Fredric E. Wondisford

Comparison of administration of recombinant human thyrotropin with withdrawal of thyroid hormone for radioactive iodine scanning in patients with thyroid carcinoma.

BACKGROUND To detect recurrent disease in patients who have had differentiated thyroid cancer, periodic withdrawal of thyroid hormone therapy may be required to raise serum thyrotropin concentrations to stimulate thyroid tissue so that radioiodine (iodine-131) scanning can be performed. However, withdrawal of thyroid hormone therapy causes hypothyroidism. Administration of recombinant human thyrotropin stimulates thyroid tissue without requiring the discontinuation of thyroid hormone therapy. METHODS One hundred twenty-seven patients with thyroid cancer underwent whole-body radioiodine scanning by two techniques: first after receiving two doses of thyrotropin while thyroid hormone therapy was continued, and second after the withdrawal of thyroid hormone therapy. The scans were evaluated by reviewers unaware of the conditions of scanning. The serum thyroglobulin concentrations and the prevalence of symptoms of hypothyroidism and mood disorders were also determined. RESULTS Sixty-two of the 127 patients had positive whole-body radioiodine scans by one or both techniques. The scans obtained after stimulation with thyrotropin were equivalent to the scans obtained after withdrawal of thyroid hormone in 41 of these patients (66 percent), superior in 3 (5 percent), and inferior in 18 (29 percent). When the 65 patients with concordant negative scans were included, the two scans were equivalent in 106 patients (83 percent). Eight patients (13 percent of those with at least one positive scan) were treated with radioiodine on the basis of superior scans done after withdrawal of thyroid hormone. Serum thyroglobulin concentrations increased in 15 of 35 tested patients: 14 after withdrawal of thyroid hormone and 13 after administration of thyrotropin. Patients had more symptoms of hypothyroidism (P<0.001) and dysphoric mood states (P<0.001) after withdrawal of thyroid hormone than after administration of thyrotropin. CONCLUSIONS Thyrotropin stimulates radioiodine uptake for scanning in patients with thyroid cancer, but the sensitivity of scanning after the administration of thyrotropin is less than that after the withdrawal of thyroid hormone. Thyrotropin scanning is associated with fewer symptoms and dysphoric mood states.

Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein.

Insulin resistance and elevated glucagon levels result in nonsuppressible hepatic glucose production and hyperglycemia in patients with type 2 diabetes. The CREB coactivator complex controls transcription of hepatic gluconeogenic enzyme genes. Here, we show that both the antidiabetic agent metformin and insulin phosphorylate the transcriptional coactivator CREB binding protein (CBP) at serine 436 via PKC iota/lambda. This event triggers the dissociation of the CREB-CBP-TORC2 transcription complex and reduces gluconeogenic enzyme gene expression. Mice carrying a germline mutation of this CBP phosphorylation site (S436A) demonstrate resistance to the hypoglycemic effect of both insulin and metformin. Obese, hyperglycemic mice display hepatic insulin resistance, but metformin is still effective in treating the hyperglycemia of these mice since it stimulates CBP phosphorylation by bypassing the block in insulin signaling. Our findings point to CBP phosphorylation at Ser436 by metformin as critical for its therapeutic effect, and as a potential target for pharmaceutical intervention.

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.

Minireview: Thyrotropin-Releasing Hormone and the Thyroid Hormone Feedback Mechanism

Thyroid hormone (TH) plays a critical role in development, growth, and cellular metabolism. TH production is controlled by a complex mechanism of positive and negative regulation. Hypothalamic TSH-releasing hormone (TRH) stimulates TSH secretion from the anterior pituitary. TSH then initiates TH synthesis and release from the thyroid gland. The synthesis of TRH and TSH subunit genes is inhibited at the transcriptional level by TH, which also inhibits posttranslational modification and release of TSH. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback at the pituitary was thought to be the primary regulator of serum TSH levels. However, study of transgenic animals showed an unexpected, dominant role for TRH in regulating the hypothalamic-pituitary-thyroid axis and an unanticipated involvement of the thyroid hormone receptor ligand-dependent activation function (AF-2) domain in TH negative regulation. These results are summarized in the review.

Divergent roles for thyroid hormone receptor β isoforms in the endocrine axis and auditory system

Thyroid hormone receptors (TRs) modulate various physiological functions in many organ systems. The TR alpha and TR beta isoforms are products of 2 distinct genes, and the beta 1 and beta 2 isoforms are splice variants of the same gene. Whereas TR alpha 1 and TR beta 1 are widely expressed, expression of the TR beta 2 isoform is mainly limited to the pituitary, triiodothyronine-responsive TRH neurons, the developing inner ear, and the retina. Mice with targeted disruption of the entire TR beta locus (TR beta-null) exhibit elevated thyroid hormone levels as a result of abnormal central regulation of thyrotropin, and also develop profound hearing loss. To clarify the contribution of the TR beta 2 isoform to the function of the endocrine and auditory systems in vivo, we have generated mice with targeted disruption of the TR beta 2 isoform. TR beta 2-null mice have preserved expression of the TR alpha and TR beta 1 isoforms. They develop a similar degree of central resistance to thyroid hormone as TR beta-null mice, indicating the important role of TR beta 2 in the regulation of the hypothalamic-pituitary-thyroid axis. Growth hormone gene expression is marginally reduced. In contrast, TR beta 2-null mice exhibit no evidence of hearing impairment, indicating that TR beta 1 and TR beta 2 subserve divergent roles in the regulation of auditory function.

Critical role for thyroid hormone receptor β2 in the regulation of paraventricular thyrotropin-releasing hormone neurons

Thyroid hormone thyroxine (T(4)) and tri-iodothyronine (T(3)) production is regulated by feedback inhibition of thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) synthesis in the pituitary and hypothalamus when T(3) binds to thyroid hormone receptors (TRs) interacting with the promoters of the genes for the TSH subunit and TRH. All of the TR isoforms likely participate in the negative regulation of TSH production in vivo, but the identity of the specific TR isoforms that negatively regulate TRH production are less clear. To clarify the role of the TR-beta2 isoform in the regulation of TRH gene expression in the hypothalamic paraventricular nucleus, we examined preprothyrotropin-releasing hormone (prepro-TRH) expression in mice lacking the TR-beta2 isoform under basal conditions, after the induction of hypothyroidism with propylthiouracil, and in response to T(3) administration. Prepro-TRH expression was increased in hypothyroid wild-type mice and markedly suppressed after T(3) administration. In contrast, basal TRH expression was increased in TR-beta2-null mice to levels seen in hypothyroid wild-type mice and did not change significantly in response to induction of hypothyroidism or T(3) treatment. However, the suppression of TRH mRNA expression in response to leptin reduction during fasting was preserved in TR-beta2-null mice. Thus TR-beta2 is the key TR isoform responsible for T(3)-mediated negative-feedback regulation by hypophysiotropic TRH neurons.

DEFECTIVE RELEASE OF COREPRESSOR BY HINGE MUTANTS OF THE THYROID HORMONE RECEPTOR FOUND IN PATIENTS WITH RESISTANCE TO THYROID HORMONE

On positive thyroid hormone response elements (pTREs), thyroid hormone receptor (TR) binding to DNA in the absence of ligand (thyroid hormone, T3) decreases transcription (silencing). Silencing is due to a family of recently described nuclear corepressor proteins (NCoR and SMRT) which bind to the CoR box in the hinge region of TR. Ligand-dependent activation of TR is associated with displacement of corepressors and recruitment of coactivating proteins. Resistance to thyroid hormone (RTH) is due to mutations in the β isoform of the thyroid hormone receptor (TR-β). To date, three RTH mutations reportedly with near-normal T3binding (A234T, R243Q, and R243W) have been described in or near the CoR box. To determine the mechanism of RTH caused by these mutants, the interaction of wild type (wt) and mutant TRs with the corepressor, NCoR, and the coactivator, SRC-1, was tested in gel-shift assays. As expected, NCoR bound wt TR in the absence of T3 and dissociated from TR with increasing T3 concentration. SRC-1 failed to bind wt TR in the absence of T3, but bound to TR with increasing avidity as T3 concentrations rose. At no T3 concentration did both NCoR and SRC-1 bind to wt TR, indicating that their binding to TR was mutually exclusive. Hinge mutants bound NCoR normally in the absence of T3; however, dissociation of NCoR and recruitment of SRC-1 was markedly impaired except at very high T3 concentrations. Importantly, hinge mutant TRs when complexed to DNA bound T3 poorly despite their near-normal T3 binding in solution. These binding studies correlated with functional assays showing defective transactivation of pTREs by hinge mutants except at high T3concentrations. Thus, we describe a novel mechanism of RTH whereby TR hinge mutants selectively affect T3 binding when complexed to DNA, and prevent NCoR dissociation from TR. Our data also suggest that solution T3 binding by RTH mutants may not accurately reflect physiologically relevant T3 binding by TR when bound to DNA.

A Unique Role of the β-2 Thyroid Hormone Receptor Isoform in Negative Regulation by Thyroid Hormone MAPPING OF A NOVEL AMINO-TERMINAL DOMAIN IMPORTANT FOR LIGAND-INDEPENDENT ACTIVATION

Negative regulation by thyroid hormone is mediated by nuclear thyroid hormone receptors (TRs) acting on thyroid hormone response elements (TREs). We examine here the role of human TR-β2, a TR isoform with central nervous system-restricted expression, in the regulation of target genes whose expression are decreased by triiodothyronine (T3). Using transient transfection studies, we found that TR-β2 achieved significantly greater ligand-independent activation on the thyrotropin-releasing hormone (TRH) and common glycoprotein α-subunit genes than either TR-β1 or TR-α1. A chimeric TR-β isoform containing the TR-β2 amino terminus linked to the TR-α1 DNA- and ligand-binding domains functioned like the TR-β2 isoform on these promoters, confirming that the amino terminus of TR-β2 was both necessary and sufficient to mediate this effect. By constructing deletion mutants of the TR-β2 amino terminus, we demonstrate that amino acids 89–116 mediate this function. This domain, important in ligand-independent activation on negative TREs, is discrete from a previously described activation domain in the amino-terminal portion of TR-β2. We conclude that the central nervous system-restricted TR-β2 isoform has a unique effect on negative regulation by T3 that can be mapped to amino acids 89–116 of the amino terminus of the human TR-β2.

Function of Nuclear Co-repressor Protein on Thyroid Hormone Response Elements Is Regulated by the Receptor A/B Domain

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.

Divergent roles of growth factors in the GnRH regulation of puberty in mice

Pubertal onset, initiated by pulsatile gonadotropin-releasing hormone (GnRH), only occurs in a favorable, anabolic hormonal milieu. Anabolic factors that may signal nutritional status to the hypothalamus include the growth factors insulin and IGF-1. It is unclear which hypothalamic neuronal subpopulation these factors affect to ultimately regulate GnRH neuron function in puberty and reproduction. We examined the direct role of the GnRH neuron in growth factor regulation of reproduction using the Cre/lox system. Mice with the IR or IGF-1R deleted specifically in GnRH neurons were generated. Male and female mice with the IR deleted in GnRH neurons displayed normal pubertal timing and fertility, but male and female mice with the IGF-1R deleted in GnRH neurons experienced delayed pubertal development with normal fertility. With IGF-1 administration, puberty was advanced in control females, but not in females with the IGF-1R deleted in GnRH neurons, in control males, or in knockout males. These mice exhibited developmental differences in GnRH neuronal morphology but normal number and distribution of neurons. These studies define the role of IGF-1R signaling in the coordination of somatic development with reproductive maturation and provide insight into the mechanisms regulating pubertal timing in anabolic states.