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.

for the Japan Thyroid Association Background: 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. Methods: 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. Results: 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 concentration > 3 mg/dL. Conclusions: 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. 1 The First Department of Medicine, Wakayama Medical University, Wakayama, Japan. 2 Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Japan. 3 Department of Medicine 2, Tokyo Women's Medical University, Tokyo, Japan. 4 Division of Endocrinology and Metabolism, Department of Internal Medicine, Fujita Health University, Toyoake, Japan. 5 Department of Internal Medicine, Keio University, Tokyo, Japan. 6 Department of Clinical and Molecular Endocrinology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan. 7 Division of Diabetes, Metabolism, and Endocrinolog...

Recently, a family of nuclear co-repressor proteins (TRACs) have been identified that interact with thyroid hormone (TR) and retinoic acid receptors to mediate ligand-independent repression of gene transcription. In this report, we have cloned and characterized a human TRAC, which when expressed as a truncated protein lacking its repressing domains, can abolish endogenous cellular TRAC activity. Use of this inhibitor has uncovered a differential function of TRACs on negative versus positive thyroid hormone response elements and has demonstrated the importance of the TR A/B domain in modulating TRAC function. Thus, isoform-specific functions of the TR may be mediated by their functional interaction with co-repressor proteins.The thyroid hormone receptor (TR), 1 a member of the steroid/ thyroid hormone receptor superfamily, mediates gene regulation through its ability to bind to specific thyroid hormone response elements (TREs) (1-4) either as a monomer, homodimer, or as a heterodimer with RXR isoforms (5-7). Through its interactions with specific TREs and other protein co-activators, the TR can either positively or negatively regulate gene expression in the presence of its ligand, triiodothyronine (T 3 ) (8 -10). The TR also has significant ligand-independent activity in both cell culture and in vitro transcription systems (11,12). On positive TREs, the TR is a potent repressor of transcription in the absence of ligand (13-15), while on negative TREs the TR activates transcription (9, 16 -18). T 3 reverses these ligand-independent activities and causes upregulation on positive TREs and down-regulation on negative TREs. Recently, two separate proteins, termed nuclear co-repressors (TRAC), have been identified which interact with the TR in the absence of ligand to mediate ligand-independent repression on positive TREs (19 -21). These proteins possess a carboxyl-terminal domain which mediates their interaction with the TR and amino-terminal domains, which presumably interact with the initiation complex to mediate ligand-independent repression. Their role in ligand-independent activation on negative TREs is unknown.Further complexity in thyroid hormone signaling is mediated by the presence of 3 separate TR isoforms (␣1, ␤1, and ␤2) which activate or repress transcription in the presence of ligand (22). The TR␤ isoforms are alternatively spliced variants which differ only in their amino terminus, while TR␣1 is the product of a separate gene but shares strong homology in its DNA and ligand binding domains but differs also in its amino terminus. Recent data from a number of different groups suggest that isoform-specific differences in both ligand-independent and -dependent activities exist on both positive and negative TREs and that these differences involve the separate amino termini which allow the TRs to either bind TREs differently, interact with members of the initiation complex with different affinities, or possess separate activation domains as is the case with the TR␤2 amino terminus (23-25). These isoforms are ...

Activation of peroxisome proliferator-activated receptor (PPAR)-g by the thiazolidinedione (TZD) class of antidiabetic drugs elicits growth inhibition in a variety of malignant tumors. We clari®ed the e ects of TZDs on growth of human non-small cell lung carcinoma (NSCLC) cells that express endogenous PPAR-g. Troglitazone and pioglitazone caused inhibition of cellular growth and induced apoptosis of NSCLC cells in a time-and dose-dependent manner. Subtraction cloning analysis identi®ed that troglitazone stimulated expression of the growth arrest and DNA-damage inducible (GADD)153 gene, and the increased expression of GADD153 mRNA was also con®rmed by an array analysis of the 160 apoptosis-related genes. Western blot analysis revealed that troglitazone also increased GADD153 protein levels in a time-dependent manner. Troglitazone did not stimulate GADD153 mRNA levels in undi erentiated 3T3-L1 cells lacking PPAR-g expression, whereas its induction was clearly observed in di erentiated adipocytes expressing PPAR-g. Activity of the GADD153 promoter occurred in a NSCLC cell line in transient transcription assays and was signi®-cantly stimulated by troglitazone, although binding of PPAR/retinoid X receptor heterodimer was not detected in the promoter region in gel retardation assays. Inhibition of GADD153 gene expression by an antisense phosphorothionate oligonucleotide attenuated the troglitazone-induced growth inhibition. These ®ndings collectively indicated that activation of PPAR-g by TZDs could cause growth inhibition and apoptosis of NSCLC cells and that GADD153 might be a candidate factor implicated in these processes.

TRH is the principal positive regulator of TSH synthesis and secretion in man. T3 is able to control TRH synthesis through feedback inhibition at the transcriptional level, presumably by binding to its receptor which interacts with one or more negative thyroid hormone response elements (TREs) present within the human TRH promoter. In the present study we have identified the specific negative TREs within the TRH promoter and characterized their ability to interact with thyroid hormone receptors (TRs), and the retinoid X receptor (RXR). Our analysis demonstrates that ligand-independent and dependent regulation of the human TRH promoter is restricted to the TR beta 1 isoform. Deletional analysis of the TRH promoter identified two discrete regions that are responsible for mediating ligand-dependent negative regulation of the TRH promoter. Mutagenesis of potential TR binding half-sites within these regions identified three separate half-sites (site 4 from -55 to -60 base pairs (bp); site 5, +14 to +19 bp; and site 6, +37 to +42 bp) which act in combination to allow for negative regulation. Mutation and/or deletion of each of these sites leads to a loss of negative regulation of the TRH promoter by T3. Gel-mobility shift assays of site 4 and its surrounding nucleotides revealed that this region of the promoter is capable of binding TR monomers, homodimers, and TR-RXR heterodimers. Mutagenesis of site 4 leads to a loss of all binding to this region. The region encompassing sites 5 and 6 binds only TR monomer, and the addition of RXR to the binding reaction leads to a loss of specific monomeric binding. To assess the functional importance of site 4 and its surrounding nucleotides we cotransfected RXR isoforms along with TR beta with TRH promoter constructs containing either site 4 or its mutant. In the presence of wild type site 4 sequence, cotransfected RXR enhanced negative regulation of the TRH promoter. Mutation and or deletion of site 4 leads to a loss of this enhancement. These data demonstrate that two structurally different negative TREs cooperate to allow for negative regulation of the human TRH promoter and that negative regulation is TR isoform-specific and modulated by the RXR-signaling pathway through a novel negative TRE.

Isoform specificity likely plays a large role in the ability of the thyroid hormone receptor (TR) to specifically regulate gene expression in both the presence and absence of its cognate ligand, triiodothyronine. To investigate further the mechanism of isoform specificity of human TRs (TR alpha 1 and TR beta 1), we have examined their functional effects on positive thyroid hormone response elements (TREs) both in the presence and absence of ligand. TR alpha 1 was greater than 2-fold more potent than TR beta 1 on both TREs studied, in terms of both ligand-independent repression and ligand-dependent stimulation. By creating a number of chimeric and mutant receptors, we have established that the increased functional potency of TR alpha 1 is due to its unique amino terminus. Deletion or substitution of the TR alpha 1 amino terminus leads to a loss of both its ligand-independent and -dependent functions on positive TREs. Furthermore, the TR alpha 1 amino terminus antagonizes homodimer formation on the positive TREs studied. TR constructs, which contain the TR alpha 1 amino terminus, are unable to form homodimers and form exclusively heterodimers with RXR alpha on direct repeat and palindromic TREs. Deletion of the amino terminus from either TR isoform leads to preferential homodimer formation, which suggests that the TR amino terminus is important for relative heterodimerization capability. From these data, we conclude that TR alpha 1 isoform specificity on positive TREs resides predominantly in its amino terminus through its ability to favor heterodimerization with the retinoid X receptor or other nuclear proteins.

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor ␤ (TR-␤) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-␤ knock-out mice, generalized expression of a non-ligand binding TR-␤ protein in this knock-in model more fully recapitulates the hypothyroid state, because the hypothyroid effect of TRs is mediated by the unliganded receptor. In the hypothyroid state, a high cholesterol diet increased serum cholesterol levels in wild-type animals (WT) but either did not change or reduced levels in mutant (MUT) mice relative to hypothyroidism alone. 7␣-Hydroxylase (CYP7A1) is the rate-limiting enzyme in cholesterol metabolism and mRNA levels were undetectable in the hypothyroid state in all animals. triiodothyronine replacement restored CYP7A1 mRNA levels in WT mice but had minimal effect in MUT mice. In contrast, a high cholesterol diet markedly induced CYP7A1 levels in MUT but not WT mice in the hypothyroid state. Elevation of CYP7A1 mRNA levels and reduced hepatic cholesterol content in MUT animals are likely because of cross-talk between TR-␤ and liver X receptor ␣ (LXR-␣), which both bind to a direct repeat ؉ 4 (DR؉4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-␤ antagonized induction of this promoter by LXR-␣. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR؉4 element in the presence of MUT but not WT TR-␤. A mechanism for cross-talk, and potential antagonism, between TR-␤ and LXR-␣ is proposed.

The patients with PA associated with autonomous cortisol secretion had high incidence of CVE, and this association may further increase the risk of CVE in patients with PA.