Circadian Gating of Thyroid Hormone Action in Hepatocytes.

Thyroid hormones, thyroxin (T4) and the biologically active triiodothyronine (T3), play important roles in liver metabolic regulation, including fatty acid biosynthesis, beta-oxidation, and cholesterol homeostasis. These functions position TH signaling as a potential target for the treatment of metabolic dysfunction-associated steatotic liver disease (MASLD). Elevated T3 levels in the circulation are associated with increased hepatic lipid turnover, which is also under the control of the circadian clock system. In this study, we developed a cell system to study the impact of hepatocyte circadian rhythms on the metabolic response to T3 treatment under control and steatotic conditions. Synchronized AML-12 circadian reporter hepatocytes were treated with T3 at different circadian phases and metabolic conditions. T3 treatment increased metabolic activity in a dose-independent fashion and had no significant effect on circadian rhythms in AML-12 cells. T3 had marked time-of-treatment-dependent effects on metabolic transcript expression. Steatosis induction altered metabolic transcript expression in AML-12 cells. In this condition, the circadian rhythm period was lengthened, and this effect was independent of T3. Under steatotic conditions, T3 had marked time-of-treatment dependent effects on metabolic transcript expression, which differed from those observed under control conditions. These findings reveal a time-of-day-dependent response of hepatocytes to T3, which is further modulated by the metabolic state. Our data suggest that time has a strong influence on liver TH action, which might be considered when treating MASLD.

Therapeutical Potential of T3 as Adjuvant Therapy in Male Alloxan-induced Diabetic Rats.

Alloxan-induced diabetic rats present with hypothyroidism. When treated with triiodothyronine (T3), glycemia and proinflammatory cytokine expression are downregulated, improving insulin sensitivity. The effectiveness of associating T3 with insulin (replacement dose [6 U] and [3 U]) in controlling glycemia was investigated in this experimental model. Male Wistar rats were made diabetic by alloxan injection and sorted into groups treated or not with insulin (3 or 6 U) associated or not with T3 (1.5 µg 100 g-1 BW) for 28 days. Nondiabetic rats constituted the control group. Fasting glycemia, glucose decay rate, and thyrotropin (TSH) were measured in the blood/serum of all animals. Immunoblotting was used to assess total GLUT4 expression in skeletal muscles and epididymal white adipose tissue. Cytokine and nuclear factor-κB (NF-κB) expression were measured in these tissues and liver. Diabetic rats presented with increased fasting glycemia, inflammatory cytokines, and NF-κB expression, TSH levels, and insulin resistance. In diabetic rats treated with T3 and/or insulin, these parameters were decreased, whereas GLUT4 and anti-inflammatory cytokine expression were increased. T3 combined with 3-U insulin restored the parameters to values of the control group and was more effective at controlling glycemia than 6-U insulin. Thus, a combination of T3 and insulin might represent a promising strategy for diabetes management since it reduces the insulin requirement by half and improves glycemic control of diabetic rats, which could postpone insulin resistance that develops with chronic insulin administration. These findings open a perspective for using thyroid analogues that provide tissue-specific effects, which might result in a potentially more effective treatment of diabetes.

Triiodothyronine enhances various forms of kidney-specific Klotho protein and suppresses the Wnt/ß-catenin pathway: Insights from in-vitro, in-vivo and in-silico investigations.

Age-related diseases are intricately linked to the molecular processes underlying aging, with the decline of the antiaging protein Klotho being a key factor. Investigating these processes is crucial for developing therapeutic strategies. The age-associated reduction in Klotho expression, coupled with a decline in the endocrine hormone triiodothyronine (T3), prompted a detailed exploration of their potential interplay. Our research, conducted through both in-vitro and in-vivo studies on BALB/c mice, unveiled a significant capacity of T3 to upregulate various forms of Klotho via ATF-3/p-c-Jun transcription factor. This effect was particularly noteworthy in aged individuals, where Klotho expression had waned compared to their younger counterparts. Importantly, T3 demonstrated a promising therapeutic impact in rejuvenating Klotho expression in this context. Further investigations elucidated the molecular mechanisms underlying T3's impact on aging-related pathways. In-vitro and in-vivo experiments established T3's ability to downregulate the Wnt/ß-Catenin pathway by enhancing Klotho expression. In-silico analyses provided insights into Klotho's intricate role, showing its capacity to inhibit Wnt ligands such as Wnt3 and Wnt8a, consequently disrupting their interaction with the Wnt receptor. Additionally, T3 was found to downregulate kidney-specific GSK-3ß expression through the augmentation of Klotho expression. The study also highlighted T3's role in maintaining calcium and phosphate homeostasis via Klotho. This comprehensive investigation not only sheds light on the intricate mechanisms governing aging processes but also presents promising avenues for therapeutic interventions targeting the Wnt/ß-Catenin pathway implicated in various age-associated diseases.

Vascular Response of Triiodothyronine on Isolated Aortic Rings: Contribution of Redox Mechanisms. / Resposta Vascular da Triiodotironina sobre Anéis de Aortas Isoladas: Contribuição de Mecanismos Redox.

BACKGROUND: Vascular dysfunction constitutes the etiology of many diseases, such as myocardial infarction and hypertension, with the disruption of redox homeostasis playing a role in the imbalance of the vasomotor control mechanism. Our group previously has shown that thyroid hormones exert protective effects on the aortic tissue of infarcted rats by improving angiogenesis signaling. OBJECTIVE: Investigate the role of triiodothyronine (T3) on vascular response, exploring its effects on isolated aortas and whether there is an involvement of vascular redox mechanisms. METHODS: Isolated aortic rings (intact- and denuded-endothelium) precontracted with phenylephrine were incubated with T3 (10-8, 10-7, 10-6, 10-5, and 10-4 M), and tension was recorded using a force-displacement transducer coupled with an acquisition system. To assess the involvement of oxidative stress, aortic rings were preincubated with T3 and subsequently submitted to an in vitro reactive oxygen species (ROS) generation system. The level of significance adopted in the statistical analysis was 5%. RESULTS: T3 (10-4 M) promoted vasorelaxation of phenylephrine precontracted aortic rings in both intact- and denuded-endothelium conditions. Aortic rings preincubated in the presence of T3 (10-4 M) also showed decreased vasoconstriction elicited by phenylephrine (1 µM) in intact-endothelium preparations. Moreover, T3 (10-4 M) vasorelaxation effect persisted in aortic rings preincubated with NG-nitro-L-arginine methylester (L-NAME, 10 µM), a nonspecific NO synthase (NOS) inhibitor. Finally, T3 (10-4 M) exhibited, in vitro, an antioxidant role by reducing NADPH oxidase activity and increasing SOD activity in the aorta's homogenates. CONCLUSION: T3 exerts dependent- and independent-endothelium vasodilation effects, which may be related to its role in maintaining redox homeostasis.

FUNDAMENTO: A disfunção vascular constitui a etiologia de diversas doenças, incluindo infarto do miocárdio e hipertensão, diante da ruptura da homeostase oxi-redutiva ("redox"), desempenhando um papel no desequilíbrio do mecanismo de controle vasomotor. Nosso grupo demonstrou anteriormente que os hormônios tireoidianos melhoram a sinalização da angiogênese, exercendo efeitos protetores sobre o tecido aórtico de ratos infartados. OBJETIVOS: Investigar o papel da triiodotironina (T3) na resposta vascular, explorando seus efeitos em aortas isoladas e a presença de mecanismos redox vasculares. MÉTODOS: Anéis aórticos isolados (endotélio intacto e desnudado) pré-contraídos com fenilefrina foram incubados com T3 (10-8, 10-7, 10-6, 10-5 e 10-4 M) e a tensão foi registrada usando um transdutor de deslocamento de força acoplado a um sistema de coleta. Para avaliar o envolvimento do estresse oxidativo, os anéis aórticos foram pré-incubados com T3 e posteriormente submetidos a um sistema de geração de espécies reativas de oxigênio (ROS) in vitro. O nível de significância adotado na análise estatística foi de 5%. RESULTADOS: A T3 (10-4 M) promoveu o vasorrelaxamento dos anéis aórticos pré-contraídos com fenilefrina em endotélio intacto e desnudado. Os anéis aórticos pré-incubados na presença de T3 (10-4 M) também mostraram diminuição da vasoconstrição provocada pela fenilefrina (1 µM) em preparações de endotélio intacto. Além disso, o efeito vasorrelaxante da T3 (10-4 M) persistiu em anéis aórticos pré-incubados com éster metílico de NG-nitro-L-arginina (L-NAME, 10 µM), um inibidor inespecífico da NO sintase (NOS). Por fim, a T3 (10-4 M) exibiu, in vitro, um papel antioxidante ao reduzir a atividade da NADPH oxidase e aumentar a atividade da SOD nos homogenatos aórticos. CONCLUSÃO: A T3 exerce efeitos dependentes e independentes de endotélio, o que pode estar relacionado ao seu papel na manutenção da homeostase redox.

Impact of the thyroid hormone T3 and its nuclear receptor TRα1 on colon cancer stem cell phenotypes and response to chemotherapies.

Colorectal cancers (CRCs) are highly heterogeneous and show a hierarchical organization, with cancer stem cells (CSCs) responsible for tumor development, maintenance, and drug resistance. Our previous studies showed the importance of thyroid hormone-dependent signaling on intestinal tumor development and progression through action on stem cells. These results have a translational value, given that the thyroid hormone nuclear receptor TRα1 is upregulated in human CRCs, including in the molecular subtypes associated with CSC features. We used an established spheroid model generated from the human colon adenocarcinoma cell line Caco2 to study the effects of T3 and TRα1 on spheroid formation, growth, and response to conventional chemotherapies. Our results show that T3 treatment and/or increased TRα1 expression in spheroids impaired the response to FOLFIRI and conferred a survival advantage. This was achieved by stimulating drug detoxification pathways and increasing ALDH1A1-expressing cells, including CSCs, within spheroids. These results suggest that clinical evaluation of the thyroid axis and assessing TRα1 levels in CRCs could help to select optimal therapeutic regimens for patients with CRC. Proposed mechanism of action of T3/TRα1 in colon cancer spheroids. In the control condition, TRα1 participates in maintaining homeostatic cell conditions. The presence of T3 in the culture medium activates TRα1 action on target genes, including the drug efflux pumps ABCG2 and ABCB1. In the case of chemotherapy FOLFIRI, the increased expression of ABC transcripts and proteins induced by T3 treatment is responsible for the augmented efflux of 5-FU and Irinotecan from the cancer cells. Taken together, these mechanisms contribute to the decreased efficacy of the chemotherapy and allow cells to escape the treatment. Created with BioRender.com .

Thyroid hormone induces ossification and terminal maturation in a preserved OA cartilage biomimetic model.

OBJECTIVE: To characterize aspects of triiodothyronine (T3) induced chondrocyte terminal maturation within the molecular osteoarthritis pathophysiology using the previously established T3 human ex vivo osteochondral explant model. DESIGNS: RNA-sequencing was performed on explant cartilage obtained from OA patients (n = 8), that was cultured ex vivo with or without T3 (10 ng/ml), and main findings were validated using RT-qPCR in an independent sample set (n = 22). Enrichment analysis was used for functional clustering and comparisons with available OA patient RNA-sequencing and GWAS datasets were used to establish relevance for OA pathophysiology by linking to OA patient genomic profiles. RESULTS: Besides the upregulation of known hypertrophic genes EPAS1 and ANKH, T3 treatment resulted in differential expression of 247 genes with main pathways linked to extracellular matrix and ossification. CCDC80, CDON, ANKH and ATOH8 were among the genes found to consistently mark early, ongoing and terminal maturational OA processes in patients. Furthermore, among the 37 OA risk genes that were significantly affected in cartilage by T3 were COL12A1, TNC, SPARC and PAPPA. CONCLUSIONS: RNA-sequencing results show that metabolic activation and recuperation of growth plate morphology are induced by T3 in OA chondrocytes, indicating terminal maturation is accelerated. The molecular mechanisms involved in hypertrophy were linked to all stages of OA pathophysiology and will be used to validate disease models for drug testing.

Triiodothyronine induces a proinflammatory monocyte/macrophage profile and impedes cardiac regeneration.

Neonatal mouse hearts can regenerate post-injury, unlike adult hearts that form fibrotic scars. The mechanism of thyroid hormone signaling in cardiac regeneration warrants further study. We found that triiodothyronine impairs cardiomyocyte proliferation and heart regeneration in neonatal mice after apical resection. Single-cell RNA-Sequencing on cardiac CD45-positive leukocytes revealed a pro-inflammatory phenotype in monocytes/macrophages after triiodothyronine treatment. Furthermore, we observed that cardiomyocyte proliferation was inhibited by medium from triiodothyronine-treated macrophages, while triiodothyronine itself had no direct effect on the cardiomyocytes in vitro. Our study unveils a novel role of triiodothyronine in mediating the inflammatory response that hinders heart regeneration.

Engineered PDGFA-ligand-modified exosomes delivery T3 for demyelinating disease targeted therapy.

Demyelination is a proper syndrome in plenty of central nervous system (CNS) diseases, which is the main obstacle to recovery and still lacks an effective treatment. To overcome the limitations of the brain-blood barrier on drug permeability, we modified an exosome secreted by neural stem cells (NSCs), which had transfected with lentivirus armed with platelet-derived growth factors A (PDGFA)-ligand. Through the in vivo and in vitro exosomes targeting test, the migration ability to the lesion areas and OPCs significantly improved after ligand modification. Furthermore, the targeted exosomes loaded with 3,5, 30-L-triiodothyronine (T3) have a critical myelination ability in CNS development, administrated to the cuprizone animal model treatment. The data shows that the novel drug vector loaded with T3 significantly promotes remyelination compared with T3 alone. At the same time, it improved the CNS microenvironment by reducing astrogliosis, inhibiting pro-inflammatory microglia, and alleviating axon damage. This investigation provides a straightforward strategy to produce a targeting exosome and indicates a possible therapeutic manner for demyelinating disease.

Quemeiteng granule relieves goiter by suppressing thyroid microvascular endothelial cell proliferation and angiogenesis via miR-217-5p-mediated targeting of FGF2-induced regulation of the ERK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Goiters are enlargements of the thyroid gland and are a global public issue. Quemeiteng granule (QMTG) is a traditional Chinese medicine (TCM) formula used to treat goiter in Yunnan Province. However, the effectiveness and underlying mechanism of these treatments have not been fully elucidated. AIM OF THE STUDY: This study aimed to investigate the therapeutic effects of QMTG on goiter and the downstream regulatory mechanisms. MATERIALS AND METHODS: In this study, we first evaluated the antigoiter efficacy of QMTG through biochemical indices [body weight, thyroid coefficient, triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), and thyroid stimulating hormone (TSH)] and hematoxylin-eosin (HE) staining in a Propylthiouracil (PTU)-induced model. Based on microRNA sequencing (miRNA-seq) and bioinformatics analysis, key miRNA was screened out. A dual-luciferase reporter assay was performed to confirm the transcriptional regulation of the target gene by the miRNA. The viability of rat thyroid microvascular endothelial cells (RTMECs) and human thyroid microvascular endothelial cells (HTMECs) was assessed using the CCK-8 assays. The migration and angiogenesis of RTMECs and HTMECs were visualized through tube formation and wound scratch assays. Proteins involved in angiogenesis and the ERK pathway were assessed via Western blotting. RESULTS: QMTG significantly increased body weight, decreased the thyroid coefficient, increased the levels of T3, T4, FT3 and FT4 and reduced TSH levels in rats with goiter. QMTG also promoted the morphological recovery of thyroid follicles. MiR-217-5p was identified as a key miRNA. Our studies revealed that miR-217-5p directly targets FGF2 and that QMTG promotes the recovery of thyroid hormone (TH) levels and morphological changes in the thyroid, suppresses thyroid microvascular endothelial cell vitality, tube formation and migration, and reduces the expression of VEGF, Ang-1 and VCAM-1 triggered by miR-217-5p, thereby inhibiting the Ras/MEK/ERK cascade through FGF2. CONCLUSIONS: Our experiments demonstrated that the QMTG had therapeutic effects on goiter. These effects were attributed to the inhibition of ERK pathway-induced proliferation and angiogenesis through the targeting of FGF2 by miR-217-5p.

The Differential Effect of a Shortage of Thyroid Hormone Compared with Knockout of Thyroid Hormone Transporters Mct8 and Mct10 on Murine Macrophage Polarization.

Innate immune cells, including macrophages, are functionally affected by thyroid hormone (TH). Macrophages can undergo phenotypical alterations, shifting between proinflammatory (M1) and immunomodulatory (M2) profiles. Cellular TH concentrations are, among others, determined by TH transporters. To study the effect of TH and TH transporters on macrophage polarization, specific proinflammatory and immunomodulatory markers were analyzed in bone marrow-derived macrophages (BMDMs) depleted of triiodothyronine (T3) and BMDMs with a knockout (KO) of Mct8 and Mct10 and a double KO (dKO) of Mct10/Mct8. Our findings show that T3 is important for M1 polarization, while a lack of T3 stimulates M2 polarization. Mct8 KO BMDMs are unaffected in their T3 responsiveness, but exhibit slight alterations in M2 polarization, while Mct10 KO BMDMs show reduced T3 responsiveness, but unaltered polarization markers. KO of both the Mct8 and Mct10 transporters decreased T3 availability and, contrary to the T3-depleted BMDMs, showed partially increased M1 markers and unaltered M2 markers. These data suggest a role for TH transporters besides transport of TH in BMDMs. This study highlights the complex role of TH transporters in macrophages and provides a new angle on the interaction between the endocrine and immune systems.

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles.

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a "molecular memory" of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3'-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka.

Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 µg/L, 1 µg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

Comparison of in vitro thyroxine (T4) metabolism between Wistar rat and human hepatocyte cultures.

In vitro assays remain relatively new in exploring human relevance of liver, in particular nuclear receptor-mediated perturbations of the hypothalamus-pituitary-thyroid axis seen in rodents, mainly in the rat. Consistent with in vivo data, we confirm that thyroid hormone thyroxine metabolism was 9 times higher in primary rat hepatocytes (PRH) than in primary human hepatocytes (PHH) cultured in a 2D sandwich (2Dsw) configuration. In addition, thyroxine glucuronide (T4-G) was by far the major metabolite formed in both species (99.1% in PRH and 69.7% in PHH) followed by thyroxine sulfate (T4-S, 0.7% in PRH and 18.1% in PHH) and triiodothyronine/reverse triiodothyronine (T3/rT3, 0.2% in PRH and 12.2% in PHH). After a 7-day daily exposure to orphan receptor-mediated liver inducers, T4 metabolism was strongly increased in PRH, almost exclusively through increased T4-G formation. These results were consistent with the inductions of glucuronosyltransferase Ugt2b1 and canalicular transporter Mrp2. PHH also responded to activation of the three nuclear receptors, with mainly induction of glucuronosyltransferase UGT1A1 and canalicular transporter MRP2. Despite this, T4 disappearance rate and secreted T4 metabolites were only slightly increased in PHH. Overall, our data highlight that cryopreserved hepatocytes in 2Dsw culture allowing long-term exposure and species comparison are of major interest in improving liver-mediated human safety assessment.

Hypothalamic Thyroid Hormone Receptor α1 Signaling Controls Body Temperature.

Background: The importance of thyroid hormones (THs) for peripheral body temperature regulation has been long recognized, as medical conditions such as hyper- and hypothyroidism lead to alterations in body temperature and energy metabolism. In the past decade, the brain actions of THs and their respective nuclear receptors, thyroid hormone receptor α1 (TRα1) and thyroid hormone receptor beta (TRß), coordinating body temperature regulation have moved into focus. However, the exact roles of the individual TR isoforms and their precise neuroanatomical substrates remain poorly understood. Methods: Here we used mice expressing a mutant TRα1 (TRα1+m) as well as TRß knockouts to study body temperature regulation using radiotelemetry in conscious and freely moving animals at different ambient temperatures, including their response to oral 3,3',5-triiodothyronine (T3) treatment. Subsequently, we tested the effects of a dominant-negative TRα1 on body temperature after adeno-associated virus (AAV)-mediated expression in the hypothalamus, a region known to be involved in thermoregulation. Results: While TRß seems to play a negligible role in body temperature regulation, TRα1+m mice had lower body temperature, which was surprisingly not entirely normalized at 30°C, where defects in facultative thermogenesis or tail heat loss are eliminated as confounding factors. Only oral T3 treatment fully normalized the body temperature profile of TRα1+m mice, suggesting that the mutant TRα1 confers an altered central temperature set point in these mice. When we tested this hypothesis more directly by expressing the dominant-negative TRα1 selectively in the hypothalamus via AAV transfection, we observed a similarly reduced body temperature at room temperature and 30°C. Conclusion: Our data suggest that TRα1 signaling in the hypothalamus is important for maintaining body temperature. However, further studies are needed to dissect the precise neuroanatomical substrates and the downstream pathways mediating this effect.

Thyroid hormone-regulated chromatin landscape and transcriptional sensitivity of the pituitary gland.

Thyroid hormone (3,5,3'-triiodothyronine, T3) is a key regulator of pituitary gland function. The response to T3 is thought to hinge crucially on interactions of nuclear T3 receptors with enhancers but these sites in pituitary chromatin remain surprisingly obscure. Here, we investigate genome-wide receptor binding in mice using tagged endogenous thyroid hormone receptor ß (TRß) and analyze T3-regulated open chromatin using an anterior pituitary-specific Cre driver (Thrbb2Cre). Strikingly, T3 regulates histone modifications and chromatin opening primarily at sites that maintain TRß binding regardless of T3 levels rather than at sites where T3 abolishes or induces de novo binding. These sites associate more frequently with T3-activated than T3-suppressed genes. TRß-deficiency blunts T3-regulated gene expression, indicating that TRß confers transcriptional sensitivity. We propose a model of gene activation in which poised receptor-enhancer complexes facilitate adjustable responses to T3 fluctuations, suggesting a genomic basis for T3-dependent pituitary function or pituitary dysfunction in thyroid disorders.

Preliminary Results of a Double-Blind Randomized Controlled Trial Evaluating the Cardiometabolic Effects of Levothyroxine and Liothyronine Compared to Levothyroxine with Placebo in Athyreotic Low-Risk Thyroid Cancer Patients.

Background: Evidence is needed on the risks and benefits of combination therapy with levothyroxine (LT4)+liothyronine (LT3) for the treatment of hypothyroidism. Objective and Methods: We performed a randomized, double-blind placebo-controlled study to assess the effects of LT4+LT3 therapy versus LT4+placebo in a homogeneous group of athyreotic patients, without cardiovascular risk factors during long-term replacement monotherapy with LT4. The primary objective of the study was to assess the effects of combination LT4+LT3 therapy on heart rate, cardiac rhythm, and sensitive cardiovascular parameters of cardiac morphology and function by means of electrocardiography and Doppler echocardiography. The secondary objective of the study was to evaluate patient compliance, tolerability, and potential adverse events. Results: Thirty-eight patients with postsurgical hypothyroidism satisfying the inclusion criteria were selected from a group of 300 patients with low-risk thyroid cancer followed for a routine follow-up; they were randomized to receive LT4+LT3 or LT4+placebo. Twenty-four patients were evaluated after 1 year of treatment. All clinical and laboratory parameters were compared with the results obtained from 50 healthy euthyroid volunteers without comorbidities, matched for gender, age, physical activity, and lifestyle. Participants and clinicians remained blinded to the treatment allocation. After 1 year of combination therapy, a significant improvement in the diastolic function, evidenced by a significant reduction in the E/e' ratio (p = 0.046) and its positive trend over time, was observed in the LT4+LT3 group versus the LT4+placebo group. In addition, the univariate analyses showed a significant relationship between free triiodothyronine (fT3) levels (in pg/mL) with Δ of variation of the E/e' ratio in the LT4+LT3 group (standardized ß coefficient = 0.603 [confidence interval: 0.001-1.248], p = 0.050) after combination therapy. No adverse events including tachycardia, arrhythmias, atrial fibrillation, or other important events occurred between the first administration and the end of the study. Conclusions: In this preliminary report, combination treatment with LT4+LT3 induced favorable changes in cardiovascular parameters of diastolic function without any adverse cardiovascular events. Trial Registration: EUDRACT number: 2017-001261-25.

A Historical Reflection on Scientific Advances in Understanding Thyroid Hormone Action.

Background: Thyroid hormone (TH) has actions in every tissue of the body and is essential for normal development, as well as having important actions in the adult. The earliest markers of TH action that were identified and monitored clinically, even before TH could be measured in serum, included oxygen consumption, basal metabolic rate, serum cholesterol, and deep tendon reflex time. Cellular, rodent, amphibian, zebrafish, and human models have been used to study TH action. Summary: Early studies of the mechanism of TH action focused on saturable-specific triiodothyronine (T3) nuclear binding and direct actions of T3 that altered protein expression. Additional effects of TH were recognized on mitochondria, stimulation of ion transport, especially the sodium potassium ATPase, augmentation of adrenergic signaling, role as a neurotransmitter, and direct plasma membrane effects. The cloning of the thyroid hormone receptor (THR) genes in 1986 and report of the THR crystal structure in 1995 produced rapid progress in understanding the mechanism of TH nuclear action, as well as the development of modified THR ligands. These findings revealed nuances of TH signaling, including the role of nuclear receptor coactivators and corepressors, repression of positively stimulated genes by the unliganded receptor, THR isoform-specific actions of TRα (THRA) and TRß (THRB), and THR binding DNA as a heterodimer with retinoid-x-receptor (RXR) for genes positively regulated by TH. The identification of genetic disorders of TH transport and signaling, especially Resistance to Thyroid Hormone (RTH) and monocarboxylate transporter 8 (Mct8) defects, has been highly informative with respect to the mechanism of TH action. Conclusions: The impact of THR isoform, post-translational modifications, receptor cofactors, DNA response element, and selective TH tissue uptake, on TH action, have clinical implications for diagnosing and treating thyroid disease. Additionally, these findings have led to the development of novel TH and TH analogue therapies for metabolic, neurological, and cardiovascular diseases.

Cell cycle activation in thyroid hormone-induced apoptosis and stem cell development during Xenopus intestinal metamorphosis.

Amphibian metamorphosis resembles mammalian postembryonic development, a period around birth when many organs mature into their adult forms and when plasma thyroid hormone (T3) concentration peaks. T3 plays a causative role for amphibian metamorphosis. This and its independence from maternal influence make metamorphosis of amphibians, particularly anurans such as pseudo-tetraploid Xenopus laevis and its highly related diploid species Xenopus tropicalis, an excellent model to investigate how T3 regulates adult organ development. Studies on intestinal remodeling, a process that involves degeneration of larval epithelium via apoptosis and de novo formation of adult stem cells followed by their proliferation and differentiation to form the adult epithelium, have revealed important molecular insights on T3 regulation of cell fate during development. Here, we review some evidence suggesting that T3-induced activation of cell cycle program is important for T3-induced larval epithelial cell death and de novo formation of adult intestinal stem cells.

Triiodothyronine positively regulates endoplasmic reticulum-associated degradation (ERAD) and promotes androgenic signaling in androgen-dependent prostate cancer cells.

Thyroid hormones (THs) play crucial roles in numerous physiological processes of nearly all mammalian tissues, including differentiation and metabolism. Deterioration of TH signaling has been associated with several pathologies, including cancer. The effect of highly active triiodothyronine (T3) has been investigated in many in vivo and in vitro cancer models. However, the role of T3 on cancerous prostate tissue is controversial today. Recent studies have focused on the characterization of the supportive roles of the endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) signaling in prostate cancer (PCa) and investigating new hormonal regulation patterns, including estrogen, progesterone and 1,25(OH)2D3. Additionally, androgenic signaling controlled by androgens, which are critical in PCa progression, has been shown to be regulated by other steroid hormones. Today, the effects of T3 on ERAD and UPR are unknown, the impact on androgenic signaling is also still not fully understood in PCa. Therefore, we aimed to investigate the molecular action of T3 on the ERAD mechanism and UPR signaling in PCa cells and also extensively examined the effect of T3 on androgenic signaling. Our data indicated that T3 tightly regulated ERAD and UPR signaling in androgen-dependent PCa cells. We also found that T3 hormone stimulated androgenic signaling by upregulating AR mRNA and protein levels and enhancing its nuclear translocation. Additionally, advanced computational studies supported the ligand binding effect of T3 on AR protein. Our data suggest that targeting thyroidal signaling should be considered in therapeutic approaches to be developed for prostate malignancy in addition to other steroidal regulations.

Primary human thyrocytes maintained the function of thyroid hormone production and secretion in vitro.

PURPOSE: Thyroid cell lines are useful tools to study the physiology and pathology of the thyroid, however, they do not produce or secrete hormones in vitro. On the other hand, the detection of endogenous thyroid hormones in primary thyrocytes was often hindered by the dedifferentiation of thyrocytes ex vivo and the presence of large amounts of exogenous hormones in the culture medium. This study aimed to create a culture system that could maintain the function of thyrocytes to produce and secrete thyroid hormones in vitro. METHODS: We established a Transwell culture system of primary human thyrocytes. Thyrocytes were seeded on a porous membrane in the inner chamber of the Transwell with top and bottom surfaces exposed to different culture components, mimicking the 'lumen-capillary' structure of the thyroid follicle. Moreover, to eliminate exogenous thyroid hormones from the culture medium, two alternatives were tried: a culture recipe using hormone-reduced serum and a serum-free culture recipe. RESULTS: The results showed that primary human thyrocytes expressed thyroid-specific genes at higher levels in the Transwell system than in the monolayer culture. Hormones were detected in the Transwell system even in the absence of serum. The age of the donor was negatively related to the hormone production of thyrocytes in vitro. Intriguingly, primary human thyrocytes cultured without serum secreted higher levels of free triiodothyronine (FT3) than free thyroxine (FT4). CONCLUSION: This study confirmed that primary human thyrocytes could maintain the function of hormone production and secretion in the Transwell system, thus providing a useful tool to study thyroid function in vitro.