatosis.

Previously we reported that the phosphorylation of Synip on serine 99 is required for Synip dissociation from Syntaxin4 and insulin-stimulated Glut4 translocation in cultured 3T3-L1 adipocytes. We also reported that the dissociated Synip remains anchored to the plasma membrane by binding to Phosphatidylinositol (3,4,5)-triphosphate. Recently Synip was reported to arrest SNARE-dependent membrane fusion as a selective t-SNARE binding inhibitor. In this study, we have found that Synip is expressed in podocytes although at a somewhat lower level than in adipocytes. To determine whether phosphorylation of Synip on serine 99 is required for insulin-stimulated Glut4 translocation and glucose uptake in podocytes we expressed a phosphorylation deficient Synip mutant (S99A-Synip) that inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake in adipocytes. We conclude that serine 99 phosphorylation of Synip is required for Glut4 translocation and glucose uptake in both adipocytes and podocytes, suggesting that defects in Synip phosphorylation may underlie insulin resistance and associated diabetic nephropathy.

Clinical evaluation was conducted to ascertain whether thyrotropin receptor antibody (TRAb) in the normal range may still be involved in the regulation of thyroid function after prolonged treatment for Graves' disease. All patients (n = 33) were treated with antithyroid drugs for an average of 10.6 years and were under euthyroid conditions in which normal blood levels of tri-iodothyronine (T3) were significantly correlated with blood thyrotropin (TSH) levels, but not with titers of TRAb. A significant correlation was observed between TRAb titer and thyroid-stimulating antibody (TSAb) activity. In contrast, this correlation was not found in normal subjects. After administration of T3 (75 microg daily for 8 days), the patients showed increased levels of T3 with concomitant suppression of TSH levels. Under these conditions, linear regression analysis showed significant correlations of TRAb titer and TSAb activity with 24-h thyroid radioiodine uptake (r = 0.641 and 0.621 respectively, P < 0.01), in contrast to declining blood thyroxine levels. Moreover, the immunoglobulin G (IgG) of the patients precipitated to a greater extent than IgG from normal subjects a peptide consisting of the amino acid sequence near the terminus of the human TSH receptor. These findings indicated that TRAb at normal levels possessed significant unremitting activities on thyroid function despite long-term treatment in euthyroid patients with Graves' disease.

Cardiovascular complications, including cardiomegaly, myocardial ischemia and left ventricular hypertrophy, are some of the major determinants of the mortality rate in patients with Cushing's syndrome. We herein report the case of a patient with Cushing's syndrome caused by an adrenal adenoma who presented with congestive heart failure secondary to dilated cardiomyopathy. Follow-up echocardiography showed a marked improvement in the left ventricular cardiac function, and the plasma B-type natriuretic peptide (BNP) levels regressed after successful treatment. "Reversible" dilated cardiomyopathy is rarely associated with Cushing's syndrome; however, it should be recognized. Administering appropriate treatment in a timely manner can reverse this cardiomyopathy along with the other symptoms of Cushing's syndrome.

Using yeast two-hybrid screen, we previously isolated HELZ2 (helicase with zinc finger 2, transcriptional coactivator) that functions as a coregulator of peroxisome proliferator-activated receptorγ (PPARγ). To further delineate its molecular function, we here identified thyroid hormone receptor-associated protein3 (THRAP3), a putative component of the Mediator complex, as a protein stably associating with HELZ2 using immunoprecipitation coupled with mass spectrometry analyses. In immunoprecipitation assays, Thrap3 could associate with endogenous Helz2 as well as Pparg in differentiated 3T3-L1 cells. HELZ2 interacts with the serine/arginine-rich domain and Bcl2 associated transcription factor1-homologous region in THRAP3, whereas THRAP3 directly binds 2 helicase motifs in HELZ2. HELZ2 and THRAP3 synergistically augment transcriptional activation mediated by PPARγ, whereas knockdown of endogenous THRAP3 abolished the enhancement by HELZ2 in reporter assays. Thrap3, similar to Helz2, is evenly expressed in the process of adipogenic differentiation in 3T3-L1 cells. Knockdown of Thrap3 in 3T3-L1 preadipocytes using short-interfering RNA did not influence the expression of Krox20, Klf5, Cebpb, or Cebpd during early stages of adipocyte differentiation, but significantly attenuated the expression of Pparg, Cebpa, and Fabp4/aP2 and accumulation of lipid droplets. Pharmacologic activation of Pparg by troglitazone could not fully restore the differentiation of Thrap3-knockdown adipocytes. In chromatin immunoprecipitation assays, endogenous Helz2 and Thrap3 could be co-recruited, in a ligand-dependent manner, to the PPARγ-response elements in Fabp4/aP2 and Adipoq gene enhancers in differentiated 3T3-L1 cells. These findings collectively suggest that Thrap3 could play indispensable roles in terminal differentiation of adipocytes by enhancing PPARγ-mediated gene activation cooperatively with Helz2.

Selective Alzheimer’s disease (AD) indicator 1 (Seladin-1) has been identified as a gene down-regulated in the degenerated lesions of AD brain. Up-regulation of Seladin-1 reduces the accumulation of β-amyloid and neuronal death. Thyroid hormone (TH) exerts an important effect on the development and maintenance of central nervous systems. In the current study, we demonstrated that Seladin-1 gene and protein expression in the forebrain was increased in thyrotoxic mice compared with that of euthyroid mice. However, unexpectedly, no significant decrease in the gene and protein expression was observed in hypothyroid mice. Interestingly, an agonist of liver X receptor (LXR), TO901317 (TO) administration in vivo increased Seladin-1 gene and protein expression in the mouse forebrain only in a hypothyroid state and in the presence of mutant TR-β, suggesting that LXR-α would compensate for TR-β function to maintain Seladin-1 gene expression in hypothyroidism and resistance to TH. TH activated the mouse Seladin-1 gene promoter (−1936/+21 bp) and site 2 including canonical TH response element (TRE) half-site in the region between −159 and −154 bp is responsible for the positive regulation. RXR-α/TR-β heterodimerization was identified on site 2 by gel-shift assay, and chromatin immunoprecipitation assay revealed the recruitment of TR-β to site 2 and the recruitment was increased upon TH administration. On the other hand, LXR-α utilizes a distinct region from site 2 (−120 to −102 bp) to activate the mouse Seladin-1 gene promoter. Taking these findings together, we concluded that TH up-regulates Seladin-1 gene expression at the transcriptional level and LXR-α maintains the gene expression.

Thyroid storm (TS) is life threatening. Its incidence is poorly defined, few series are available, and population-based diagnostic criteria have not been established. We surveyed TS in Japan, defined its characteristics, and formulated diagnostic criteria, FINAL-CRITERIA1 and FINAL-CRITERIA2, for two grades of TS, TS1, and TS2 respectively.We first developed diagnostic criteria based on 99 patients in the literature and 7 of our patients (LIT-CRITERIA1 for TS1 and LIT-CRITERIA2 for TS2). Thyrotoxicosis was a prerequisite for TS1 and TS2 as well as for combinations of the central nervous system manifestations, fever, tachycardia, congestive heart failure (CHF), and gastrointestinal (GI)/hepatic disturbances. We then conducted initial and follow-up surveys from 2004 through 2008, targeting all hospitals in Japan, with an eight-layered random extraction selection process to obtain and verify information on patients who met LIT-CRITERIA1 and LIT-CRITERIA2.We identified 282 patients with TS1 and 74 patients with TS2. Based on these data and information from the Ministry of Health, Labor, and Welfare of Japan, we estimated the incidence of TS in hospitalized patients in Japan to be 0.20 per 100,000 per year. Serum-free thyroxine and free triiodothyroine concentrations were similar among patients with TS in the literature, Japanese patients with TS1 or TS2, and a group of patients with thyrotoxicosis without TS (Tox-NoTS). The mortality rate was 11.0% in TS1, 9.5% in TS2, and 0% in Tox-NoTS patients. Multiple organ failure was the most common cause of death in TS1 and TS2, followed by CHF, respiratory failure, arrhythmia, disseminated intravascular coagulation, GI perforation, hypoxic brain syndrome, and sepsis. Glasgow Coma Scale results and blood urea nitrogen (BUN) were associated with irreversible damages in 22 survivors. The only change in our final diagnostic criteria for TS as compared with our initial criteria related to serum bilirubin concentration3 mg/dL.TS is still a life-threatening disorder with more than 10% mortality in Japan. We present newly formulated diagnostic criteria for TS and clarify its clinical features, prognosis, and incidence based on nationwide surveys in Japan. This information will help diagnose TS and in understanding the factors contributing to mortality and irreversible complications.

Thyrotropin-releasing hormone (TRH) is a major stimulator of thyrotropin-stimulating hormone (TSH) synthesis in the anterior pituitary, though precisely how TRH stimulates the TSHβ gene remains unclear. Analysis of TRH-deficient mice differing in thyroid hormone status demonstrated that TRH was critical for the basal activity and responsiveness to thyroid hormone of the TSHβ gene. cDNA microarray and K-means cluster analyses with pituitaries from wild-type mice, TRH-deficient mice and TRH-deficient mice with thyroid hormone replacement revealed that the largest and most consistent decrease in expression in the absence of TRH and on supplementation with thyroid hormone was shown by the TSHβ gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSHβ gene. Immunohistochemical analysis demonstrated that NR4A1 was expressed not only in ACTH- and FSH- producing cells but also in thyrotrophs and the expression was remarkably reduced in TRH-deficient pituitary. Furthermore, experiments in vitro demonstrated that incubation with TRH in GH4C1 cells increased the endogenous NR4A1 mRNA level by approximately 50-fold within one hour, and this stimulation was inhibited by inhibitors for PKC and ERK1/2. Western blot analysis confirmed that TRH increased NR4A1 expression within 2 h. A series of deletions of the promoter demonstrated that the region between bp -138 and +37 of the TSHβ gene was responsible for the TRH-induced stimulation, and Chip analysis revealed that NR4A1 was recruited to this region. Conversely, knockdown of NR4A1 by siRNA led to a significant reduction in TRH-induced TSHβ promoter activity. Furthermore, TRH stimulated NR4A1 promoter activity through the TRH receptor. These findings demonstrated that 1) TRH is a highly specific regulator of the TSHβ gene, and 2) TRH mediated induction of the TSHβ gene, at least in part by sequential stimulation of the NR4A1-TSHβ genes through a PKC and ERK1/2 pathway.

Nesfatin-1 is a novel anorexic peptide that reduces the food intake of rodents when administered either intraventricularly or intraperitoneally. However, the molecular mechanism of intracellular signaling via Nesfatin-1 is yet to be resolved. In the current study, we investigated the ability of different neuronal cell lines to respond to Nesfatin-1 and further elucidated the signal transduction pathway of Nesfatin-1. To achieve this, we transfected several cell lines with various combinations of reporter vectors containing different kinds of response elements and performed reporter assays with Nesfatin-1, its active midsegment encoding 30 amino acid residues (M30) and M30-derived mutants. Notably, we found that both Nesfatin-1 as well as M30, significantly increased cAMP response element (CRE) reporter activity in a mouse neuroblastoma cell line, NB41A3. An antagonist of Melanocortin 3/4 receptor, SHU9119, aborted the promoter activity, and a mutant M30, which exerts no anorexic effect in vivo did not induce the CRE reporter activity in NB41A3 cells. Western blotting analyses revealed that Nesfatin-1 and M30 significantly increased the phosphorylation levels of CRE-binding protein (CREB), without altering the intracellular cAMP levels. Further, our study showed that a mitogen-activated protein kinase (MAPK) kinase inhibitor and an L-type Calcium (Ca(2+)) channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that (125)I-Nesfatin-1 binds in a saturable fashion to the membrane fractions of the mouse hypothalamus and NB41A3 cells, with Kd values of 0.79 nM and 0.17 nM, respectively. Collectively, our findings indicate the presence of a Nesfatin-1-specific receptor on the cell surface of NB41A3 cells and mouse hypothalamus. Our study highlights that Nesfatin-1, via its receptor, induces the phosphorylation of CREB, thus activating the intracellular signaling cascade in neurons.

The insulin responsive Glut4 transport vesicles contain the v-SNARE protein Vamp2 that associate with the plasma membrane t-SNARE protein Syntaxin 4 to drive insulin-stimulated Glut4 translocation in skeletal muscle and adipocytes. The syntaxin 4 interacting protein (Synip) binds to syntaxin 4 in the basal state and dissociates in the insulin-stimulated state allowing for the subsequent binding of Vamp2 containing Glut4 vesicles and fusion with the plasma membrane. In this study, we have found that Synip binds phosphatidylinositol 3,4,5-triphosphate (PIP3), but not phosphatidylinositol 3 phosphate (PIP) or phosphatidylinositol 3,4-biphosphate (PIP2) through the Synip WW domain as deletion of this domain (Synip ΔWW) failed to bind PIP3. Over-expressed Synip ΔWW in 3T3L1 adipocytes reduced the basal levels of Glut4 at the plasma membrane with no effect on the binding to syntaxin 4 in vitro. Subcellular fractionation demonstrated that the amount of Synip ΔWW at the PM was decreased in response to insulin in 3T3L1 adipocytes whereas the amount of Synip WT increased. These data suggest that in the presence of insulin, the dissociated Synip remains anchored to the plasma membrane by binding to PIP3.

Purpose: Mixed lineage leukemia (MLL) is a histone methyltransferase that activates gene transcription and associates with menin. In multiple endocrine neoplasia type 1 (Men1), a mutation of menin caused decreased expression of the p27Kip1 and p18Ink4C genes and deregulated cell growth. We hypothesized that the same pathway might be involved in sporadic pituitary adenomas. Experimental Design: mRNA levels for MLL, Men1, p27Kip1, and p18Ink4C were measured in specimens of several sporadic pituitary adenomas, and a search for clinical parameters revealed that octreotide treatment affected the level of expression of some genes tested. To study molecular mechanisms, we cloned and characterized the MLL promoter region and used small interfering RNA for MLL and specific inhibitors for signal transduction pathways. Results: A strong correlation between MLL and p27Kip1 mRNA levels was observed in prolactinomas and growth hormone–secreting adenomas, and these levels were attenuated except in growth hormone–secreting adenomas treated with a somatostatin analogue, octreotide. Conversely, the patients treated with octreotide showed high levels of MLL-p27Kip1 mRNA. Experiments in vitro showed that octreotide increased MLL and p27Kip1 protein and mRNA levels, and overexpression of MLL induced a marked increase in p27Kip1promoter activity. Furthermore, octreotide stimulated the promoter activity of the MLL gene through phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. In addition, incubation with an inhibitor for methyltransferase, MTA, and knockdown of MLL completely inhibited the octreotide-induced expression of p27Kip1. Conclusions: The MLL-p27Kip1 pathway was down-regulated in the pituitary adenomas, and octreotide increased the p27Kip1 level, at least in part, by sequential transcriptional stimulation of the MLL and p27Kip1 genes through phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways.

The 26S proteasome, which degrades ubiquitinated proteins, appears to contribute to the cyclical loading of androgen receptor (AR) to androgen response elements of target gene promoters; however, the mechanism whereby the 26S proteasome modulates AR recruitment remains unknown. Using yeast two-hybrid screening, we previously identified Tat-binding protein-1 (TBP-1), an adenosine triphosphatase of 19S regulatory particles of the 26S proteasome, as a transcriptional coactivator of thyroid hormone receptor. Independently, TBP-1-interacting protein (TBPIP) was also identified as a coactivator of several nuclear receptors, including AR. Here, we investigated whether TBP-1 could interact with and modulate transcriptional activation by AR cooperatively with TBPIP. TBP-1 mRNA was ubiquitously expressed in human tissues, including the testis and prostate, as well as in LNCaP cells. TBP-1 directly bound TBPIP through the amino-terminal domain possessing the leucine zipper structure. AR is physically associated with TBP-1 and TBPIP in vitro and in LNCaP cells. TBP-1 similarly and additively augmented AR-mediated transcription upon coexpression with TBPIP, and the ATPase domain, as well as leucine zipper structure in TBP-1, was essential for transcriptional enhancement. Overexpression of TBP-1 did not alter AR protein and mRNA levels. In the chromatin immunoprecipitation assay, TBP-1 was transiently recruited to the proximal androgen response element of the prostate-specific antigen gene promoter in a ligand-dependent manner in LNCaP cells. These findings suggest that a component of 19S regulatory particles directly binds AR and might participate in AR-mediated transcriptional activation in cooperation with TBPIP.

Objective: CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP)-10/growth arrest and DNA damage 153 is a dominant-negative member of the C/EBP transcription family and inhibits adipogenesis in vitro. The study was undertaken to determine the role of CHOP in obesity in vivo. Research Methods and Procedures: Changes in daily food consumption and body weight were measured. The weight of white and brown adipose tissue was compared between Chop(+/+) and (−/−) mice fed normal chow or a high-fat diet. Glucose and insulin tolerance tests were done, and serum adipocytokine was measured to determine their metabolic state. Fat cell size of subcutaneous and mesenteric adipose tissue was microscopically observed. C/EBP expression in white adipose tissue was examined by Western blot. Results: Female Chop(−/−) mice had significantly greater body weight and adiposity than Chop(+/+) mice, although daily food intake and rectal temperature did not differ. In comparison with Chop(+/+) mice, glucose tolerance and insulin sensitivity did not differ in female Chop(−/−) mice, but levels of plasma leptin and adiponectin were higher. High-fat diet feeding resulted in obesity in female Chop(+/−) and (−/−) mice, although caloric intake did not differ from that of Chop(+/+) mice. Fat cell area was larger in mesenteric fat but not in subcutaneous fat in Chop(−/−) mice fed a high-fat diet. C/EBPβ and the 30-kDa form of C/EBPα expressions were increased in parametrial fat of Chop(−/−) mice, but the 42-kDa form of C/EBPα expression was lower than in Chop(+/+) mice. Discussion: CHOP deficiency causes obesity in female animals without severe metabolic disorders, and C/EBP's expression may be considered to participate in the process.