Massimo Tonacchera

Tissue Distribution and Cardiac Metabolism of 3-Iodothyronamine

3-iodothyronamine (T1AM) is a novel relative of thyroid hormone, able to interact with specific G protein-coupled receptors, known as trace amine-associated receptors. Significant functional effects are produced by exogenous T1AM, including a negative inotropic and chronotropic effect in cardiac preparations. This work was aimed at estimating endogenous T1AM concentration in different tissues and determining its cardiac metabolism. A novel HPLC tandem mass spectrometry assay was developed, allowing detection of T1AM, thyronamine, 3-iodothyroacetic acid, and thyroacetic acid. T1AM was detected in rat serum, at the concentration of 0.3±0.03 pmol/ml, and in all tested organs (heart, liver, kidney, skeletal muscle, stomach, lung, and brain), at concentrations significantly higher than the serum concentration, ranging from 5.6±1.5 pmol/g in lung to 92.9±28.5 pmol/g in liver. T1AM was also identified for the first time in human blood. In H9c2 cardiomyocytes and isolated perfused rat hearts, significant Na+-dependent uptake of exogenous T1AM was observed, and at the steady state total cellular or tissue T1AM concentration exceeded extracellular concentration by more than 20-fold. In both preparations T1AM underwent oxidative deamination to 3-iodothyroacetic acid. T1AM deamination was inhibited by iproniazid but not pargyline or semicarbazide, suggesting the involvement of both monoamine oxidase and semicarbazide-sensitive amine oxidase. Thyronamine and thyroacetic acid were not detected in heart. Finally, evidence of T1AM production was observed in cardiomyocytes exposed to exogenous thyroid hormone, although the activity of this pathway was very low.

Differential effects of NaCl concentration on the constitutive activity of the thyrotropin and the luteinizing hormone/chorionic gonadotropin receptors

The TSH receptor (TSHR) and the receptor (LHR) are members of the family of G protein‐coupled receptors. Recently, point mutations conferring constitutive activity to the TSHR and LHR have been observed as a cause of toxic adenoma and familial/sporadic male pseudo‐precocious puberty, respectively. When evaluated by transfection in COS‐7 cells the wildtype (wt) TSHR displays definite constitutive activity towards Gs‐dependent adenylylcyclase stimulation, while available evidence shows that the LHR does not. In order to compare the constitutive activity of both receptors, we performed functional studies in COS‐7 cells using different assay conditions. Human TSHR and LHR cDNAs subcloned in the expression vector pSVL were transiently expressed in COS‐7 cells and cAMP production was determined following incubation in a medium containing physiological concentration of NaCl [isotonic (NaCl)] or in the same medium without NaCl [hypotonic (NaCl−)] or where NaCl was replaced by an isoosmolar concentration of sucrose [isotonic (sucrose)]. Cells transfected with the TSHR showed higher basal cAMP levels over cells transfected with pSVL in all conditions tested. The effect was stronger when cells were incubated in isotonic (sucrose) buffer. Cells expressing LHR exhibited a minimal increase of cAMP levels over cells transfected with pSVL in isotonic (NaCl) buffer; however, a marked increase in basal cAMP levels was observed when cells were assayed in hypotonic (NaCl−) or isotonic (sucrose) buffers. Varying the pH or incubation temperature was without effect on the results obtained with both receptors. Our data show that despite extensive sequence similarity, the LH and TSH receptors differ markedly in their basal activity. The differential sensitivity of both receptors to low NaCl concentrations, suggests that the unliganded TSH receptor is less constrained than its LH homolog and may be more susceptible to activation by a wide spectrum of mutations.

TSH receptor and disease

The TSH, LH/CG and FSH receptors belong to a subfamily of G protein-coupled receptors. As such, their primary structures deduced from the sequence of the corresponding cDNA (Parmentieret al., 1989; Nagayama et al., 1989; Libertet al., 1989; Minegishiet al., 1990; 1991), predict the existence of seven segments with hydropathy compatible with a transmembrane location. The glycoprotein hormone receptor subfamily (TSH, LH/CG, FSH) share characteristics that distinguish them from the other G protein-coupled receptors. They contain a signal peptide (20 amino acids for the TSH receptor) and they have a long extracellular aminoterminal domain (398 aminoacids for the TSH receptor) comprising the loose repetition of a motif of 25 residues rich in leucine (Parmentieret al., 1989; McFarlandet al., 1989). Similar leucine-rich motifs are also found in a number of widely different proteins (Roth, 1991), in which they are believed to confer the ability to interact with other proteins. The threedimensional structure of one such protein, the ribonuclease inhibitor, has been determined (Kobe & Deisenhofer, 1993), and provides a basis for the modelling of other leucine-rich protein segments. Site-directed mutagenesis studies involving chimeric receptors have clearly shown that the binding specificity and the effector properties of the glycoprotein hormone receptors are encoded in separate domains of the proteins (Xie et al., 1990; Braunet al., 1991; Nagayamaet al., 1991; Vassart & Dumont, 1992); the extracellular domain mediates the binding specificities and the ‘serpentine’ portion with the seven transmembrane segments displays the effector properties triggering G protein activation. This duality is reflected at the genomic level where a single exon encodes the serpentine portion of the receptors and many exons (nine for the TSH receptor) encode the extracellular domain (Gross et al., 1991). When aligned, the three glycoprotein hormone receptors show stronger conservation in the serpentine domain (approximately 70% similarity) than in the extracellular domain (approximately 40% similarity). A peculiarity of the TSH receptor with no counterpart in the FSH or LH/CG receptors is a 50-residue insert upstream from the hinge between the aminoterminal extracellular portion and the first transmembrane segment. A first model for the three-dimensional structure of the aminoterminal extracellular segment of the thyrotrophin receptor has been recently proposed (Kajavaet al., 1995) (Fig. 1). It is based on the known structure of the ribonuclease inhibitor (Kobe & Deisenhofer, 1993).

Thyroid autoimmunity and female gender

Sexual dimorphism exists in regard to the immune response between women and men, and it accounts for the greater prevalence of thyroid autoimmunity in women. Similarly to the human situation a sex-related susceptibility to autoimmune thyroiditis is evident in animal models. A direct influence of genes on sex chromosomes (X or Y) on the immune response has been postulated in some models of autoimmune thyroiditis in rats. On the other hand sex hormones have been implicated to explain the majority of sex differences in the autoimmune response against the thyroid. A state of immune suppression during pregnancy influences the clinical course of autoimmune thyroid diseases, in that a typical amelioration during pregnancy is accompanied by aggravation following delivery. This immmunologic rebound phenomenon may also underly the post partum thyroid dysfunction in otherwise healthy women with a genetic predisposition to autoimmune thyroid disease. Thyroid autoimmunity also interferes with the female reproductive function. Hypothyroidism and less frequently hyperthyroidism due to thyroid autoimmune disorders may produce menstrual dysfunction, anovulation and eventually infertility. Maternal hyper-or hypothyroidism can affect the outcome of pregnancy, producing a higher incidence of miscarriages, maternal complications, and congenital malformations. Untreated maternal hypothyroidism produced by Hashimoto’s disease during pregnancy can impair the neurological development of the fetus due to a reduced availability of maternal thyroxine during early gestation. More specifically, fetal and/or neonatal hypo- or hyperthyroidism produced by the transplacental passage of maternal thyroid autoantibodies can impair growth and neuropsychological development of affected children.

In vitro assay of thyroid disruptors affecting TSH-stimulated adenylate cyclase activity

Several natural or synthetic chemicals have been indicated as potential thyroid disruptors. The development of in vitro assays has been recommended to comprehensively assess the potential thyroid disrupting activity of a substance or a complex mixture. In this study, 12 substances suspected for acting as thyroid disruptors were tested for their ability to inhibit TSH-stimulated cAMP production in vitro. Those substances producing an inhibition were further studied to establish the level at which they interfere with this step of thyroid cell function. Using Chinese hamster ovary cells (CHO) transfected with the recombinant human TSH receptor, a dose-dependent inhibition of TSH-stimulated adenylate cyclase activity was produced by 1,1-bis-(4-chlorphenyl)-2,2,2-trichloroethan (DDT), Aroclor 1254 and Melissa Officinalis. All three substances also inhibited the cAMP production stimulated by TSH receptor antibody. Melissa Officinalis produced a significant inhibition of TSH binding to its receptor and of antibody binding to TSH, while no significant changes were produced by Aroclor 1254 or DDT in these assays. These data suggest that principles contained in Melissa Officinalis may block the binding of TSH to its receptor by acting both on the hormone and the receptor itself, while DDT and Aroclor 1254 affect cAMP production mainly at post-receptor step. In conclusion, we have developed a set of in vitro assays that allow investigation into the effect of thyroid disruptors on the TSH-mediated activation of the cAMP cascade. These assays may be useful to identify the mechanism of action of thyroid disruptors, coming beside and supporting animal studies or epidemiological surveys.

Thyroid Hormone Action in the Adult Brain: Gene Expression Profiling of the Effects of Single and Multiple Doses of Triiodo-L-Thyronine in the Rat Striatum

Thyroid hormones have profound effects on mood and behavior, but the molecular basis of thyroid hormone action in the adult brain is relatively unknown. In particular, few thyroid hormone-dependent genes have been identified in the adult brain despite extensive work carried out on the developing brain. In this work we performed global analysis of gene expression in the adult rat striatum in search for genomic changes taking place after administration of T(3) to hypothyroid rats. The hormone was administered in two different schedules: 1) a single, large dose of 25 microg per 100 g body weight (SD) or 2) 1.5 microg per 100 g body weight once daily for 5 d (RD). Twenty-four hours after the single or last of multiple doses, gene expression in the striatum was analyzed using Codelink microarrays. SD caused up-regulation of 149 genes and down-regulation of 88 genes. RD caused up-regulation of 18 genes and down-regulation of one gene. The results were confirmed by hybridization to Affymetrix microarrays and by TaqMan PCR. Among the genes identified are genes involved in circadian regulation and the regulation of signaling pathways in the striatum. These results suggest that thyroid hormone is involved in regulation of striatal physiology at multiple control points. In addition, they may explain the beneficial effects of large doses of thyroid hormone in bipolar disorders.

Genetics and phenomics of hypothyroidism and thyroid dys- and agenesis due to PAX8 and TTF1 mutations

Thyroid dysgenesis (TD) is the most common cause of congenital hypothyroidism (CH), a relatively frequent endocrine disease in newborns (1 in 3000-4000 live births). TD is a defect in the organogenesis of the gland resulting in hypoplastic, ectopic or absent-thyroid gland. TD is usually sporadic but mutations in transcription factors (PAX8, TTF1, FOXE1 and NKX2-5) involved in thyroid development have been shown to cause a minority of cases transmitted as Mendelian diseases. This review focuses on the genetics and phenomics of hypothyroidism and TD due to PAX8 and TTF1 mutations.

Thyrotropin-Stimulating Hormone Receptor Gene Analysis in Pediatric Patients with Non-Autoimmune Subclinical Hypothyroidism

CONTEXT Mutations in TSH receptor (TSHR) are notoriously associated with congenital hypothyroidism as well as with non-autoimmune subclinical hypothyroidism, a mild form of TSH resistance that is not as well characterized by diagnostic procedures. OBJECTIVE The genetic analysis of the TSHR gene was performed to determine the prevalence of TSHR gene mutations in non-autoimmune subclinical hypothyroidism during the pediatric age. The new mutations were studied for genotypic-phenotypic correlation. PATIENTS Thirty-eight children (ages 0.5-18.0 yr) affected by non-autoimmune subclinical hypothyroidism diagnosed in our center (follow-up from 1 to 11.5 yr) and normal at neonatal screening were enrolled in the genetic study. In 11 cases, the relatives were included in the genetic analysis. RESULTS Eleven different mutations of the TSHR gene were identified in 11 of the 38 patients. Two are new: the nonsense mutation C31X and the missense mutation P68S, which shows a decrease in TSH binding capacity but not in biological activity. In all cases the carrier parent was identified. CONCLUSIONS To date, this study demonstrates the highest prevalence (29%) of TSHR gene mutations in children and adolescents with non-autoimmune subclinical hypothyroidism not selected by neonatal screening. Functional studies show that some mutations cause a slight inactivation of the TSHR. This reveals a possible limit of the in vitro study or of the knowledge of mechanisms involving TSHR, or that other candidate genes must be considered.

Detection of 3-Iodothyronamine in Human Patients: A Preliminary Study

CONTEXT AND OBJECTIVE The primary purpose of this study was to detect and quantify 3-iodothyronamine (T(1)AM), an endogenous biogenic amine related to thyroid hormone, in human blood. DESIGN T(1)AM, total T(3), and total T(4) were assayed in serum by a novel HPLC tandem mass spectrometry assay, which has already been validated in animal investigations, and the results were related to standard clinical and laboratory variables. SETTING AND PATIENTS The series included one healthy volunteer, 24 patients admitted to a cardiological ward, and 17 ambulatory patients suspected of thyroid disease, who underwent blood sampling at admission for routine diagnostic purposes. Seven patients were affected by type 2 diabetes, and six patients showed echocardiographic evidence of impaired left ventricular function. INTERVENTIONS No intervention or any patient selection was performed. MAIN OUTCOME MEASURES serum T(1)AM, total and free T(3) and T(4), routine chemistry, routine hematology, and echocardiographic parameters were measured. RESULTS T(1)AM was detected in all samples, and its concentration averaged 0.219 ± 0.012 pmol/ml. The T(1)AM concentration was significantly correlated to total T(4) (r = 0.654, P < 0.001), total T(3) (r = 0.705, P < 0.001), glycated hemoglobin (r = 0.508, P = 0.013), brain natriuretic peptide (r = 0.543, P = 0.016), and γ-glutamyl transpeptidase (r = 0.675, P < 0.001). In diabetic vs. nondiabetic patients T(1)AM concentration was significantly increased (0.232 ± 0.014 vs. 0.203 ± 0.006 pmol/ml, P = 0.044), whereas no significant difference was observed in patients with cardiac dysfunction. CONCLUSIONS T(1)AM is an endogenous messenger that can be assayed in human blood. Our results are consistent with the hypothesis that circulating T(1)AM is produced from thyroid hormones and encourage further investigations on the potential role of T(1)AM in insulin resistance and heart failure.

The Effect of Voluntary Iodine Prophylaxis in a Small Rural Community: The Pescopagano Survey 15 Years Later

CONTEXT Iodine deficiency disorders are a major public health problem, and programs have been implemented to improve iodine nutrition. OBJECTIVE The objective of the study was to verify the effects of voluntary iodine prophylaxis in a small rural community (Pescopagano, Italy). DESIGN The design of the study was the evaluation of the prevalence of thyroid disorders 15 years after a previous survey conducted before iodine prophylaxis. SETTING The setting for this study was a general community survey. PARTICIPANTS One thousand one hundred forty-eight residents were examined in 2010 and 1411 in 1995. RESULTS In 2010, 757 of 1148 subjects (65.9%) routinely used iodized salt, urinary iodine excretion being significantly higher than in 1955 (median 98.0 μg/L, vs 55.0 μg/L, P < .0001). The prevalence of goiter was lower in 2010 than in 1995 (25.8% vs 46.1%, P < .0001), mainly due to the reduction of diffuse goiter (10.3% vs 34.0%, P < .0001). In 2010 vs 1995, thyroid autonomy in subjects younger than 45 years old (3 of 579, 0.5% vs 25 of 1010, 2.5% P = .004) and nonautoimmune hyperthyroidism in subjects older than 45 years old (8 of 569, 1.4% vs 18 of 401, 4.5%, P = .03) were less frequent. The prevalence of hypothyroidism was higher in 2010 vs 1995 (5.0% vs 2.8%, P = .005), mainly because of an increased frequency of subclinical hypothyroidism in subjects younger than 15 years old (7 of 83, 8.4% vs 0 of 419, 0.0%, P < .0001). Accordingly, serum thyroid autoantibodies (19.5% vs 12.6%; P < .0001) and Hashimotos thyroiditis (14.5% vs 3.5%; P < .0001) were more frequent in 2010 than in 1995. CONCLUSIONS In the present work, the role of voluntary iodine prophylaxis was assessed in a small rural community relatively segregated, in which genetic and other environmental factors have not substantially changed between the 2 surveys. Iodine intake strongly affected the pattern of thyroid diseases, but the benefits of correcting iodine deficiency (decreased prevalence of goiter and thyroid autonomy in younger subjects and reduced frequency of nonautoimmune hyperthyroidism in older subjects) far outweighs the risk of development of thyroid autoimmunity and mild hypothyroidism in youngsters.