Koshi Hashimoto

An unliganded thyroid hormone receptor causes severe neurological dysfunction.

Congenital hypothyroidism and the thyroid hormone (T3) resistance syndrome are associated with severe central nervous system (CNS) dysfunction. Because thyroid hormones are thought to act principally by binding to their nuclear receptors (TRs), it is unexplained why TR knock-out animals are reported to have normal CNS structure and function. To investigate this discrepancy further, a T3 binding mutation was introduced into the mouse TR-β locus by homologous recombination. Because of this T3 binding defect, the mutant TR constitutively interacts with corepressor proteins and mimics the hypothyroid state, regardless of the circulating thyroid hormone concentrations. Severe abnormalities in cerebellar development and function and abnormal hippocampal gene expression and learning were found. These findings demonstrate the specific and deleterious action of unliganded TR in the brain and suggest the importance of corepressors bound to TR in the pathogenesis of hypothyroidism.

Peripheral Administration of Nesfatin-1 Reduces Food Intake in Mice: The Leptin-Independent Mechanism

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.

Expression and Mutations of KCNJ5 mRNA in Japanese Patients with Aldosterone-Producing Adenomas

CONTEXT Mutations of the KCNJ5 gene have recently been identified in patients with aldosterone-producing adenomas (APA). OBJECTIVE Our objective was to investigate the expression and mutations of the KCNJ5 gene in Japanese patients with APA. DESIGN AND PATIENTS We sequenced KCNJ5 cDNA and measured KCNJ5 mRNA levels in 23 patients with APA operated on at Gunma University Hospital. MAIN OUTCOME MEASURES Mutations and mRNA levels of the KCNJ5 gene were examined and compared to those in cortisol-producing adenomas (Cushings syndrome) and pheochromocytomas. RESULTS 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.451G>A, and four with c.451G>C) and three cases of p.L168R (c.503T>G). 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.503T>G 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. CONCLUSION 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.

Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo.

Thyroid hormone action is mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. DNA-binding is presumed to be essential for all nuclear actions of thyroid hormone. To test this hypothesis in vivo, the DNA-binding domain of TR-beta was mutated within its P-box (GS mutant) using gene targeting techniques. This mutation in vitro completely abolishes TR-beta DNA-binding, while preserving ligand (T3) and cofactor interactions with the receptor. Homozygous mutant (TR-betaGS/GS) mice displayed abnormal T3 regulation of the hypothalamic-pituitary-thyroid axis and retina identical to abnormalities previously observed in TR-beta KO (TR-beta-/-) mice. However, TR-betaGS/GS mutant mice maintained normal hearing at certain frequencies and did not display significant outer hair cell loss, in contrast to TR-beta-/- mice. DNA-binding, therefore, is essential for many functions of the TR, including retinal development and negative feedback regulation by thyroid hormone of the hypothalamic-pituitary-thyroid axis. Inner ear development, although not completely normal, can occur in the absence of TR DNA-binding, suggesting that an alternative and perhaps novel thyroid hormone-signaling pathway may mediate these effects.

THE SATIETY MOLECULE NESFATIN-1 IS CO-EXPRESSED WITH MELANIN CONCENTRATING HORMONE IN TUBERAL HYPOTHALAMIC NEURONS OF THE RAT

Overlapped in the tuberal hypothalamic area (THA), melanin-concentrating hormone (MCH) and hypocretin (Hcrt) neurons contribute to the integrated regulation of food intake, energy regulation and sleep. Recently, physiological role in appetite suppression has been defined for a novel hypothalamic molecule, nesfatin-1. Acute i.c.v. infusion of nesfatin-1 (nesf-1) promotes anorexia whereas chronic treatment reduces body weight in rats. This satiety molecule is expressed in neurons from areas prominently involved in appetite regulation including THA. We therefore sought functionally relevant to determine whether nesf-1 might be a reliable signaling marker for a new cell contingent within THA, in addition to MCH and Hcrt neurons. Thus, we completed a detailed topographical mapping of neurons immunostained for nesf-1 (nesf-1+) together with cell quantification in each discrete nucleus from THA in the rat. We further combined the immunodetection of nesf-1 with that of MCH or Hcrt to assess possible co-expression. More than three quarters of the nesf-1+ neurons were encountered in nuclei from the lateral half of THA. By double immunofluorescent staining, we showed that all neurons immunoreactive for melanin concentrating hormone (MCH+) neurons depicted nesf-1 immunoreactivity and approximately 80% of the nesf-1+ neurons were labeled for MCH. Maximal co-expression rates were observed in the lateral THA containing approximately 86% of the double-labeled neurons plotted in THA. The present data suggest that nesf-1 co-expressed in MCH neurons may play a complex role not only in food intake regulation but also in other essential integrative brain functions involving MCH signaling, ranging from autonomic regulation, stress, mood, cognition to sleep.

Novel insight from transgenic mice into thyroid hormone resistance and the regulation of thyrotropin

Patients with resistance to thyroid hormone (RTH) exhibit elevated thyroid hormone levels and inappropriate thyrotropin (thyroid-stimulating hormone, or TSH) production. The molecular basis of this disorder resides in the dominant inhibition of endogenous thyroid hormone receptors (TRs) by a mutant receptor. To determine the relative contributions of pituitary versus hypothalamic resistance to the dysregulated production of thyroid hormone in these patients, we developed a transgenic mouse model with pituitary-specific expression of a mutant TR (Delta337T). The equivalent mutation in humans is associated with severe generalized RTH. Transgenic mice developed profound pituitary resistance to thyroid hormone, as demonstrated by markedly elevated baseline and non-triodothyronine (T3)-suppressible serum TSH and pituitary TSH-beta mRNA. Serum thyroxine (T4) levels were only marginally elevated in transgenic mice and thyrotropin-releasing hormone (TRH) gene expression in the paraventricular hypothalamus was downregulated. After TRH administration, T4 concentrations increased markedly in transgenic, but not in wild-type mice. Transgenic mice rendered hypothyroid exhibited a TSH response that was only 30% of the response observed in wild-type animals. These findings indicate that pituitary expression of this mutant TR impairs both T3-mediated suppression and T3-independent activation of TSH production in vivo. The discordance between basal TSH and T4 levels and the reversal with TRH administration demonstrates that resistance at the level of both the thyrotroph and the hypothalamic TRH neurons are required to elevate thyroid hormone levels in patients with RTH.

Fasting concentrations of nesfatin-1 are negatively correlated with body mass index in non-obese males

Background  We recently identified a novel anorexigenic protein, nesfatin‐1, which is processed from nesfatin/nucleobindin‐2 (NUCB2). However, the clinical importance of this protein has not been determined.

Dominant Role of Thyrotropin-releasing Hormone in the Hypothalamic-Pituitary-Thyroid Axis

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the β isoforms of TH receptors (TRβ KO), or both (double KO). TRβ knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.

Thyroid Hormone-independent Interaction between the Thyroid Hormone Receptor β2 Amino Terminus and Coactivators

Thyroid hormone receptors (TRs) mediate hormone action by binding to DNA response elements (TREs) and either activating or repressing gene expression in the presence of ligand, T3. Coactivator recruitment to the AF-2 region of TR in the presence of T3 is central to this process. The different TR isoforms, TR-β1, TR-β2, and TR-α1, share strong homology in their DNA- and ligand-binding domains but differ in their amino-terminal domains. Because TR-β2 exhibits greater T3-independent activation on TREs than other TR isoforms, we wanted to determine whether coactivators bound to TR-β2 in the absence of ligand. Our results show that TR-β2, unlike TR-β1 or TR-α1, is able to bind certain coactivators (CBP, SRC-1, and pCIP) in the absence of T3 through a domain which maps to the amino-terminal half of its A/B domain. This interaction is specific for certain coactivators, as TR-β2 does not interact with other co-factors (p120 or the CBP-associated factor (pCAF)) in the absence of T3. The minimal TR-β2 domain for coactivator binding is aa 21–50, although aa 1–50 are required for the full functional response. Thus, isoform-specific regulation by TRs may involve T3-independent coactivator recruitment to the transcription complex via the AF-1 domain.

Cross-talk between Thyroid Hormone Receptor and Liver X Receptor Regulatory Pathways Is Revealed in a Thyroid Hormone Resistance Mouse Model

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor β (TR-β) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-β knock-out mice, generalized expression of a non-ligand binding TR-β 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. 7α-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-β and liver X receptor α (LXR-α), which both bind to a direct repeat + 4(DR+4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-β antagonized induction of this promoter by LXR-α. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR+4 element in the presence of MUT but not WT TR-β. A mechanism for cross-talk, and potential antagonism, between TR-β and LXR-α is proposed.