Thyroid Hormone Derivative Research Articles

The induction of myeloma cell death and DNA damage by tetrac, a thyroid hormone derivative

Multiple myeloma (MM) is a plasma cell malignancy in which involvement of the thyroid hormone-integrin αvβ3 pathway was shown, and pharmacologic inhibition of this pathway is a rational approach to disease management. A thyroid hormone derivative, tetraiodothyroacetic acid (tetrac), which inhibits l-thyroxine (T4) and 3,5,3'-triiodo-l-thyronine (T3) binding to αvβ3 integrin, was studied in five MM cell lines and primary bone marrow (BM) MM cells. Tetrac inhibited MM cell proliferation (absolute cell number/viability) and induced caspase-dependent apoptosis (annexin-V/PI and cell cycle). Activation of caspase-9 and caspase-3 was further demonstrated. Moreover, DNA damage markers, ataxia-telangiectasia-mutated (ATM) kinase, poly ADP-ribose polymerase (PARP-1) and histone γH2AX were induced by tetrac. The various tetrac-initiated effects were attenuated by Arg-Gly-Asp (RGD) peptide, suggesting integrin involvement. Primary BM mononuclear cells were harvested from MM patients (n = 39) at various disease stages. Tetrac-induced apoptosis (12/17 samples) and sensitized the cytotoxic action of bortezomib (6/9 samples). Lastly, expression of plasma membrane integrin αvβ3 was shown not only in the malignant plasma clone, but also in other cell populations within the BM samples (n = 25). Tetrac is anti-proliferative and pro-apoptotic in MM and cells may offer a therapeutic approach for this disease.

Both 3,5-Diiodo-L-Thyronine and 3,5,3'-Triiodo-L-Thyronine Prevent Short-term Hepatic Lipid Accumulation via Distinct Mechanisms in Rats Being Fed a High-Fat Diet.

3,3′,5-triiodo-L-thyronine (T3) improves hepatic lipid accumulation by increasing lipid catabolism but it also increases lipogenesis, which at first glance appears contradictory. Recent studies have shown that 3,5-diiodothyronine (T2), a natural thyroid hormone derivative, also has the capacity to stimulate hepatic lipid catabolism, however, little is known about its possible effects on lipogenic gene expression. Because genes classically involved in hepatic lipogenesis such as SPOT14, acetyl-CoA-carboxylase (ACC), and fatty acid synthase (FAS) contain thyroid hormone response elements (TREs), we studied their transcriptional regulation, focusing on TRE-mediated effects of T3 compared to T2 in rats receiving high-fat diet (HFD) for 1 week. HFD rats showed a marked lipid accumulation in the liver, which was significantly reduced upon simultaneous administration of either T3 or T2 with the diet. When administered to HFD rats, T2, in contrast with T3, markedly downregulated the expression of the above-mentioned genes. T2 downregulated expression of the transcription factors carbohydrate-response element-binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) involved in activation of transcription of these genes, which explains the suppressed expression of their target genes involved in lipogenesis. T3, however, did not repress expression of the TRE-containing ChREBP gene but repressed SREBP-1c expression. Despite suppression of SREBP-1c expression by T3 (which can be explained by the presence of nTRE in its promoter), the target genes were not suppressed, but normalized to HFD reference levels or even upregulated (ACC), partly due to the presence of TREs on the promoters of these genes and partly to the lack of suppression of ChREBP. Thus, T2 and T3 probably act by different molecular mechanisms to achieve inhibition of hepatic lipid accumulation.

3-Iodothyronamine, a Novel Endogenous Modulator of Transient Receptor Potential Melastatin 8?

The decarboxylated and deiodinated thyroid hormone (TH) derivative, 3-iodothyronamine (3-T1AM), is suggested to be involved in energy metabolism and thermoregulation. G protein-coupled receptors (GPCRs) are known as the main targets for 3-T1AM; however, transient receptor potential channels (TRPs) were also recently identified as new targets of 3-T1AM. This article reviews the current knowledge of a putative novel role of 3-T1AM in the modulation of TRPs. Specifically, the TRP melastatin 8 (TRPM8) was identified as a target of 3-T1AM in different cell types including neoplastic cells, whereby 3-T1AM significantly increased cytosolic Ca2+ through TRPM8 activation. Similarly, the β-adrenergic receptor is involved in 3-T1AM-induced Ca2+ influx. Therefore, it has been suggested that 3-T1AM-induced Ca2+ mobilization might be due to β-adrenergic receptor/TRPM8 channel interaction, which adds to the complexity of GPCR regulation by TRPs. It has been revealed that TRPM8 activation leads to a decline in TRPV1 activity, which may be of therapeutic benefit in clinical circumstances such as treatment of TRPV1-mediated inflammatory hyperalgesia, colitis, and dry eye syndrome. This review also summarizes the inverse association between changes in TRPM8 and TRPV1 activity after 3-T1AM stimulation. This finding prompted further detailed investigations of the interplay between 3-T1AM and the GPCR/TRPM8 axis and indicated the probability of additional GPCR/TRP constellations that are modulated by this TH derivative.

Abstract LB-129: NDAT and P-bi-TAT formulations of tetraiodothyroacetic acid (tetrac) induce devascularization, necrosis and apoptosis in glioblastoma xenografts

Abstract Thyroid hormone as L-thyroxine (T4) has been reported to stimulate proliferation of multiple glioma cell lines in vitro and there is clinical evidence that medical induction of hypothyroidism slows growth of glioblastoma multiforme (GBM). The proliferative action of T4 on glioma cells is initiated at a cell surface receptor for thyroid hormone on the extracellular domain of integrin αvβ3. Tetraiodothyroacetic acid (tetrac) is a thyroid hormone (T4) derivative that blocks T4 action at αvβ3 and has anticancer and anti-angiogenic activity. Tetrac has been covalently bonded via a linker to a nanoparticle (Nanotetrac, Nano-diamino-tetrac, NDAT) or to two polyethylene glycol (PEG) molecules (P-bi-TAT) that increase the potency of tetrac and broadens the anticancer properties of the drug. In the present studies of U87 MG GBM xenografts, NDAT administered daily for 10 days s.c. as 1 mg tetrac equivalent/kg reduced tumor xenograft weight at animal sacrifice by 50%, compared to untreated control lesions (p <0.01). Histopathological analysis of tumors revealed a 95% loss of the vascularity of treated tumors compared to controls at 10 days (p <0.001). There was no intratumoral hemorrhage. There was extensive cellular necrosis in treated tumors attributable to devascularization. Cells not necrotic in treated xenografts exhibited apoptosis, so that 80% of cells were either necrotic or apoptotic, compared to controls (p <0.001). Induction of apoptosis in cancer cells is a well-described quality of NDAT. P-bi-TAT treatment resulted in similar results after 20 days of treatment, but included a 21-day off-treatment follow-up after therapy. Xenografts achieved a 95% volume loss in the off-treatment, follow-up phase (P < 0.001). In summary, systemic NDAT and P-bi-TAT have been shown to be effective by multiple mechanisms in treatment of GBM xenografts. Citation Format: Paul J. Davis, Sudha Thangirala, Stewart Sell, Aleck Hercbergs, Shaker A. Mousa. NDAT and P-bi-TAT formulations of tetraiodothyroacetic acid (tetrac) induce devascularization, necrosis and apoptosis in glioblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-129. doi:10.1158/1538-7445.AM2017-LB-129

Halogen-Bonding Interactions of Polybrominated Diphenyl Ethers and Thyroid Hormone Derivatives: A Potential Mechanism for the Inhibition of Iodothyronine Deiodinase.

Polybrominated diphenyl ethers (PBDEs) increase flame resistance in consumer goods, but these compounds and their hydroxylated derivatives (OH-BDEs) impair normal thyroid function. Halogen bonding (XB) of PBDEs to an active site selenocysteine may prevent iodothyronine deiodinase(Dio)-catalyzed activation/deactivation of thyroid hormone (TH) derivatives. In this study, we compare the strength of the XB interactions of TH derivatives, iodine-based contrast agents and PBDEs/OH-BDEs with a methylselenolate model of the Dio active site using density functional theory calculations. The strength of the XB interaction depends upon the acceptor halide, the position of the halide, the number of ring substituents, and the proximity of hydroxyl groups to the XB site. The weaker Se⋅⋅⋅Br interactions relative to Se⋅⋅⋅I interactions are consistent with a model of competitive inhibition that blocks binding of THs at elevated PBDE/OH-BDE concentrations. XB interactions were generally more favorable at ortho and meta positions and in substrates with more electron-withdrawing substituents. PBDEs/OH-BDEs that mimic the binding behavior of THs, that is, containing ortho and meta bromides and adjacent hydroxyl groups, may be the most effective inhibitors. Highly-brominated PBDEs/OH-BDEs have comparable interaction energies to THs and may undergo debromination. These results may also suggest that XB strength must exceed a threshold value in order for PBDEs/OH-BDEs to undergo nucleophilic attack by Dio.

Nanoparticulate Tetrac Inhibits Growth and Vascularity of Glioblastoma Xenografts

Thyroid hormone as L-thyroxine (T4) stimulates proliferation of glioma cells in vitro and medical induction of hypothyroidism slows clinical growth of glioblastoma multiforme (GBM). The proliferative action of T4 on glioma cells is initiated nongenomically at a cell surface receptor for thyroid hormone on the extracellular domain of integrin αvβ3. Tetraiodothyroacetic acid (tetrac) is a thyroid hormone derivative that blocks T4 action at αvβ3 and has anticancer and anti-angiogenic activity. Tetrac has been covalently bonded via a linker to a nanoparticle (Nanotetrac, Nano-diamino-tetrac, NDAT) that increases the potency of tetrac and broadens the anticancer properties of the drug. In the present studies of human GBM xenografts in immunodeficient mice, NDAT administered daily for 10 days subcutaneously as 1 mg tetrac equivalent/kg reduced tumor xenograft weight at animal sacrifice by 50%, compared to untreated control lesions (p < 0.01). Histopathological analysis of tumors revealed a 95% loss of the vascularity of treated tumors compared to controls at 10 days (p < 0.001), without intratumoral hemorrhage. Up to 80% of tumor cells were necrotic in various microscopic fields (p < 0.001 vs. control tumors), an effect attributable to devascularization. There was substantial evidence of apoptosis in other fields (p < 0.001 vs. control tumors). Induction of apoptosis in cancer cells is a well-described quality of NDAT. In summary, systemic NDAT has been shown to be effective by multiple mechanisms in treatment of GBM xenografts.

3-Iodothyroacetic acid lacks thermoregulatory and cardiovascular effects in vivo.

3-Iodothyronamine (3-T1 AM) is an endogenous thyroid hormone derivative reported to induce strong hypothermia and bradycardia within minutes upon injection in rodents. Although 3-T1 AM is rapidly converted to several other metabolites in vivo, these strong pharmacological responses were solely attributed to 3-T1 AM, leaving potential contributions of downstream products untested. We therefore examined the cardiometabolic effects of 3-iodothyroacetic acid (TA1 ), the main degradation product of 3-T1 AM. We used a sensitive implantable radiotelemetry system in C57/Bl6J mice to study the effects of TA1 on body temperature and heart rate, as well as other metabolic parameters. Interestingly, despite using pharmacological TA1 doses, we observed no effects on heart rate or body temperature after a single TA1 injection (50 mg·kg(-1) , i.p.) compared to sham-injected controls. Repeated administration of TA1 (5 mg·kg(-1) , i.p. for 7 days) likewise did not alter body weight, food and water intake, heart rate, blood pressure, brown adipose tissue (BAT) thermogenesis or body temperature. Moreover, mRNA expression of tissue specific genes in heart, kidney, liver, BAT and lung was also not altered by TA1 compared to sham-injected controls. Our data therefore conclusively demonstrate that TA1 does not contribute to the cardiovascular or thermoregulatory effects observed after 3-T1 AM administration in mice, suggesting that the oxidative deamination constitutes an important deactivation mechanism for 3-T1 AM with possible implications for cardiovascular and thermoregulatory functions.

Evolutionary Conservation of 3-Iodothyronamine as an Agonist at the Trace Amine-Associated Receptor 1

Objectives: The trace amine-associated receptor 1 (Taar1) is a G_s protein-coupled receptor activated by trace amines, such as β-phenylethylamine (β-PEA) and 3-iodothyronamine (T₁AM). T₁AM is an endogenous biogenic amine and thyroid hormone derivative that exerts several biological functions. However, the physiological relevance of T₁AM acting via Taar1 is still under discussion. Therefore, we studied the structural and functional evolution of Taar1 in vertebrates to provide evidence for a conserved Taar1-mediated T₁AM function. Study Design: We searched public sequence databases to retrieve Taar1 sequence information from vertebrates. We cloned and functionally characterized Taar1 from selected vertebrate species using cAMP assays to determine the evolutionary conservation of T₁AM action at Taar1. Results: We found intact open reading frames of Taar1 in more than 100 vertebrate species, including mammals, sauropsids and amphibians. Evolutionary conservation analyses of Taar1 protein sequences revealed a high variation in amino acid residues proposed to be involved in agonist binding, especially in rodent Taar1 orthologs. Functional characterization showed that T₁AM, β-PEA and p-tyramine (p-Tyr) act as agonists at all tested orthologs, but EC₅₀ values of T₁AM at rat Taar1 differed significantly when compared to all other tested vertebrate Taar1. Conclusions: The high structural conservation of Taar1 throughout vertebrate evolution highlights the physiological relevance of Taar1, but species-specific differences in T₁AM potency at Taar1 orthologs suggest a specialization of rat Taar1 for T₁AM recognition. In contrast, β-PEA and p-Tyr potencies were rather conserved throughout all tested Taar1 orthologs. We provide evidence that the observed differences in potency are related to differences in constraint during Taar1 evolution.

3,5-Diiodo-L-thyronine activates brown adipose tissue thermogenesis in hypothyroid rats.

3,5-diiodo-l-thyronine (T2), a thyroid hormone derivative, is capable of increasing energy expenditure, as well as preventing high fat diet-induced overweight and related metabolic dysfunction. Most studies to date on T2 have been carried out on liver and skeletal muscle. Considering the role of brown adipose tissue (BAT) in energy and metabolic homeostasis, we explored whether T2 could activate BAT thermogenesis. Using euthyroid, hypothyroid, and T2-treated hypothyroid rats (all maintained at thermoneutrality) in morphological and functional studies, we found that hypothyroidism suppresses the maximal oxidative capacity of BAT and thermogenesis, as revealed by reduced mitochondrial content and respiration, enlarged cells and lipid droplets, and increased number of unilocular cells within the tissue. In vivo administration of T2 to hypothyroid rats activated BAT thermogenesis and increased the sympathetic innervation and vascularization of tissue. Likewise, T2 increased BAT oxidative capacity in vitro when added to BAT homogenates from hypothyroid rats. In vivo administration of T2 to hypothyroid rats enhanced mitochondrial respiration. Moreover, UCP1 seems to be a molecular determinant underlying the effect of T2 on mitochondrial thermogenesis. In fact, inhibition of mitochondrial respiration by GDP and its reactivation by fatty acids were greater in mitochondria from T2-treated hypothyroid rats than untreated hypothyroid rats. In vivo administration of T2 led to an increase in PGC-1α protein levels in nuclei (transient) and mitochondria (longer lasting), suggesting a coordinate effect of T2 in these organelles that ultimately promotes net activation of mitochondrial biogenesis and BAT thermogenesis. The effect of T2 on PGC-1α is similar to that elicited by triiodothyronine. As a whole, the data reported here indicate T2 is a thyroid hormone derivative able to activate BAT thermogenesis.

Analysis of human TAAR8 and murine Taar8b mediated signaling pathways and expression profile.

The thyroid hormone derivative 3-iodothyronamine (3-T1AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs.

3-Iodothyronamine-mediated metabolic suppression increases the phosphorylation of AMPK and induces fuel choice toward lipid mobilization.

Despite broad medical application, induction of artificial hypometabolism in vitro and its biochemical consequence have been rarely addressed. This study aimed to elucidate whether 3-iodothyronamine (T1AM) induces hypometabolism in an in vitro model with activation of AMP-activated protein kinase (AMPK) and whether it leads to a switch in primary fuel from carbohydrates to lipids as observed in in vivo models. Mouse C2C12 myotube and T1AM, a natural derivative of thyroid hormone, were used in this study. The oxygen consumption rate (OCR) decreased in a dose-dependent manner in response to 0-100 μM T1AM for up to 10 h. Upon 6-h of exposure to 75 μM T1AM, the OCR was reduced to 60 vs. ~ 95% for the control. The intracellular [AMP]/[ATP] was 1.35-fold higher in T1AM-treated cells. RT-PCR and immunoblotting analyses revealed that treated cells had upregulated p-AMPK/AMPK (1.8-fold), carnitine palmitoyl transferase 1 mRNA, and pyruvate dehydrogenase kinase, and downregulated acetyl CoA carboxylase (0.4-fold) and pyruvate dehydrogenase phosphatase. The treated cells had darker periodic acid-Schiff staining with 1.2-fold greater glycogen content than controls. Taken together, the hypometabolic response of myotubes to T1AM was dramatic and accompanied by increases in both the relative abundance of AMP and AMPK activation, and fuel choice favoring lipids over carbohydrates. These results are consistent with the general trends observed for rodent models and true hibernators.

3,3'-Diiodothyronine Concentrations in Hospitalized or Thyroidectomized Patients: Results from a Pilot Study

To determine if various medical conditions affect the serum concentrations of 3,3'-diiodothyronine (3,3'-T2). A total of 100 patients who were recruited from a group of inpatients and outpatients with a diverse range of medical conditions, donated a single blood sample that was assayed for thyroid hormone derivatives using liquid-chromatography tandem mass spectrometry (LC-MS/MS). The associations between 3,3'-T2 concentrations and physiologic data and medical conditions were assessed. Higher quartiles of 3,3'-T2 concentrations (quartile 1: 2.01-7.48, quartile 2: 7.74-12.4, quartile 3: 12.5-17, quartile 4: 17.9-45.8 pg/mL) were associated with decreasing occurrence of critical illness (58%, 11%, 0%, 8%), stroke (29%, 7.7%, 4%, 0%), critical care unit hospitalization (75%, 39%, 8.3 %, 12%), and inpatient status (83%, 42%, 8%, 12%) (all P<.001). The same quartiles were associated with increasing frequency of thyroidectomy (4%, 12%, 17%, 60%). In multivariate analyses, after adjustment for age and sex, inpatient status was associated with decreasing concentrations of 3,3'-T2 (46% decrease for inpatients with 95% confidence interval [CI] 32-57%, P<.0001). Thyroidectomy was associated with increasing concentrations of 3,3'-T2 (29% increase (CI 0.5-66%, P = .049). We observed associations between inpatient status and reduced 3,3'-T2 concentrations. This appears to be a global change associated with illness, rather than an association with specific medical conditions. We also observed higher 3,3'-T2 concentrations in athyreotic outpatients receiving thyroid-stimulating hormone (TSH) suppression therapy. This demonstrates that there is production of 3,3'-T2 from levothyroxine (LT4) in extrathyroidal tissues. Conversion of thyroxine (T4) to 3,3'-T2 via both triiodothyronine (T3) and reverse triiodothyronine (rT3) pathways may prevent excessive T3 concentrations in such patients.

Regioselective deiodination of iodothyronamines, endogenous thyroid hormone derivatives, by deiodinase mimics.

Iodothyronine deiodinases (IDs) are mammalian selenoenzymes that play an important role in the activation and inactivation£ of thyroid hormones. It is known that iodothyronamines (TnAMs), produced by the decarboxylation of thyroid hormones, act as substrates for deiodinases. To understand whether decarboxylation alters the rate and/or regioselectivity of deiodination by using synthetic deiodinase mimics, we studied the deiodination of different iodothyronamines. The triiodo derivative 3,3',5-triiodothyronamine (T3 AM) is deiodinated at the inner ring by naphthyl-based deiodinase mimics, which is similar to the deiodination of 3,3',5-triiodothyronine (T3). However, T3 AM undergoes much slower deiodination than T3. Detailed experimental and theoretical investigations suggest that T3 AM forms a weaker halogen bond with selenium donors than T3. Kinetic studies and single-crystal X-ray structures of T3 and T3 AM reveal that intermolecular I⋅⋅⋅I interactions may play an important role in deiodination. The formation of hydrogen- and halogen-bonding assemblies, which leads to the formation of a dimeric species of T3 in solution, facilitates the interactions between the selenium and iodine atoms. In contrast, T3 AM, which does not have I⋅⋅⋅I interactions, undergoes much slower deiodination.

Aspects of 3-iodothyronamine (3T1AM) induced signaling by human and mouse trace amine-associated receptor 5 (TAAR5)

The thyroid hormone derivative 3-iodothyronamine (3T1AM) is an agonist for trace amine-associated receptor 1 (TAAR1). Application of 3T1AM in rodents results in a variety of effects, including decreased body temperature. Taar1-/- mice still show these effects, excluding Taar1 as primary mediator of 3T1AM induced action in vivo. Because of its role as potential treatment option for stroke patients, possible targets and effects of 3T1AM in humans are of medical interest. Here we investigated the human (hTAAR5) and mouse Taar5 (mTaar5) as potential target to explain 3T1AM effects in Taar1-/- mice. In situ hybridization for mTaar1 and mTaar5 revealed overlapping expression profiles in the amygdala and VMH. Next, we investigated several signaling pathways of mTaar5 and hTAAR5. We confirmed previously reported data that the volatile amine dimethylethylamine is an agonist for Gs activation by mTaar5, however, not for hTAAR5. Further investigations revealed a basal tone of ERK and Gq activation for hTAAR5 and mTaar5 and additional basal Gs activity for mTaar5 mediating cAMP accumulation. In response to 3T1AM, no signaling via Gs, Gq, Gi, G12/13 and ERK was observed for hTAAR5 and mTaar5, while 3T1AM acted as inverse agonist on hTAAR5 by decreasing the basal tone of IP3 accumulation and ERK. To elucidate the particular determinants involved in signaling differences between hTAAR5 and mTaar5 we created chimeric receptors, aiming to transfer murine signaling properties to hTAAR5. We identified 6 amino acids in the ligand binding and G protein-coupling region to be involved. Our data demonstrated that caution is needed when transferring signaling properties of mTaar5 to the human situation. While observed pharmacological effects of 3T1AM on Taar1-/- mice are likely not related to mTaar5, according to our in vitro data, hTAAR5 functions as a target for 3T1AM and exhibits inhibitory 3T1AM effects on Gq and ERK signaling. Supported by DFG-SPP1629 ThyroidTransAct

Elucidating the actions of 3-Iodothyroacetic acid in thermoregulation and cardiovascular function

Introduction: 3-Iodo-L-thyronamine (3-T1AM) is an endogenous thyroid hormone derivative linking endocrinology, cardiovascular function and metabolism. A single injection of 3-T1AM induces hypothermia and bradycardia within minutes upon injection. Until now, the biosynthesis of thyronamines as well as the molecular mechanisms behind the pharmacological effects are still unknown. Recently, it was shown that a single i.p. injection of 3-T1AM in mice lead rapidly to the formation of the corresponding thyroacetic acid (TA1); however, it is unclear if the acetic form contributes to the pharmacological effects observed after 3-T1AM administration. This project therefore aims to determine if TA1 plays a role in the control of thermoregulation and cardiovascular function.

Schilddrüsenhormonwirkung – jenseits klassischer Konzepte

Thyroid hormones are of crucial importance for the function of nearly all organ systems. In case of dysfunction of thyroid hormone production and function many organ systems may be affected. The estimation of normal thyroid function is based on determination of TSH and the thyroid hormones T3 and T4. However, international conventions about the normal TSH range are still lacking which bears consequences for patient`s treatment. Hence not unexpected, many patients complain although their thyroid hormone status is in the normal range by clinical estimation. Here, more precise parameters are needed for a better definition of the healthy thyroid status of an individual. Recently, new key players in the system of thyroid hormone action were detected, like specific transporters for uptake of thyroid hormones and thyroid hormone derivatives. DFG, the German Research Foundation supports the priority program Thyroid Trans Act to find answers to the main question: what defines the healthy thyroid status of an individual. The overall aim of this interdisciplinary research consortium is to specify physiological and pathophysiological functions of thyroid hormone transporters and thyroid hormone derivative as new players in thyroid regulation in order to better evaluate, treat, and prevent thyroid-related disease.

Effects of acute microinjections of the thyroid hormone derivative 3-iodothyronamine to the preoptic region of adult male rats on sleep, thermoregulation and motor activity

The decarboxylated thyroid hormone derivative 3-iodothyronamine (T1AM) has been reported as having behavioral and physiological consequences distinct from those of thyroid hormones. Here, we investigate the effects of T1AM on EEG-defined sleep after acute administration to the preoptic region of adult male rats. Our laboratory recently demonstrated a decrease in EEG-defined sleep after administration of 3,3′,5-triiodo-l-thyronine (T3) to the same brain region. After injection of T1AM or vehicle solution, EEG, EMG, activity, and core body temperature were recorded for 24h. Sleep parameters were determined from EEG and EMG data. Earlier investigations found contrasting systemic effects of T3 and T1AM, such as decreased heart rate and body temperature after intraperitoneal T1AM injection. However, nREM sleep was decreased in the present study after injections of 1 or 3μg T1AM, but not after 0.3 or 10μg, closely mimicking the previously reported effects of T3 administration to the preoptic region. The biphasic dose–response observed after either T1AM or T3 administration seems to indicate shared mechanisms and/or functions of sleep regulation in the preoptic region. Consistent with systemic administration of T1AM, however, microinjection of T1AM decreased body temperature. The current study is the first to show modulation of sleep by T1AM, and suggests that T1AM and T3 have both shared and independent effects in the adult mammalian brain.

The Synthesis of 13C9-15N-Labeled 3,5-Diiodothyronine and Thyroxine

Thyroid hormones undergo extensive metabolism to regulate hormone activity. A labeled thyroid hormone would be useful to track hormone metabolism through various pathways. While radiolabeled thyroid hormones have been synthesized and used for in vivo studies, a stable isotope labeled form of thyroid hormone is required for studying thyroid hormone metabolism by LC-MS/MS, an analytical technique that has certain advantages without the complications of radioactivity. Here we report the synthesis of 13C9-15N-T2 and 13C9-15N-T4, two labeled thyroid hormone derivatives suitable for in vivo LC-MS/MS studies. Supplemental materials are available for this article. Go to the publisher's online edition of Synthetic Communications® to view the free supplemental file.

Rapid and Comprehensive Impurity Profiling of Synthetic Thyroxine by Ultrahigh-Performance Liquid Chromatography–High-Resolution Mass Spectrometry

Rapid and efficient quality control according to the public authority regulations is mandatory to guarantee safety of the pharmaceuticals and to save resources in the pharmaceutical industry. In the case of so-called "grandfather products" like the synthetic thyroid hormone thyroxine, strict regulations enforce a detailed chemical analysis in order to characterize potentially toxic or pharmacologically relevant impurities. We report a straightforward workflow for the comprehensive impurity profiling of synthetic thyroid hormones and impurities employing ultrahigh-performance liquid chromatography (UHPLC) hyphenated to high-resolution mass spectrometry (HRMS). Five different batches of synthetic thyroxin were analyzed resulting in the detection of 71 impurities within 3 min total analysis time. Structural elucidation of the compounds was accomplished via a combination of accurate mass measurements, computer based calculations of molecular formulas, multistage high-resolution mass spectrometry (HRMS(n)), and nuclear magnetic resonance spectroscopy, which enabled the identification of 71 impurities, of which 47 have been unknown so far. Thirty of the latter were structurally elucidated, including products of deiodination, aliphatic chain oxidation, as well as dimeric compounds as new class of thyroid hormone derivatives. Limits of detection for the thyroid compounds were in the 6 ng/mL range for negative electrospray ionization mass spectrometric detection in full scan mode. Within day and day-to-day repeatabilities of retention times and peak areas were below 0.5% and 3.5% R.SD. The performance characteristics of the method in terms of robustness and information content clearly show that UHPLC-HRMS is adequate for the rapid and reliable detection, identification, and semiquantitative determination of trace levels of impurities in synthetic pharmaceuticals.

Biosynthesis of 3-Iodothyronamine (T1AM) Is Dependent on the Sodium-Iodide Symporter and Thyroperoxidase but Does Not Involve Extrathyroidal Metabolism of T4

3-Iodothyronamine (T(1)AM) is an endogenous thyroid hormone derivative with unknown biosynthetic origins. Structural similarities have led to the hypothesis that T(1)AM is an extrathyroidal metabolite of T(4). This study uses an isotope-labeled T(4) [heavy-T(4) (H-T(4))] that can be distinguished from endogenous T(4) by mass spectrometry, which allows metabolites to be identified based on the presence of this unique isotope signature. Endogenous T(1)AM levels depend upon thyroid status and decrease upon induction of hypothyroidism. However, in hypothyroid mice replaced with H-T(4), the isotope-labeled H-T(3) metabolite is detected, but no isotope-labeled T(1)AM is detected. These data suggest that T(1)AM is not an extrathyroidal metabolite of T(4), yet is produced by a process that requires the same biosynthetic factors necessary for T(4) synthesis.