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The thyroid hormone metabolite 3-iodothyronamine (3-T1AM) exerts diverse physiological reactions such as a decrease of body temperature, and negative inotropic and chronotropic effects. This observed pleomorphic effect in physiology can be barely explained by interaction with only one target protein such as the trace-amine receptor 1 (TAAR1), a class A G-protein coupled receptor (GPCR). Moreover, Taar1 knock-out mice still react to 3-T1AM through physiological responses with a rapid decrease in body temperature. These facts propelled our group and others to search for further targets for this molecule.The group of TAARs evolved early in evolution and, according to sequence similarities, they are closely related to adrenoceptors and other aminergic receptors. Therefore, several of these receptors were characterized by their potential to interplay with 3-T1AM. Indeed, 3-T1AM acts as a positive allosteric modulator on the beta2-adrenoceptor (ADRB2) and as a biased agonist on the serotonin receptor 1B (5HT1b) and the alpha2-adrenoceptor (ADRA2A). In addition, 3-T1AM was reported to be a weak antagonist at a non-aminergic muscarinic receptor (M3).These findings impressively reflect that such trace amines can unselectively and simultaneously function at different receptors expressed by one cell or at different tissues. In conclusion, the role of 3-T1AM is hypothesized to concert the fine-tuning of specific cell reactions by the accentuation of certain pathways dependent on distinct receptors. 3-T1AM acts as a regulator of signals by blocking, modulating, or inducing simultaneously distinct intracellular signaling cascades via different GPCRs.

The rediscovery of the group of thyronamines (TAMs), especially the first detailed description of their most prominent congener 3-iodothyronamine (3T1AM) 14 years ago, boosted research on this thyroid hormone metabolite tremendously. TAMs exert actions partly opposite to and distinct from known functions of thyroid hormones. These fascinating metabolic, anapyrexic, cytoprotective, and brain effects quickly evoked the hope to use hormone-derived TAMs as a therapeutic option. The G protein-coupled receptor (GPCR) TAAR1, a member of the trace amine-associated receptor (TAAR) family, was identified as the first target and effector of TAM action. The initial enthusiasm on pharmacological actions of exogenous TAMs elicited many questions, such as sites of biosynthesis, analytics, modes of action, inactivation, and role of TAMs in (patho)physiology. Meanwhile, it became clear that TAMs not only interact with TAAR1 or other TAAR family members but also with several aminergic receptors and non-GPCR targets such as transient receptor potential channels, mitochondrial proteins, and the serum TAM-binding protein apolipoprotein B100, thus classifying 3T1AM as a multitarget ligand. The physiological mode of action of TAMs is still controversial because regulation of endogenous TAM production and the sites of its biosynthesis are not fully elucidated. Methods for 3T1AM analytics need further validation, as they revealed different blood and tissue concentrations depending on detection principles used such as monoclonal antibody-based immunoassay vs liquid chromatography- matrix-assisted laser desorption/ionization mass spectrometry or time-of-flight mass spectrometry. In this review, we comprehensively summarize and critically evaluate current basic, translational, and clinical knowledge on 3T1AM and its main metabolite 3-iodothyroacetic acid, focusing on endocrine-relevant aspects and open but highly challenging issues.

Background Coronavirus disease 2019 (COVID-19) has been shown to affect almost every organ throughout the body. However, it is not clear whether the thyroid gland is impaired in COVID-19 patients. Euthyroid sick syndrome (ESS) is usually associated with the disease severity and deterioration prognosis in critical illness. In this study, the thyroid function of COVID-19 patients was assessed and factors associated with outcomes were analyzed to determine the potential predictive value of ESS. Methods Clinical and laboratory data of COVID-19 patients with or without ESS in Changsha, China, were collected and analyzed on admission. Kaplan-Meier curve and cox regression model were utilized to determine the correlation between ESS and the endpoints. Subsequently, a receiver operating characteristic (ROC) curve was plotted to evaluate the predictive performances of FT3 and C-reactive protein (CRP) in the disease severity. Results Forty-one (27.52%) cases of COVID-19 patients diagnosed with ESS. ESS patients had higher proportions of fever, shortness of breath, hypertension, diabetes, and severe events than those of non-ESS patients. The levels of erythrocyte sedimentation rate and C-reactive protein, and the positive rate of procalcitonin were significantly higher, whereas the lymphocyte count was apparently lower in ESS patients than in non-ESS patients. The regression analysis showed that ESS was significantly associated with the disease severity of COVID-19 (HR = 2.515, 95% CI: 1.050–6.026, P = 0.039). The areas under the curve (AUCs) for predicting the severe disease were [0.809 (95% CI 0.727–0.892), P < 0.001] and [0.792 (95% CI 0.689–0.895), P < 0.001] for FT3 and CRP, respectively. Conclusion ESS was significantly associated with the disease severity and inflammatory parameters in COVID-19 patients.

We investigated the effect of 3-iodothyronamine (T1AM) on thermogenic substrates in brown adipocytes (BAs). BAs isolated from the stromal fraction of rat brown adipose tissue were exposed to an adipogenic medium containing insulin in the absence (M) or in the presence of 20 nM T1AM (M+T1AM) for 6 days. At the end of the treatment, the expression of p-PKA/PKA, p-AKT/AKT, p-AMPK/AMPK, p-CREB/CREB, p-P38/P38, type 1 and 3 beta adrenergic receptors (β1–β3AR), GLUT4, type 2 deiodinase (DIO2), and uncoupling protein 1 (UCP-1) were evaluated. The effects of cell conditioning with T1AM on fatty acid mobilization (basal and adrenergic-mediated), glucose uptake (basal and insulin-mediated), and ATP cell content were also analyzed in both cell populations. When compared to cells not exposed, M+T1AM cells showed increased p-PKA/PKA, p-AKT/AKT, p-CREB/CREB, p-P38/P38, and p-AMPK/AMPK, downregulation of DIO2 and β1AR, and upregulation of glycosylated β3AR, GLUT4, and adiponectin. At basal conditions, glycerol release was higher for M+T1AM cells than M cells, without any significant differences in basal glucose uptake. Notably, in M+T1AM cells, adrenergic agonists failed to activate PKA and lipolysis and to increase ATP level, but the glucose uptake in response to insulin exposure was more pronounced than in M cells. In conclusion, our results suggest that BAs conditioning with T1AM promote a catabolic condition promising to fight obesity and insulin resistance.

The development of thyroid hormone (TH) analogues was prompted by the attempt to exploit the effects of TH on lipid metabolism, avoiding cardiac thyrotoxicosis. Analysis of the relative distribution of the α and β subtypes of nuclear TH receptors (TRα and TRβ) showed that TRα and TRβ are responsible for cardiac and metabolic responses, respectively. Therefore, analogues with TRβ selectivity were developed, and four different compounds have been used in clinical trials: GC-1 (sobetirome), KB-2115 (eprotirome), MB07344/VK2809, and MGL-3196 (resmetirom). Each of these compounds was able to reduce low-density lipoprotein cholesterol, but a phase 3 trial with eprotirome was interrupted because of a significant increase in liver enzymes and the contemporary report of cartilage side effects in animals. As a consequence, the other projects were terminated as well. However, in recent years, TRβ agonists have raised new interest for the treatment of nonalcoholic fatty liver disease (NAFLD). After obtaining excellent results in experimental models, clinical trials have been started with MGL-3196 and VK2809, and the initial reports are encouraging. Sobetirome turned out to be effective also in experimental models of demyelinating disease. Aside TRβ agonists, TH analogues include some TH metabolites that are biologically active on their own, and their synthetic analogues. 3,5,3'-triiodothyroacetic acid has already found clinical use in the treatment of some cases of TH resistance due to TRβ mutations, and interesting results have recently been reported in patients with the Allan-Herndon-Dudley syndrome, a rare disease caused by mutations in the TH transporter MCT8. 3,5-diiodothyronine (T2) has been used with success in rat models of dyslipidemia and NAFLD, but the outcome of a clinical trial with a synthetic T2 analogue was disappointing. 3-iodothyronamine (T1AM) is the last entry in the group of active TH metabolites. Promising results have been obtained in animal models of neurological injury induced by β-amyloid or by convulsive agents, but no clinical data are available so far.

Objectives l-dopa decarboxylase (DDC) is responsible for the synthesis of dopamine. Dopamine, which binds to the D2-dopamine receptor (D2R), plays an important role in the maintenance of pregnancy. Aim of our study was the analysis of DDC and D2R expression in placentas of spontaneous miscarriages (SMs) and recurrent miscarriages (RMs) in comparison to healthy controls. Methods Patients with SM (n = 15) and RM (n = 15) were compared with patients from healthy pregnancies (n = 15) (pregnancy weeks 7–13 each). Placental tissue has been collected from SMs and RMs from the first trimester (Department of Gynaecology and Obstetrics, LMU Munich) and from abruptions (private practice, Munich). Placental cell lines, BeWo- and JEG-3 cells, were stimulated with the trace amines T0AM and T1AM in vitro. Results Levels of DDC and D2R in trophoblasts and the decidua were lower in RMs in comparison to healthy controls. Stimulation of BeWo cells with T1AM significantly reduced DDC mRNA and protein levels. Via double-immunofluorescence, a DDC-positive cell type beneath decidual stromal cells and foetal EVT in the decidua could be detected. Conclusions Downregulation of DDC and D2R in trophoblasts of RMs reflects a reduced signal cascade of catecholamines on the foetal side.

Purpose 3-iodothyronamine (3-T1AM), a decarboxylated and deiodinated thyroid hormone metabolite, leads at pharmacological doses to hypoinsulinemia, hyperglucagonemia and hyperglycemia in vivo. As the pancreatic Langerhans islets express thyroid hormone transmembrane transporters (THTT), we tested the hypothesis that not only plasma membrane-mediated 3-T1AM binding to and activation of G-protein coupled receptors, but also 3-T1AM metabolite(s) generated by 3-T1AM uptake and metabolism might decrease glucose-stimulated insulin secretion (GSIS). Methods Murine pancreatic β-cells MIN6 were characterized for gene expression of THTT, deiodinases and monoamine oxidases. 3-T1AM uptake and intracellular metabolism to the corresponding 3-iodothyroacetic acid were analysed by liquid-chromatography tandem mass spectrometry (LC-MS/MS) at different time points in cells as well as the conditioned medium. Mitochondrial activity, especially ATP-production, was monitored real-time after 3-T1AM application using Seahorse Bioanalyzer technique. Effect of 3-T1AM on GSIS into the culture medium was assayed by ELISA. Results MIN6 cells express classical THTT, proposed to transport 3-T1AM, as well as 3-T1AM metabolizing enzymes comparable to murine primary pancreatic islets. 3-T1AM accumulates in MIN6 cells and is metabolized by intracellular MaoB to 3-iodothyroacetic, which in turn is rapidly exported. 3-T1AM decreases mitochondrial ATP-production concentration dependently. GSIS is diminished by 3-T1AM treatment. Using LC-MS/MS, no further 3-T1AM metabolites except 3-iodothyroacetic were detectable. Conclusions This data provides a first link between cellular 3-T1AM uptake and regulation of mitochondrial energy metabolism in s-cells, resulting in reduced insulin secretion. We conclude that MIN6 is an appropriate cell model to study 3-T1AM-dependent (intra-)cellular biochemical mechanisms affecting insulin production in vitro.

We previously demonstrated that 3-iodothyronamine (T1AM), a by-product of thyroid hormone metabolism, pharmacologically administered to mice acutely stimulated learning and memory acquisition and provided hyperalgesia with a mechanism which remains to be defined. We now aimed to investigate whether the T1AM effect on memory and pain was maintained in mice pre-treated with scopolamine, a non-selective muscarinic antagonist expected to induce amnesia and, possibly, hyperalgesia. Mice were pre-treated with scopolamine and, after 20 min, injected intracerebroventricularly (i.c.v.) with T1AM (0.13, 0.4, 1.32 μg/kg). 15 min after T1AM injection, the mice learning capacity or their pain threshold were evaluated by the light/dark box and by the hot plate test (51.5 °C) respectively. Experiments in the light/dark box were repeated in mice receiving clorgyline (2.5 mg/kg, i.p.), a monoamine oxidase (MAO) inhibitor administered 10 min before scopolamine (0.3 mg/kg). Our results demonstrated that 0.3 mg/kg scopolamine induced amnesia without modifying the murine pain threshold. T1AM fully reversed scopolamine-induced amnesia and produced hyperalgesia at a dose as low as 0.13 μg/kg. The T1AM anti-amnestic effect was lost in mice pre-treated with clorgyline. We report that the removal of muscarinic signalling increases T1AM pro learning and hyperalgesic effectiveness suggesting T1AM as a potential treatment as a “pro-drug” for memory dysfunction in neurodegenerative diseases.

3-Iodothyronamine (T1AM) is an endogenous metabolite of thyroid hormone with noticeable metabolic and neurological effects. Alteration of feeding behaviour by this compound was different at various doses in experimental models and it is not clear whether this effect is partially accounted by changes in neuropeptide secretion. In this study, we attempted to find out whether chronic low dose 3-iodothyronamine treatment could modulate some food intake regulatory neuropeptides such as leptin, ghrelin, and galanin in mice brain. Eighteen male mice were divided randomly into control (n = 8) and treatment (n = 10) groups. The experimental procedure was applied for 7 days during which treatment group received T1AM (i.p) whereas the control group received DMSO and normal saline. The brain was analyzed for leptin, ghrelin, and galanin concentrations. There were significant differences in leptin concentration (1.75 ± 0.05 versus 2.9 ± 0.07 ng/ml) and ghrelin concentration (8.4 ± 0.35 versus 5 ± 0.08 ng/ml) between control and treatment groups (P < 0.05). There was no significant difference in galanin concentration (745.87 ± 34.91 ng/l) in control group compared with the treatment group (698.05 ± 66.88 ng/l). Interestingly, the treatment group mice lost weight (~1 g) whereas non-significant increase in weight mean was seen in control group before (day 1) and after the procedure (day 8). Clearly, further works in this area will be required to delineate the central role of T1AM, but based on our findings described here, we propose that some of peripheral metabolic effects of this compound may be accomplished by brain peptide regulation.

Background Classical thyroid hormones have an established necessary role in the normal development of the central nervous system, and they have been recently considered as decisive factors influencing cognitive functions in the adult brain and involved in the development of Alzheimer's disease. The picture summarizing thyroid hormone effects on the adult brain, however, does not only include classical thyroid hormones but also the products of their peripheral metabolism. These latter have been considered as inactive breakdown products for long but recently were proved to produce significant biological effects. Objective In this review article we presented recent evidence supporting the hypothesis that thyroid hormones exert a neuroprotective effect in the brain areas involved in learning and memory. Moreover, we summarized the evidence that suggests that non-classical thyroid hormones produce significant neurological effects in the adult brain. We also discussed the possible role of thyroid hormones in the cognitive impairment, typical of Alzheimer's disease. Methods A comprehensive review of the literature based on the current knowledge of the effects of classical and nonclassical thyroid hormones on the adult brain and their role in Alzheimer's disease was performed. Results The available literature suggests that both classical and non-classical thyroid hormones act as neuroprotective agents in the brain areas related to learning and memory. Their role in these areas supports the idea that they may be involved in the development of Alzheimer's disease. Conclusion Thyroid hormones produce significant neurological effects, act as neuroprotective agents and might be considered as future diagnostic and therapeutic tools for Alzheimer's disease.

Background: A novel form of thyroid hormone (TH) signaling is represented by 3-iodothyronamine (T1AM), an endogenous TH derivative that interacts with specific molecular targets, including trace am...

This study was undertaken to determine if crosstalk among the transient receptor potential (TRP) melastatin 8 (TRPM8), TRP vanilloid 1 (TRPV1), and vascular endothelial growth factor (VEGF) receptor triad modulates VEGF-induced Ca2+ signaling in human corneal keratocytes. Using RT-PCR, qPCR and immunohistochemistry, we determined TRPV1 and TRPM8 gene and protein coexpression in a human corneal keratocyte cell line (HCK) and human corneal cross sections. Fluorescence Ca2+ imaging using both a photomultiplier and a single cell digital imaging system as well as planar patch-clamping measured relative intracellular Ca2+ levels and underlying whole-cell currents. The TRPV1 agonist capsaicin increased both intracellular Ca2+ levels and whole-cell currents, while the antagonist capsazepine (CPZ) inhibited them. VEGF-induced Ca2+ transients and rises in whole-cell currents were suppressed by CPZ, whereas a selective TRPM8 antagonist, AMTB, increased VEGF signaling. In contrast, an endogenous thyroid hormone-derived metabolite 3-Iodothyronamine (3-T1AM) suppressed increases in the VEGF-induced current. The TRPM8 agonist menthol increased the currents, while AMTB suppressed this response. The VEGF-induced increases in Ca2+ influx and their underlying ionic currents stem from crosstalk between VEGFR and TRPV1, which can be impeded by 3-T1AM-induced TRPM8 activation. Such suppression in turn blocks VEGF-induced TRPV1 activation. Therefore, crosstalk between TRPM8 and TRPV1 inhibits VEGFR-induced activation of TRPV1.

Obesity is a complex disease associated with environmental and genetic factors. 3-Iodothyronamine (T1AM) has revealed great potential as an effective weight loss drug. We used metabolomics and associated transcriptional gene and protein expression analysis to investigate the tissue specific metabolic reprogramming effects of subchronic T1AM treatment at two pharmacological daily doses (10 and 25 mg/kg) on targeted metabolic pathways. Multi-analytical results indicated that T1AM at 25 mg/kg can act as a novel master regulator of both glucose and lipid metabolism in mice through sirtuin-mediated pathways. In liver, we observed an increased gene and protein expression of Sirt6 (a master gene regulator of glucose) and Gck (glucose kinase) and a decreased expression of Sirt4 (a negative regulator of fatty acids oxidation (FAO)), whereas in white adipose tissue only Sirt6 was increased. Metabolomics analysis supported physiological changes at both doses with most increases in FAO, glycolysis indicators and the mitochondrial substrate, at the highest dose of T1AM. Together our results suggest that T1AM acts through sirtuin-mediated pathways to metabolically reprogram fatty acid and glucose metabolism possibly through small molecules signaling. Our novel mechanistic findings indicate that T1AM has a great potential as a drug for the treatment of obesity and possibly diabetes.

The endogenous metabolite 3-iodothyronamine (3-T1AM) induces strong hypothermia and bradycardia at pharmacological doses. Although its biosynthesis from thyroid hormone precursors appears likely, the sequence and sites of reactions are still controversial: studies in T4-substituted thyroid cancer patients lacking functional thyroid tissue suggested extrathyroidal 3-T1AM production, whereas studies using labeled T4 in mice indicated intrathyroidal formation. However, because the patients received T4 orally, whereas the mice were injected ip, we hypothesized that 3-T1AM synthesis requires the intestinal passage of T4. Using the everted gut sac model in combination with mass spectrometry, we demonstrate 3-T1AM production from T4 in mouse intestine via several deiodination and decarboxylation steps. Gene expression analysis confirmed the expression of all 3 deiodinases as well as ornithine decarboxylase (ODC) in intestine. Subsequent experiments employing purified human ODC revealed that this enzyme can in fact mediate decarboxylation of 3,5-T2 and T4 to the respective thyronamines (TAMs), demonstrating that the intestine expresses the entire molecular machinery required for 3-T1AM biosynthesis. Interestingly, TAM production was strongly affected by the antithyroid treatment methimazole and perchlorate independently of thyroid status, limiting the validity of the respective mouse models in this context. Taken together, our data demonstrate intestinal 3-T1AM biosynthesis from T4 involving decarboxylation through ODC with subsequent deiodination, and explain the apparent discrepancy between 3-T1AM serum levels in patients substituted orally and mice injected ip with T4. Identifying ODC as the first enzyme capable of decarboxylating thyroid hormone, our findings open the path to further investigations of TAM metabolism on molecular and cellular levels.

Objectives Thyronamines are decarboxylated and deiodinated metabolites of thyroid hormones (THs). Of all possible thyronamine variants, only 3-iodothyronamine (3-T1AM) and iodine-free thyronamine (T0AM) have been detected in vivo. While intensive research has been done on the (patho-)physiological action of 3-T1AM, the role of T0AM has been studied less intensively. Study design We determined whether a single pharmacological dose (50 mg/kg, i.p.) or repeated administration (5 mg/kg/day, i.p., for 7 days) of T0AM affects metabolism, cardiovascular function, or thermoregulation in male C57BL/6J mice. Since selenium (Se) is important for proper TH function and Se metabolism is affected by TH, we additionally analyzed Se concentrations in liver, serum, and kidney using total reflection X-ray analysis. Results A single injection of T0AM had no effect on heart rate, temperature, or activity as assessed by radio telemetry. Likewise, daily administration of T0AM did not alter body weight, food or water intake, heart rate, blood pressure, brown adipose tissue thermogenesis, or body temperature, and no significant differences in hepatic glycogen content or mRNA expression of genes involved in cardiovascular function or metabolic control were determined. Also, the X-ray analysis of Se concentrations revealed no significant changes. However, hepatic T0AM was significantly increased in the treated animals. Conclusions In summary, our data demonstrate that T0AM elicits no obvious metabolic, cardiovascular, or thermoregulatory activities in mice. As T0AM does also not interfere with TH or Se metabolism, we conclude that the deiodination of 3-T1AM to T0AM constitutes an efficient inactivation mechanism, terminating the actions of the more powerful precursor.

Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and tentatively attributed to problems in extraction or other pre-analytical steps. For this reason, even cell culture experiments have often be performed with unphysiological protein-free media. The aim of this study was to evaluate the recovery of exogenous T1AM added to a standard cell culture medium, namely Dulbecco's minimum essential medium (DMEM) supplemented with fetal bovine serum (FBS), and to other biological matrixes.Cell culture media (Krebs-Ringer buffer, DMEM, FBS, DMEM + FBS, used either in the absence or in the presence of NG108-15 cells) and other biological matrixes (rat brain and liver homogenates, human plasma, and blood) were spiked with T1AM and/or deuterated T1AM (d4-T1AM) and incubated for times ranging from 0 to 240 minutes. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry in order to assay T1AM and its metabolites, namely 3-iodothyroacetic acid (TA1), thyronamine, thyroacetic acid, N-acetyl-T1AM, and T1AM esters.In FBS-containing buffers, T1AM decreased exponentially over time, with a half-life of 6-17 minutes, depending on FBS content, and after 60 minutes, it averaged 0-10% of the baseline. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4-T1AM decreased over time at a much lower rate, reaching 50-70% of the baseline at 60 minutes. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 minutes, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human blood.These results suggest binding and sequestration of T1AM and/or its aldehyde derivative by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenous T1AM.

The decarboxylated and deiodinated thyroid hormone 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 add 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 thyroid hormone derivative.

3-iodothyroacetic acid (TA1) is among the by-products of thyroid hormone metabolism suspected to mediate the non-genomic effects of the hormone (T3). We aim to investigate whether TA1 systemically administered to mice stimulated mice wakefulness, an effect already described for T3 and for another T3 metabolite (i.e. 3-iodothryonamine; T1AM), and whether TA1 interacted at GABA-A receptors (GABA-AR). Mice were pre-treated with either saline (vehicle) or TA1 (1.32, 4 and 11 μg/kg) and, after 10 min, they received ethanol (3.5 g/kg, i.p.). In another set of experiments, TA1 was administered 5 min after ethanol. The latency of sleep onset and the time of sleep duration were recorded. Voltage-clamp experiments to evaluate the effect of 1 μM TA1 on bicuculline-sensitive currents in acute rat hippocampal slice neurons and binding experiments evaluating the capacity of 1, 10, 100 μM TA1 to displace [3H]flumazenil from mice brain membranes were also performed. 4 μg/kg TA1 increases the latency of onset and at 1.32 and 4 μg/kg it reduces the duration of ethanol-induced sleep only if administered before ethanol. TA1 does not functionally interact at GABA-AR. Overall these results indicate a further similarity between the pharmacological profile of TA1 and that of T1AM.

Background and Purpose 3-Iodothyroacetic acid (TA1) is an end product of thyroid hormone metabolism. So far, it is not known if TA1 is present in mouse brain and if it has any pharmacological effects. Experimental Approach TA1 levels in mouse brain were measured by HPLC coupled to mass spectrometry. After i.c.v. administration of exogenous TA1 (0.4, 1.32 and 4 μg·kg−1) to mice, memory acquisition-retention (passive avoidance paradigm with a light-dark box), pain threshold to thermal stimulus (51.5°C; hot plate test) and plasma glucose (glucorefractometer) were evaluated. Similar assays were performed in mice pretreated with s.c. injections of the histamine H1 receptor antagonist pyrilamine (10 mg·kg−1) or the H2 receptor antagonist zolantidine (5 mg·kg−1). TA1 (1.32 and 4 μg·kg−1) was also given i.c.v. to mice lacking histidine decarboxylase (HDC−/−) and the corresponding WT strain. Key Results TA1 was found in the brain of CD1 but not of HDC mice. Exogenous TA1 induced amnesia (at 0.4 μg·kg−1), stimulation of learning (1.32 and 4 μg·kg−1), hyperalgesia (0.4, 1.32 and 4 μg·kg−1) and hyperglycaemia (1.32 and 4 μg·kg−1). All these effects were modulated by pyrilamine and zolantidine. In HDC−/− mice, TA1 (1.32 and 4 μg·kg−1) did not increase plasma glucose or induce hyperalgesia. Conclusions and Implications Behavioural and metabolic effects of TA1 disclosed interactions between the thyroid and histaminergic systems.

Objective 3-Iodothyronamine (T1AM), an analog of thyroid hormone, is a recently discovered fast-acting endogenous metabolite. Single high-dose treatments of T1AM have produced rapid short-term effects, including a reduction of body temperature, bradycardia, and hyperglycemia in mice. Design and Methods The effect of daily low doses of T1AM (10 mg/kg) for 8 days on weight loss and metabolism in spontaneously overweight mice was monitored. The experiments were repeated twice (n = 4). Nuclear magnetic resonance (NMR) spectroscopy of plasma and real-time analysis of exhaled 13CO2 in breath by cavity ring down spectroscopy (CRDS) were used to detect T1AM-induced lipolysis. Results CRDS detected increased lipolysis in breath shortly after T1AM administration that was associated with a significant weight loss but independent of food consumption. NMR spectroscopy revealed alterations in key metabolites in serum: valine, glycine, and 3-hydroxybutyrate, suggesting that the subchronic effects of T1AM include both lipolysis and protein breakdown. After discontinuation of T1AM treatment, mice regained only 1.8% of the lost weight in the following 2 weeks, indicating lasting effects of T1AM on weight maintenance. Conclusions CRDS in combination with NMR and 13C-metabolic tracing constitute a powerful method of investigation in obesity studies for identifying in vivo biochemical pathway shifts and unanticipated debilitating side effects.

3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone (TH)-derived metabolite that induces severe hypothermia in mice after systemic administration; however, the underlying mechanisms have remained enigmatic. We show here that the rapid 3-T1AM-induced loss in body temperature is a consequence of peripheral vasodilation and subsequent heat loss (e.g., over the tail surface). The condition is subsequently intensified by hypomotility and a lack of brown adipose tissue activation. Although the possible 3-T1AM targets trace amine-associated receptor 1 or α2a-adrenergic receptor were detected in tail artery and aorta respectively, myograph studies did not show any direct effect of 3-T1AM on vasodilation, suggesting that its actions are likely indirect. Intracerebroventricular application of 3-T1AM, however, replicated the phenotype of tail vasodilation and body temperature decline and led to neuronal activation in the hypothalamus, suggesting that the metabolite causes tail vasodilation through a hypothalamic signaling pathway. Consequently, the 3-T1AM response constitutes anapyrexia rather than hypothermia and closely resembles the heat-stress response mediated by hypothalamic temperature-sensitive neurons. Our results thus underline the well-known role of the hypothalamus as the body's thermostat and suggest an additional molecular link between TH signaling and the central control of body temperature.

Thyroid hormones (THs) have a major role in regulating cardiac function. Their classical mechanism of action is genomic. Recent findings have broadened our knowledge about the (patho)physiology of cardiac regulation by THs, to include non-genomic actions of THs and their metabolites (THM). This review provides an overview of classical and non-classical cardiac effects controlled by: i) iodothyronines (thyroxine, T4; 3,5,3’-triiodothyronine,T3; 3, 5-diiodothyronine, T2); ii) thyronamines (thyronamine, T0AM; 3-iodothyronamine, T1AM); and iii) iodothyroacetic acids (3, 5, 3′, 5’-tetraiodothyroacetic acid, tetrac; 3, 5, 3’-triiodothyroacetic acid, triac; 3-iodothyroacetic acid, TA1). Whereas iodothyronines enhance both diastolic and systolic function and heart rate, thyronamines were observed to have negative inotropic and chronotropic effects and might function as a brake with respect to THs, although their physiological role is unclear. Moreover, thyronamines showed a cardioprotective effect at physiological concentrations. The cardiac effects of iodothyroacetic acids seem to be limited and need to be elucidated.

Thyronamines (3-T1AM, T0AM) are endogenous compounds probably derived from L-thyroxine or its intermediate metabolites. Combined activities of intestinal deiodinases and ornithine decarboxylase generate 3-T1AM in vitro. Alternatively, 3-T1AM might be formed by the thyroid gland and secreted into the blood. 3-T1AM and T0AM concentrations have been determined by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) in tissues, serum, and cell lines. However, large variations of 3-T1AM concentrations in human serum were reported by LC-MS/MS compared with a monoclonal antibody-based immunoassay. These differences might be caused by strong binding of the highly hydrophobic 3-T1AM to apolipoprotein B100. Pharmacological administration of 3-T1AM results in dose-dependent reversible effects on body temperature, cardiac function, energy metabolism, and neurological functions. The physiological relevance of these actions is unclear, but may occur at tissue concentrations close to the estimated endogenous concentrations of 3-T1AM or its metabolites T0AM or thyroacetic acid (TA1). A number of putative receptors, binding sites, and cellular target molecules mediating actions of the multi-target ligand 3-T1AM have been proposed. Among those are members of the trace amine associated receptor family, the adrenergic receptor ADRα2a, and the thermosensitive transient receptor potential melastatin 8 channel. Preclinical studies employing various animal experimental models are in progress, and more stable receptor-selective agonistic and antagonistic analogues of 3-T1AM are now available for testing. The potent endogenous thyroid hormone-derived biogenic amine 3-T1AM exerts marked cryogenic, metabolic, cardiac and central actions and represents a valuable lead compound linking endocrine, metabolic, and neuroscience research to advance development of new drugs.

It has been known for a long time that changes in cardiac function are a major component of the clinical presentation of thyroid disease. Increased heart rate and hyperdynamic circulation are hallmarks of hyperthyroidism, while bradycardia and decreased contractility characterize hypothyroidism. Recent findings have provided novel insights in the physiology and pathophysiology of heart regulation by thyroid hormones. In this review, we summarize the present knowledge on thyroxine (T4) transport and metabolism and on the biochemical pathways leading to genomic and non-genomic effects produced by 3,5,3'-triiodothyronine (T3) and by its active metabolites, particularly 3,5-diiodothyronine (T2) and 3-iodothyronamine (T1AM). On this basis, specific issues of special interest for cardiology are discussed, namely (1) relevance of the regulation of proteins involved in the control of calcium homeostasis and in pacemaker cell activity, due to non-genomic as well as to classical genomic effects; (2) stimulation of fatty acid oxidation by T2 and T1AM, the latter also causing a negative inotropic and chronotropic action at micromolar concentrations; (3) induction of D3 deiodinase in heart failure, potentially causing selective cardiac hypothyroidism, whose clinical implications are still controversial; and (4) cardioprotective effect of T1AM, possibly occurring at physiological concentrations, and relevance of T3 and of thyroid hormone receptor α1 in post-infarction repair.

Background and Purpose 3-Iodothyronamine (T1AM), an endogenous derivative of thyroid hormones, is regarded as a rapid modulator of behaviour and metabolism. To determine whether brain thyroid hormone levels contribute to these effects, we investigated the effect of central administration of T1AM on learning and pain threshold of mice either untreated or pretreated with clorgyline (2.5 mg·kg−1, i.p.), an inhibitor of amine oxidative metabolism. Experimental Approach T1AM (0.13, 0.4, 1.32 and 4 μg·kg−1) or vehicle was injected i.c.v. into male mice, and after 30 min their effects on memory acquisition capacity, pain threshold and curiosity were evaluated by the following tests: passive avoidance, licking latency on the hot plate and movements on the hole-board platform. Plasma glycaemia was measured using a glucorefractometer. Brain levels of triiodothyroxine (T3), thyroxine (T4) and T1AM were measured by HPLC coupled to tandem MS. ERK1/2 activation and c-fos expression in different brain regions were evaluated by Western blot analysis. Results T1AM improved learning capacity, decreased pain threshold to hot stimuli, enhanced curiosity and raised plasma glycaemia in a dose-dependent way, without modifying T3 and T4 brain concentrations. T1AM effects on learning and pain were abolished or significantly affected by clorgyline, suggesting a role for some metabolite(s), or that T1AM interacts at the rapid desensitizing target(s). T1AM activated ERK in different brain areas at lower doses than those effective on behaviour. Conclusions And Implications T1AM is a novel memory enhancer. This feature might have important implications for the treatment of endocrine and neurodegenerative-induced memory disorders.

3-Iodothyronamine (T1AM) is an endogenous thyroid hormone derivative with unknown biosynthetic origins. Structural similarities have led to the hypothesis that T1AM is an extrathyroidal metabolite of T4. This study uses an isotope-labeled T4 [heavy-T4 (H-T4)] that can be distinguished from endogenous T4 by mass spectrometry, which allows metabolites to be identified based on the presence of this unique isotope signature. Endogenous T1AM levels depend upon thyroid status and decrease upon induction of hypothyroidism. However, in hypothyroid mice replaced with H-T4, the isotope-labeled H-T3 metabolite is detected, but no isotope-labeled T1AM is detected. These data suggest that T1AM is not an extrathyroidal metabolite of T4, yet is produced by a process that requires the same biosynthetic factors necessary for T4 synthesis.

3-Iodothyronamine (T(1)AM) is a naturally occurring thyroid hormone metabolite with distinct biological effects that are opposite those of thyroid hormone. The known molecular targets of T(1)AM include both plasma membrane and intracellular proteins, suggesting that intracellular transport of T(1)AM may be an important component of its action, although no uptake mechanism has yet been described. Using various human cell lines, we show that, indeed, cellular uptake of T(1)AM occurs in multiple cell types and that this process involves specific, saturable, and inhibitable transport mechanisms. These mechanisms are sodium and chloride independent, pH dependent, thyronamine specific, and do not involve the likely candidate transporters of other monoamines, organic cations, or thyroid hormones. A large-scale RNA interference screen targeting the entire solute carrier superfamily of transporter genes reveals that the transport of T(1)AM into cells involves multiple transporters, and we identify eight transporters that may contribute to the uptake of T(1)AM in HeLa cells. This type of transporter small interfering RNA screening approach can be used in general to identify the constellation of transporters that participate in the intracellular disposition of compounds.

3-Iodothyronamine (T1AM) is an endogenous compound with chemical features that are similar to thyroid hormone. T1AM has a carbon skeleton identical to that of T4 and contains a single carbon-iodine bond. Theoretically, T1AM could be produced from T4 by enzymatic decarboxylation and deiodination. Recent studies show that T1AM and higher iodinated thyronamines are subject to similar metabolic processing as iodothyronines such as T4, suggesting a biological linkage between iodothyronines and iodothyronamines. In addition, single doses of T1AM administered to rodents induce a hypometabolic state that in certain ways resembles hibernation and is opposite to the effects of excess T4. This review will discuss the latest developments on this recently discovered thyroid hormone derivative.3-Iodothyronamine is a biologically active, endogenous derivative of thyroid hormone that may represent a new arm of the thyroid endocrine system.

3-Iodothyronamine (3-T 1 AM) and thyronamine (T AM) are novel endogenous signaling molecules that exhibit great structural similarity to thyroid hormones but apparently antagonize classical thyroid hormone (T(3)) actions. Their proposed biosynthesis from thyroid hormones would require decarboxylation and more or less extensive deiodination. Deiodinases (Dio1, Dio2, and Dio3) catalyze the removal of iodine from their substrates. Because a role of deiodinases in thyronamine biosynthesis requires their ability to accept thyronamines as substrates, we investigated whether thyronamines are converted by deiodinases. Thyronamines were incubated with isozyme-specific deiodinase preparations. Deiodination products were analyzed using a newly established method applying liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phenolic ring deiodinations of 3,3',5'-triiodothyronamine (rT3AM), 3',5'-diiodothyronamine (3',5'-T2AM), and 3,3'-diiodothyronamine (3,3'-T2AM) as well as tyrosyl ring deiodinations of 3,5,3'-triiodothyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) were observed with Dio1. These reactions were completely inhibited by the Dio1-specific inhibitor 6n-propyl-2-thiouracil (PTU). Dio2 containing preparations also deiodinated rT(3)AM and 3',5'-T2AM at the phenolic rings but in a PTU-insensitive fashion. All thyronamines with tyrosyl ring iodine atoms were 5(3)-deiodinated by Dio3-containing preparations. In functional competition assays, the newly identified thyronamine substrates inhibited an established iodothyronine deiodination reaction. By contrast, thyronamines that had been excluded as deiodinase substrates in LC-MS/MS experiments failed to show any effect in the competition assays, thus verifying the former results. These data support a role for deiodinases in thyronamine biosynthesis and contribute to confining the biosynthetic pathways for 3-T 1 AM and T 0 AM.

Sulfotransferases (SULTs) catalyze the sulfation of many endogenous compounds that include monoamine neurotransmitters, such as dopamine (DA), and thyroid hormones (iodothyronines). Decarboxylation of iodothyronines results in formation of thyronamines. In the mouse, thyronamines act rapidly in a nongenomic fashion to initiate hypothermia and decrease cardiac output and heart rate. These effects are attenuated after 1-4 h, and metabolism of thyronamines via sulfation may be a mechanism for termination of thyronamine action. We carried out this study to test thyronamine (T0AM), 3-iodothyronamine (T1AM), 3,5-diiodothyronamine (T2AM), and 3,5,3'-triiodothyronamine (T3AM) as substrates for human liver and cDNA-expressed SULT activities. We characterized several biochemical properties of SULTs using the thyronamines that acted as substrates for SULT activities in a human liver high-speed supernatant pool (n=3). T1AM led to the highest SULT activity. Activities with T0AM and T3AM were 10-fold lower, and there was no detectable activity with T2AM. Thyronamines were then tested as substrates with eight cDNA-expressed SULTs (1A1, 1A2, 1A3, 1C2, 1E1, 2A1, 2B1a, and 2B1b). Expressed SULT1A3 had the greatest activity with T0AM, T1AM, and T3AM, whereas SULT1A1 showed similar activity only with T3AM. Expressed SULT1E1 had low activity with each substrate. T1AM, the most active thyronamine pharmacologically, was associated with the greatest SULT activity of the thyronamines tested in the liver pool and in both the expressed SULT1A3 and SULT1E1 preparations. Our results support the conclusion that sulfation contributes to the metabolism of thyronamines in human liver and that SULT activities may regulate the physiological effects of endogenous thyronamines.

Mostin vivoeffects of 3-iodothyronamine (3-T1AM) have been thus far thought to be mediated by binding at the trace amine-associated receptor 1 (TAAR1). Inconsistently, the 3-T1AM-induced hypothermic effect still persists inTaar1knockout mice, which suggests additional receptor targets. In support of this general assumption, it has previously been reported that 3-T1AM also binds to the α-2A-adrenergic receptor (ADRA2A), which modulates insulin secretion. However, the mechanism of this effect remains unclear. We tested two different scenarios that may explain the effect: the sole action of 3-T1AM at ADRA2A and a combined action of 3-T1AM at ADRA2A and TAAR1, which is also expressed in pancreatic islets. We first investigated a potential general signaling modification using the label-free EPIC technology and then specified changes in signaling by cAMP inhibition and MAPKs (ERK1/2) determination. We found that 3-T1AM induced Gi/oactivation at ADRA2A and reduced the norepinephrine (NorEpi)-induced MAPK activation. Interestingly, in ADRA2A/TAAR1 hetero-oligomers, application of NorEpi resulted in uncoupling of the Gi/osignaling pathway, but it did not affect MAPK activation. However, 3-T1AM application in mice over a period of 6 days at a daily dose of 5 mg/kg had no significant effects on glucose homeostasis. In summary, we report an agonistic effect of 3-T1AM on the ADRA2A-mediated Gi/opathway but an antagonistic effect on MAPK induced by NorEpi. Moreover, in ADRA2A/TAAR1 hetero-oligomers, the capacity of NorEpi to stimulate Gi/osignaling is reduced by co-stimulation with 3-T1AM. The present study therefore points to a complex spectrum of signaling modification mediated by 3-T1AM at different G protein-coupled receptors.

Application of 3-iodothyronamine (3-T(1)AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T(1)AM. However, 3-T(1)AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T(1)AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct G(s)-, G(i/o)-, G(12/13)-, G(q/11)- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T(1)AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the G(q/11) pathway but show differences in the basal activity in G(s) and MAP kinase signaling. In contrast to mTaar5, 3-T(1)AM application at hTAAR5 resulted in significant reduction in basal IP3 formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T(1)AM, exhibiting inhibitory effects on IP3 formation and MAP kinase signaling pathways, but does not mediate Gs signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T(1)AM-mediated effects may differ between rodents and humans

We have previously shown that several thyronamines, decarboxylated and deiodinated metabolites of the thyroid hormone, potently activate an orphan G protein-coupled receptor in vitro (TAAR1) and induced hypothermia in vivo on a rapid time scale [Scanlan, T. S.; Suchland, K. L.; Hart, M. E.; Chiellini, G.; Huang, Y.; Kruzich, P. J.; Frascarelli, S.; Crossley, D. A.; Bunzow, J. R.; Ronca-Testoni, S.; Lin, E. T.; Hatton, D.; Zucchi, R.; Grandy, D. K. 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nat. Med. 2004, 10 (6), 638-642]. Herein, we report the synthesis of these thyronamines. Additionally, a large number of thyroamine derivatives were synthesized in an effort to understand the molecular basis of TAAR1 activation and hypothermia induction. Several derivatives were found to potently activate both rTAAR1 and mTAAR1 in vitro (compounds 77, 85, 91, and 92). When administered to mice at a 50 mg/kg dose, these derivatives all induced significant hypothermia within 60 min and exhibited a hypothermic induction profile analogous to 3-iodothyronamine (1, T(1)AM) except 91, which proved to be more efficacious. On the basis of this result, a dose-dependent profile for 91 was generated and an ED(50) of 30 mumol/kg was calculated. Compound 91 proved to be more potent than T(1)AM for TAAR1 activation and exhibits increased potency and efficacy for hypothermia induction. These data further strengthen the pharmacological correlation linking TAAR1 activation by thyronamines and hypothermia induction in mice.

In this study, we attempt to find out whether chronic low dose 3-Iodothyronamine (an endogenous metabolite of thyroid hormone) administration could modulate liver de novo cholesterol synthesis, the same as thyroid hormones. Eighteen male mice were divided randomly into treatment (n=10) and control (n=8) groups. The experimental procedure was applied for 7 days during which test group received T1AM whereas the control group received dimethyl sulfoxide and normal saline. The liver was analyzed for HMG-CoA reductase concentration and hepatic lipase activity whiles cholesterol, LDL and HDL concentrations were measured in the blood serum. There was non-significant decrease in HMG-CoA reductase concentration (224±21.2 versus 187±32.5) in test group compared to control. Interestingly LDL and cholesterol concentrations exhibited significant decrease in test group versus the control. There was non-significant decrease in hepatic lipase activity (771±316 versus 645±317) in test group versus the control. It appears that T1AM reduced serum LDL and cholesterol just like T 3 , in contrast, it decreased liver cholesterol biosynthesis contrary to THs.

3-iodothyronamine (T1AM) is an endogenous amine, that has been detected in many rodent tissues, and in human blood. It has been hypothesized to derive from thyroid hormone metabolism, but this hypothesis still requires validation. T1AM is not a ligand for nuclear thyroid hormone receptors, but stimulates with nanomolar affinity trace amine-associated receptor 1 (TAAR1), a G protein-coupled membrane receptor. With a lower affinity it interacts with alpha2A adrenergic receptors. Additional targets are represented by apolipoprotein B100, mitochondrial ATP synthase, and membrane monoamine transporters, but the functional relevance of these interactions is still uncertain. Among the effects reported after administration of exogenous T1AM to experimental animals, metabolic and neurological responses deserve special attention, because they were obtained at low dosages, which increased endogenous tissue concentration by about one order of magnitude. Systemic T1AM administration favored fatty acid over glucose catabolism, increased ketogenesis and increased blood glucose. Similar responses were elicited by intracerebral infusion, which inhibited insulin secretion and stimulated glucagon secretion. However, T1AM administration increased ketogenesis and gluconeogenesis also in hepatic cell lines and in perfused liver preparations, providing evidence for a peripheral action, as well. In the central nervous system, T1AM behaved as a neuromodulator, affecting adrenergic and/or histaminergic neurons. Intracerebral T1AM administration favored learning and memory, modulated sleep and feeding, and decreased the pain threshold. In conclusion T1AM should be considered as a component of thyroid hormone signaling and might play a significant physiological and/or pathophysiological role. T1AM analogs have already been synthetized and their therapeutical potential is currently under investigation. 3-iodothyronamine (T1AM) is a biogenic amine whose structure is closely related to that of thyroid hormone (3,5,3′-triiodothyronine, or T3). The differences with T3 are the absence of the carboxylate group and the substitution of iodine with hydrogen in 5 and 3′ positions (Figure 1). In this paper we will review the evidence supporting the hypothesis that T1AM is a chemical messenger, namely that it is an endogenous substance able to interact with specific receptors producing significant functional effects. Special emphasis will be placed on neurological and metabolic effects, which are likely to have physiological and pathophysiological importance.

3-Iodothyronamine (T1AM), an endogenous thyroid hormone (TH) metabolite, induces numerous responses including a spontaneously reversible body temperature decline. As such an effect is associated in the eye with increases in basal tear flow and thermosensitive transient receptor potential melastatin 8 (TRPM8) channel activation, we determined in human conjunctival epithelial cells (IOBA-NHC) if T1AM also acts as a cooling agent to directly affect TRPM8 activation at a constant temperature. RT-PCR and quantitative real-time PCR (qPCR) along with immunocytochemistry probed for TRPM8 gene and protein expression whereas functional activity was evaluated by comparing the effects of T1AM with those of TRPM8 mediators on intracellular Ca(2+) ([Ca(2+)]i) and whole-cell currents. TRPM8 gene and protein expression was evident and icilin (20μM), a TRPM8 agonist, increased Ca(2+) influx as well as whole-cell currents whereas BCTC (10μM), a TRPM8 antagonist, suppressed these effects. Similarly, either temperature lowering below 23°C or T1AM (1μM) induced Ca(2+) transients that were blocked by this antagonist. TRPM8 activation by both 1µM T1AM and 20μM icilin prevented capsaicin (CAP) (20μM) from inducing increases in Ca(2+) influx through TRP vanilloid 1 (TRPV1) activation, whereas BCTC did not block this response. CAP (20μM) induced a 2.5-fold increase in IL-6 release whereas during exposure to 20μM capsazepine this rise was completely blocked. Similarly, T1AM (1μM) prevented this response. Taken together, T1AM like icilin is a cooling agent since they both directly elicit TRPM8 activation at a constant temperature. Moreover, there is an inverse association between changes in TRPM8 and TRPV1 activity since these cooling agents blocked both CAP-induced TRPV1 activation and downstream rises in IL-6 release.

Objectives: T1AM subchronic low doses cause rapid lipolysis and weight loss with no change in food consumption. We investigated tissue specific effects of T1AM, a natural thyroid hormone analog, on metabolic pathways and associated transcriptional gene signaling and protein profiles. Methods: Three groups (n=5), randomly selected spontaneously obese female CD -1 mice were injected once daily for 7 days with saline, 10 mg/kg T1AM, or 25 mg/kg T1AM. Blood drawn on days -3, 4, and 7, was analyzed by 1H-NMR. Mice were sacrificed on day 7 and the organs collected for real-time PCR and protein profiles. Results: Multivariate analysis of the 1H-NMR data revealed increased 3-hydroxybutyrate and acetate and no differences in plasma glucose levels in T1AM-treated vs. control mice, . Mice treated with high dose T1AM show significant changes in tissue specific gene expression. The livers had increased expression of SIRT6 and GCK, and decreased expression of SIRT4, but in adipose tissue only SIRT6 increased. Protein e...

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

This study used an optical technique to measure the effects of treating low (10 mg/kg) and high (25 mg/kg) doses of 3-iodothyronamine (T1AM) on the metabolism in the kidney and heart of mice. The ratio of two intrinsic fluorophores in tissue, (NADH/FAD), called the NADH redox ratio (NADH RR), is a marker of the metabolic state of the tissue. A cryofluorescence imaging instrument was used to provide a quantitative assessment of NADH RR in both kidneys and hearts in mice treated with 3-iodothyronamine. We compared those results to corresponding tissues in control mice. In the kidneys of mice treated with a high dose T1AM, the mean values of the maximum projection of NADH RR were 2.6 ± 0.6 compared to 3.20 ± 0.03 in control mice, indicating a 19% ( ± 0.4) significant increase in oxidative stress (OS) in the high dose-treated kidneys ( P = 0.047). However, kidneys treated with a low dose of T1AM showed no difference in NADH RR compared to the kidneys of control mice. Furthermore, low versus high dose treatment of T1AM showed different responses in the heart than in the kidneys. The mean value of the maximum projection of NADH RR in the heart changed from 3.0 ± 0.3 to 3.2 ± 0.6 for the low dose and the high dose T1AM-treated mice, respectively, as compared to 2.8 ± 0.7 in control mice. These values correspond to a 9% (±0.5) ( P = 0.045) and 14% (±0.5) ( P = 0.008) significant increase in NADH RR in the T1AM-treated hearts, indicating that the high dose T1AM-treated tissues have reduced OS compared to the low dose-treated tissues or the control tissues. These results suggest that while T1AM at a high dose increases oxidative response in kidneys, it has a protective effect in the heart and may exert its effect through alternative pathways at different doses and at tissue specific levels.

3-Iodothyronamine (T1AM), produced from thyroid hormones (TH) through decarboxylation and deiodination, is a potent agonist of trace amine-associated receptor 1 (TAAR1), a G protein-coupled receptor belonging to the family of TAARs.In vivoT1AM induces functional effects opposite to those produced on a longer time scale by TH and might represent a novel branch of TH signaling. In this study, we investigated the action of T1AM on thyroid and determined its uptake and catabolism using FRTL5 cells. The expression of TAAR1 was determined by PCR and western blot in FRTL5 cells, and cAMP, iodide uptake, and glucose uptake were measured after incubation with increasing concentrations of T1AM for different times. T1AM and its catabolites thyronamine (T0AM), 3-iodothyroacetic acid (TA1), and thyroacetic acid (TA0) were analyzed in FRTL5 cells by HPLC coupled to tandem mass spectrometry. The product of amplification ofTAAR1gene and TAAR1 protein was demonstrated in FRTL5 cells. No persistent and dose-dependent response to T1AM was observed after treatment with increasing doses of this substance for different times in terms of cAMP production and iodide uptake. A slight inhibition of glucose uptake was observed in the presence of 100 μM T1AM after 60 and 120 min (28 and 32% respectively), but the effect disappeared after 18 h. T1AM was taken up by FRTL5 cells and catabolized to T0AM, TA1, and TA0confirming the presence of deiodinase and amine oxidase activity in thyroid. In conclusion, T1AM determined a slight inhibition of glucose uptake in FRTL5 cells, but it was taken up and catabolized by these cells.

Thyronamines T 0 AM and T 1 AM are naturally occurring decarboxylated thyroid hormone derivatives. Their in vivo administration induces effects opposite to those induced by thyroid hormone, including lowering of body temperature. Since the mitochondrial energy-transduction apparatus is known to be a potential target of thyroid hormone and its derivatives, we investigated the in vitro effects of T 0 AM and T 1 AM on the rates of O 2 consumption and H 2 O 2 release by rat liver mitochondria. Hypothyroid animals were used because of the low levels of endogenous thyronamines. We found that both compounds are able to reduce mitochondrial O 2 consumption and increase H 2 O 2 release. The observed changes could be explained by a partial block, operated by thyronamines, at a site located near the site of action of antimycin A. This hypothesis was confirmed by the observation that thyronamines reduced the activity of Complex III where the site of antimycin action is located. Because thyronamines exerted their effects at concentrations comparable to those found in hepatic tissue, it is conceivable that they can affect in vivo mitochondrial O 2 consumption and H 2 O 2 production acting as modulators of thyroid hormone action.

Thyroid hormones play a wide range of important physiological activities in almost all organism. As changes in these hormones levels-observed in hypothyroidism and hyperthyroidism-promote serious derangements of the cardiovascular system, it is important to know their mechanisms of action. Although the classic genomic actions which are dependent on interaction with nuclear receptors to modulate cardiac myocytes genes expression, there is growing evidence about T(3) and T(4)-triggered nongenomic pathways, resulted from their binding to plasma membrane, cytoplasm, or mitocondrial receptors that leads to a rapidly regulation of cardiac functions. Interestingly both actions converge to amplify thyroid hormone effects on cardiovascular system. T(3) and T(4) nongenomic actions modify inotropic and chronotropic effects, cardiac action potential duration, cardiac growth, and myocyte shape by protein translation through protein kinases-dependent signaling cascades, which include PKA, PKC, PI3K, and MAPK, and changes on ion channels and pumps activity. In respect to the decreased systemic vascular resistance seen in hyperthyroidism, T(3) appears to activate NOS or ATP-sensitive K(+) channels. In addition, a novel biologically active T(4)-derived metabolite has been described, 3-iodothyronamine, T(1)AM, which also acts through membrane receptors to mediate nongenomic cardiac effects. This metabolite influences the physiological manifestations of thyroid hormone actions by inducing opposite effects from those stimulated by T(3) and T(4), such as negative inotropic and chronotropic effects. Therefore, beyond genomic and nongenomic effects of thyroid hormones, it is crucial for there to be an equilibrium between T(3) or T(4) and T(1)AM levels for maintaining cardiac homeostasis.

3-Iodothyronamine (T1AM) is a metabolite of thyroid hormone. It is an agonist at trace amine-associated receptor 1 (TAAR1), a recently identified receptor involved in monoaminergic regulation and a potential novel therapeutic target. Here, T1AM was studied using rhesus monkey TAAR1 and/or human dopamine transporter (DAT) co-transfected cells, and wild-type (WT) and TAAR1 knock-out (KO) mice. The IC(50) of T1AM competition for binding of the DAT-specific radio-ligand [(3)H]CFT was highly similar in DAT cells, WT striatal synaptosomes and KO striatal synaptosomes (0.72-0.81 microM). T1AM inhibition of 10 nM [(3)H]dopamine uptake (IC(50): WT, 1.4 + or - 0.5 microM; KO, 1.2 + or - 0.4 microM) or 50 nM [(3)H]serotonin uptake (IC(50): WT, 4.5 + or - 0.6 microM; KO, 4.7 + or - 1.1 microM) in WT and KO synaptosomes was also highly similar. Unlike other TAAR1 agonists that are DAT substrates, TAAR1 signaling in response to T1AM was not enhanced in the presence of DAT as determined by CRE-luciferase assay. In vivo, T1AM induced robust hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/kg at 60 min: WT -6.0 + or - 0.4, KO -5.6 + or - 1.0; and 25 mg/kg at 30 min: WT -2.7 + or - 0.4, KO -3.0 + or - 0.2). Other TAAR1 agonists including beta-phenylethylamine (beta-PEA), MDMA (3,4-methylenedioxymethamphetamine) and methamphetamine also induced significant, time-dependent thermoregulatory responses that were alike in WT and KO mice. Therefore, TAAR1 co-expression does not alter T1AM binding to DAT in vitro nor T1AM inhibition of [(3)H]monoamine uptake ex vivo, and TAAR1 agonist-induced thermoregulatory responses are TAAR1-independent. Accordingly, TAAR1-directed compounds will likely not affect thermoregulation nor are they likely to be cryogens.