Thyronamines—Past, Present, and Future

Abstract

After their initial discovery in the early 1950's, thyronamines (TAM), a class of endogenous signalling compounds exhibiting structural similarity to the thyroid hormone L-thyroxine, are now back in the focus of basic and clinical research. Numerous prompt pharmacological effects, such as metabolic depression, hypothermia, negative chronotropy, negative inotropy, hyperglycemia, reduction of the respiratory quotient, ketonuria and reduction of fat mass as well as promising therapeutic potential in the experimental prophylaxis and treatment of stroke have already been demonstrated in rodent experimental models. This review article summarizes the currently still somewhat scattered data on TAM, trying to yield a complete and updated picture of the current state of TAM research, which addresses issues on TAM biosynthesis, receptors, signalling and therapeutically relevant targets such as energy metabolism and the cardiovascular system.

Thyronamines (TAMs) are a newly identified class of endogenous signaling compounds. Their structure is identical to that of thyroid hormone and deiodinated thyroid hormone derivatives, except that TAMs do not possess a carboxylate group.

Despite some initial publications dating back to the 1950s, TAMs did not develop into an independent area of research until 2004, when they were rediscovered as potential ligands to a class of G protein-coupled receptors called trace-amine associated receptors. Since this discovery, two representatives of TAMs, namely 3-iodothyronamine (3-T₁AM) and thyronamine (T₀AM), have been detected in vivo. Intraperitoneal or central injection of 3-T₁AM or T₀AM into mice, rats, or Djungarian hamsters caused various prompt effects, such as metabolic depression, hypothermia, negative chronotropy, negative inotropy, hyperglycemia, reduction of the respiratory quotient, ketonuria, and reduction of fat mass. Although their physiological function remains elusive, 3-T₁AM and T₀AM have already revealed promising therapeutic potential because they represent the only endogenous compounds inducing hypothermia as a prophylactic or acute treatment of stroke and might thus be expected to cause fewer side effects than synthetic compounds.

This review article summarizes the still somewhat scattered data on TAMs obtained both recently and more than 20 yr ago to yield a complete and updated picture of the current state of TAM research.

• Introduction

• Structure

• In Vivo Detection

• Systemic and Local Effects

  • Reduction of metabolic rate by 3-T₁AM

  • Hypothermia

  • Negative chronotropy and negative inotropy of 3-T₁AM

  • Negative chronotropy of T₀AM

  • Hyperglycemia

  • Reduction of the respiratory quotient

  • Ketonuria and reduction of fat mass

  • 3-T₁AM does not induce torpor

  • Application routes other than ip and doses other than 50 mg/kg body weight

  • Inhibition of monoamine transporters

• Receptors

  • Early descriptions of thyronamine binding sites

  • Receptors for thyroid hormones and biogenic amines

  • Trace amine-associated receptor 1

  • Alpha_2A adrenergic receptor

  • Concerted function of TAAR-1 and Adra_2A?

• Signal Transduction Pathways Activated by Thyronamines

• Biosynthesis and Deiodination Reactions

• Metabolism

  • First findings

  • Sulfation

  • Oxidative deamination

• Cellular Uptake and Transport

• Therapeutic Applications

• Thyronamines in Comparison to Other Recently Characterized TH Metabolites and Their Targets

  • Mitochondrial TRα1 and effects of 3,5,-T₂

  • Plasma membrane-mediated effects of T₄, T₃, and Tetrac

• Novel Synthetic Analogs of Thyroid Hormones, Specific Ligands, DITPA, and Eprotirome, Specific Ligands to T₃ Receptor Isoforms

• Conclusions

I. Introduction

The history of thyronamine (TAM) research appears peculiar. From 1951 to 1984, a few papers were published now and then, but most lacked any topical consistency. In 2004, after a 20-yr gap in TAM research, TAMs were rediscovered as potential ligands to a new class of G protein-coupled receptors (GPCRs) now referred to as trace-amine-associated receptors (TAARs) (1–3). Since this discovery, the field of TAM research has regained considerable momentum.

II. Structure

TAMs are a novel class of endogenous signaling compounds. In terms of structure, they differ from thyroid (pro-)hormone L-T₄ (T₄) and deiodinated thyronine (TH) derivatives only concerning the absence of the carboxylate group of the alanine side chain. THs and TAMs are designated T_x and T_xAM, respectively, with “x” indicating the number of iodine atoms per molecule, thus following the same rules for nomenclature. As illustrated in Fig. 1, there are nine THs and TAMs that differ by either the number or the position of the iodine atoms.

Fig. 1

A, Structure of THs. B, Structure and nomenclature of TAMs. R, Variable residue.

So far, only the three-dimensional structure of T₃AM has been published (4). In an attempt to compare the conformational properties of T₃AM with those of the corresponding thyronine T₃ and to examine the relative influence of the ethylamine side chain on the diphenylether conformation, the borosalicylate salt of T₃AM was subjected to crystallographic analysis. The experiments revealed that the overall molecular conformation of T₃AM was similar to that of T₃, displaying a twist-skewed conformation of the diphenylether portion and a proximal orientation of the 3′-iodine atom. These observations suggested that the amino group is of key importance in the determination of the conformation of T₃AM.

III. In Vivo Detection

So far, only two representatives of TAMs, namely 3-iodothyronamine (3-T₁AM) and thyronamine (T₀AM), have been detected in vivo using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (3, 5). Both compounds were detected in blood, heart, liver, adipose tissue, thyroid, and brain of adult male C57BL/6 mice, as well as in brain of several other species, such as Long-Evans rats and guinea pigs (3). 3-T₁AM was also detected in blood and brain of Djungarian hamsters (6).

The presence of the other TAMs in vivo has not been reported yet. In the LC-MS/MS studies by Scanlan et al. (3), the samples were simultaneously monitored for T₀AM, T₁AM, and T₂AM presence, but no T₂AM isomer was detected in any of the tissues. Likewise, T₃AM has not been detected from biological sources so far (7, 8). In early experiments applying butanolic extraction and subsequent paper chromatographic separation, ¹³¹I-labeled T₄AM was found in plasma and in the thyroid glands, but not in the thyroid-derived thyroglobulin fractions of rats treated with ¹³¹I (7). Furthermore, ¹³¹I-labeled T₄AM, but not T₃AM, was detected in plasma of two patients who suffered from metastasizing thyroid tumors and were treated with high doses of ¹³¹I. Based on these observations, T₄AM was postulated to be present in vivo at a serum concentration equaling 1 to 2% of the serum T₄ concentration (7, 9). However, T₄AM biosynthesized from endogenous nonradioactive iodine precursors has not been described yet.

Hitherto, several publications have reported on endogenous TAM concentrations in tissues. 3-T₁AM was estimated to be present in brain of Long-Evans rats at subpicomole per gram quantities (3). This concentration is lower compared with 1 to 6 pmol/g T₄ reported for rat brain using a RIA technique (10). Although this concentration seems low at first glance, it was emphasized that the cell type-specific and subcellular distributions of TAMs are unknown. Therefore, their effective concentrations within a given cell type and in specific cellular compartments might be significantly higher than the average whole tissue concentrations measured. In the hearts of male Wistar rats, the endogenous 3-T₁AM concentration averaged 68 pmol/g wet weight and showed a wide range of variation from 1 to 120 pmol/g wet weight (11). Interestingly, the average value of 68 pmol/g wet weight corresponds to the cardiac content of classical modulators of heart function, such as adrenaline, dopamine, and adenosine (11). Moreover, it exceeds the endogenous intracardial T₃ and T₄ concentrations by a factor of 20 and 2, respectively (11). Even if it was not stated whether the hearts had been perfused, these findings indicate that TAMs can override TH concentrations locally in the heart. In serum of Djungarian hamsters, endogenous 3-T₁AM was detected at a concentration of roughly 6 nm (6).

In 2008, Geraci et al. (12) showed at the American Thyroid Association (ATA) meeting in Chicago, Illinois, the first endogenous 3-T₁AM concentration in human tissues (higher concentrations in thyroid, skeletal muscle, adipose tissue, and prostate, ∼60 nm; n = 2–5) and serum (∼60 nm; n = 16) using a solid phase extraction and LC-MS/MS method described by DeBarber et al. (5). Independently of the Scanlan group (12), Soldin et al. (13) described at the 2009 ATA meeting in Palm Beach, Florida, a novel analytical method applying LC-MS/MS for the simultaneous identification and quantification of THs and TAMs in biological samples using today's most sensitive triple quadrupole mass spectrometer (API 5000 LC/MS/MS System). Using an existing procedure for preanalytical extraction of THs and TAMs from mouse tissues and a previously validated LC-MS/MS analytical method published by Piehl et al. (14), mouse tissue 3-T₁AM levels in the range of 1 to 20 pmol/g were determined (F. Wohlgemuth, C. S. Hoefig, and J. Köhrle, unpublished data).

But there are still controversial discussions in the scientific community about the detection and quantification of endogenous 3-T₁AM. Part of this controversy might be related to the strong binding of 3-T₁AM to apolipoprotein B100 in human serum, which leads to very low free TAM concentrations in the range around 60 nm or lower (Ref. 12, and our unpublished data). Surprisingly, 3-T₁AM and T₀AM appear to prefer different serum proteins for avid binding compared with THs, which strongly bind to T₄ binding globulin, transthyretin, albumin, and to some extent also to lipoproteins (15). Currently, no validated and peer-reviewed procedure has been published that allows for the concomitant and simultaneous determination of both the TH and the TAM profiles from one whole blood, serum, or plasma sample using an efficient and reproducible extraction procedure and LC-MS/MS analytics.

IV. Systemic and Local Effects

The physiological roles of TAMs remain elusive. The first documented biological activity of TAMs was reported for their remarkable sensitization of the inhibitory effect of 10⁻⁶/cm³ adrenaline on the isolated intestine of thyroidectomized rabbits. Surprisingly, triiodothyronamine (probably 3,3′,5-triiodothyronamine) was the most potent compound (10⁻¹⁸) compared with thyroxamine (10⁻¹⁴) and diiodothyronamine (10⁻⁵), but unfortunately it has not been clearly specified whether rT₃AM or T₃AM was used and which of the three T2-thyronamine isomers was actually tested (16). Compared with the thyronines T₄ (10⁻¹¹) and triiodothyronine (10⁻¹⁵), the TAMs were more potent and acted immediately without the latency period observed for the THs (16). These authors already proposed an in situ decarboxylation of THs to TAMs by hypothyroid rabbit intestinal tissue. In further studies from the 1950s, T₄AM and T₃AM were only 10% as potent as T₄ in terms of goiter prevention and stimulation of oxygen consumption in rats (17, 18). However, T₃AM was 36 times as potent as T₄ at inducing tadpole metamorphosis (18). In 1974, it was suggested that TAMs might be derived from THs in adrenergic neurons to act as “thyronergic” neurotransmitters (19). This suggestion was based on preliminary evidence that some enzymes involved in catecholamine biosynthesis, including aromatic L-amino acid decarboxylase, might use T₄ as an alternate substrate.

In more recent studies, ip injection of 3-T₁AM or T₀AM into C57BL/6 wild-type mice or Djungarian hamsters caused various prompt effects, such as metabolic depression, hypothermia, negative chronotropy, negative inotropy, hyperglycemia, reduction of the respiratory quotient (RQ), ketonuria, and reduction of fat mass (3, 6). In most of these studies, a dose of 50 mg 3-T₁AM or T₀AM/kg body weight was injected (∼140 μmol 3-T₁AM/kg body weight and ∼218 μmol T₀AM/kg body weight). In Djungarian hamsters, this dose leads to an approximately 10-fold increase in 3-T₁AM blood concentrations from approximately 6 nm to roughly 56 nm (6). Currently, it is not known whether such changes in TAM blood and tissue concentrations have to be considered as physiological or pharmacological. Therefore, the effects of TAMs described so far will conditionally be classified and described as pharmacological effects herein (Table 1).

Table 1

Summary of the pharmacological effects of TAMs identified so far

	Experimental model	Mechanism and/or interpretation	Refs.
Central nervous effects
Hypothermia	C57BL/6 mice, Djungarian hamsters	Due to a decrease in metabolic rate	3, 6, 20
Inhibition of monoamine transporters	Transfected cell lines	Hypothesized action as a physiologically relevant neuromodulator	31
Cardiac effects
Negative chronotropy,a negative inotropy	C57BL/6 mice, isolated rat hearts	Due to effects on intracellular calcium homeostasis	3, 11, 22–24
Effects on plasma hormone concentrations
Hyperglycemia, hypoinsulinemia, hyperglucagonemia	Primary human and murine pancreatic β-cells, male Wistar rats, ROSA26PTX mice	Hypothesized central action increasing the sympathetic tone on pancreatic islets	27, 28
Hypothalamic-pituitary-thyroid-axis: TSH ↓, T4 ↓, T3 ↓	Male Wistar rats	Causing a state reminiscent of the nonthyroidal illness	28
Metabolic effects
Metabolic rate ↓, RQ ↓, lipid utilization ↑, fat mass ↓, ketonuria	C57BL/6 mice, Djungarian hamsters	Hypothesized action as a physiologically relevant modulator of metabolism: change in metabolic fuel utilization from carbohydrates to mainly lipids	6

↓, Reduction; ↑, increase.

^a

Conflicting data have been reported for the cardiac effects of T₀AM (11, 25, 26).

The majority of the pharmacological effects of 3-T₁AM described so far were detected within minutes after injection and displayed roughly similar time courses. However, the respective amplitudes of metabolic depression, negative chronotropy and inotropy, hypothermia, hyperglycemia, and RQ reduction were reached at different time points, namely at 5 min, less than 10 min, 30 min, 2.5 h, and 3 to 4.5 h after injection, respectively (3, 6, 11). Furthermore, whereas the metabolic rate, heart activity, body temperature, and blood glucose levels were normalized 6 to 8 h after injection, the reduced RQ was maintained for more than 24 h (6). However, ketonuria and the reduction of fat mass were detected and reversed much later, compared with the aforementioned acute effects of 3-T₁AM injection.

A. Reduction of metabolic rate by 3-T₁AM

Intraperitoneal injection of 3-T₁AM into C57BL/6 wild-type mice and Djungarian hamsters caused a reduction of metabolic rate measured as a decrease in VO₂ from 2.5 to 1.8 ml/g · h within 5 min (6).

B. Hypothermia

Upon ip injection into C57BL/6 wild-type mice, 3-T₁AM and T₀AM reduced the body temperature within 30 min by roughly 8 C at EC₅₀ of 59 and 178 μmol/kg body weight, respectively (Fig. 2) (3, 20, 21). The mice became inactive but reflexes were retained, indicating that they were not anesthetized. Furthermore, their skin got cool to the touch, but they did not display any compensatory reactions, such as shivering, huddling, or piloerection. The effect was completely reversible after 6 to 8 h and, so far, no side effects were reported even after repeated treatments over a period of 2 months (3).

Fig. 2

Pharmacological effects of 3-T₁AM and T₀AM on body temperature (left) and cardiac output (right) in mice. ‡, indicates significant difference between two treatment groups. [Modified with permission from T. S. Scanlan et al.: Nat Med 10:638–642, 2004 (3). Copyright Nature Medicine.]

Although less pronounced, this hypothermic effect was also observed after ip injection of 50 mg (∼140 μmol) 3-T₁AM/kg body weight into Djungarian hamsters (6).

In both wild-type C57BL/6 mice and Djungarian hamsters, the amplitude of hypothermia caused by 3-T₁AM injection (30 min) was preceded by the amplitude of the reduction in metabolic rate (5 min) (6). Because the extent in hypothermia and the amount of metabolic reduction were highly correlated, the hypothermic effect was interpreted as a result of the reduced metabolic rate (6).

C. Negative chronotropy and negative inotropy of 3-T₁AM

The hypothermic and metabolic effects of 3-T₁AM ip injected into wild-type C57BL/6 mice were accompanied by reversible bradycardia (3). The amplitude of the effect was measured 1 h after injection when the heart rate had dropped from approximately 600 to approximately 350 beats per minute. Bradycardia reversed to normal 6–8 h after injection. This negative chronotropic effect of 3-T₁AM was found to be direct by perfusing an ex vivo adult rat denervated working heart preparation with increasing 3-T₁AM concentrations. 3-T₁AM reduced the heart rate within minutes at a half maximal IC₅₀ of 38 μm. The effect persisted throughout the experiment (60 min) and was reversed by removing 3-T₁AM from the perfusion medium (11). In addition, 3-T₁AM was even more potent (IC₅₀ = 29 μm) at decreasing the cardiac output of the perfused rat hearts (Fig. 2). The amplitude of this negative inotropic effect was measured 10 min after application. Consistent with the negative chronotropic effect observed, the negative inotropy also persisted throughout the experiment (1 h) (3) and was reversible by removing 3-T₁AM (11). Taken together, 3-T₁AM elicits chronotropic and inotropic effects on the heart with different potencies (3), which is remarkable considering that only a few endogenous negative inotropic agents such as adenosine, TNFα, and IL-6 have been identified so far (11). Using the ex vivo adult rat denervated working heart preparation, 3-T₁AM also inhibited all further cardiac parameters measured, including systolic aortic pressure (3) and coronary flow (11).

Studies employing isolated perfused rat hearts revealed that 20 μm 3-T₁AM decreased cardiac contractility but did not alter glucose uptake or oxygen consumption compared with mechanical work (22). Only at 25 μm 3-T₁AM, oxygen consumption decreased. Patch clamp analyses on adult rat cardiomyocytes demonstrated altered calcium and potassium homeostasis, including a diminished calcium pool in the sarcoplasmic reticulum (22). Twenty micromoles of 3-T₁AM increased the diastolic leak through the ryanodine receptor leading to the negative inotropic action. Furthermore, a longer duration of the action potential was observed, which might be related to 3-T₁AM inhibiting transient outward I_to and I_K1 background currents. 3-T₁AM decreased both the amplitude and the duration of depolarization-induced calcium transients at these pharmacological concentrations. However, no evidence for 3-T₁AM effects on protein kinase A activity was observed in these studies (22).

The negative chronotropic and inotropic effects of exogenous 3-T₁AM as well as the increased resistance to ischemic injury, possibly due to its actions on intracellular calcium homeostasis, have been reviewed comprehensively elsewhere (23, 24).

D. Negative chronotropy of T₀AM

In the perfused, denervated rat heart, T₀AM decreased the cardiac output within minutes at an IC₅₀ of 83 μm, whereas no effect on heart rate was observed (Fig. 2) (3). Because the effect was not abolished by pacing the hearts, it was concluded that T₀AM is a negative inotropic agent but (in contrast to 3-T₁AM) not a negative chronotropic agent (11).

Those results are inconsistent with a number of earlier studies using different models. T₀AM (9.4 to 94 μm) displayed positive inotropic effects and did not modify the heart rate in an isolated guinea pig atria model (25). In anesthetized rats, 3 to 30 mg T₀AM/kg body weight (13 to 131 μmol T₀AM/kg body weight) decreased the heart rate significantly (25). Upon iv injection into anesthetized dogs, T₀AM increased the cardiac output (25, 26). However, T₀AM induced concomitant negative chronotropy in one study (25) but positive chronotropy in the other study (26).

In summary, conflicting data have been reported for the cardiac effects of T₀AM.

E. Hyperglycemia

In addition to hypothermia and metabolic suppression, C57BL/6 wild-type mice ip injected with 50 mg (∼140 μmol) 3-T₁AM/kg body weight displayed an increase in blood glucose level (27). This hyperglycemic effect was detectable within minutes after treatment, reaching a maximum of 250% above basal 2 h after 3-T₁AM injection and was completely reversed 8 h after injection. It was accompanied by a decrease in plasma insulin levels and an increase in plasma glucagon levels. Insulin applied exogenously 2 h after 3-T₁AM injection normalized the blood glucose levels, indicating that the peripheral tissues had remained sensitive to insulin during the 3-T₁AM treatment (27). Consistent with these findings, 3-T₁AM inhibited the glucose-stimulated insulin release from primary murine and human pancreatic β-cells in vitro (27).

In a more recent study, ip injection of 50 mg (∼218 μmol) T₀AM/kg body weight into rats was also reported to increase the plasma concentrations of glucose and glucagon, albeit to a lesser extent than 3-T₁AM (28).

F. Reduction of the respiratory quotient

Also within minutes after ip injection of 3-T₁AM, the RQ decreased from 0.9 to 0.7 in both C57BL/6 wild-type mice and Djungarian hamsters (6). This was indicative of a rapid change in metabolic fuel utilization from predominantly carbohydrates (RQ, ∼0.9) before treatment to primarily lipids (RQ, ∼0.7) after treatment (6). Minimal RQ values were obtained 3 to 4.5 h after treatment. The reduced RQ was maintained for more than 24 h, i.e., even after recovery of the metabolic rate, and it only returned to normothermia and normalization of cardiac function much later. This indicated that treated animals required several hours to readjust their metabolic machinery to normal catabolism (6).

G. Ketonuria and reduction of fat mass

The assumption that energy requirements were primarily covered by lipid utilization in response to 3-T₁AM treatment was confirmed by two distinct observations. First, ketone bodies were detected in the urine of Djungarian hamsters from 8 to 28 h after injection (6). Second, a significant loss of body weight due to a loss of fat mass was measured from d 1 to 4 and 6 in summer-acclimatized [long photoperiod (LP)] and winter-acclimatized [short photoperiod (SP)] hamsters, respectively (6).

H. 3-T₁AM does not induce torpor

The initial reports on profound hypothermia, bradycardia, and reduced cardiac output caused by injection of 3-T₁AM into C57BL/6 wild-type mice prompted several researchers to question whether 3-T₁AM acted as a hormonal cue for torpor initiation. To address this question, the effects of 3-T₁AM injection were compared between C57BL/6 wild-type mice, SP and LP Djungarian hamsters. Although mice and LP hamsters do not display spontaneous torpor (29, 30), SP hamsters are seasonally prepared to enter torpor spontaneously, even if fed ad libitum (6). However, the metabolic depression via 3-T₁AM was greater in mice than in hamsters and also more pronounced in LP hamsters compared with SP hamsters. Furthermore, the amplitudes of metabolic reduction and hypothermia triggered by 3-T₁AM were less pronounced compared with torpor. Because additionally the time course of the decrease in metabolic rate and RQ mediated by 3-T₁AM were inconsistent with those observed in torpor, 3-T₁AM was excluded as the major hormonal stimulus for spontaneous daily torpor (6).

I. Application routes other than ip and doses other than 50 mg/kg body weight

A recent study demonstrated that compared with the regular ip injection of 50 mg 3-T₁AM or T₀AM/kg body weight, intracerebroventricular (icv) infusion of a 100-fold lower dose of 3-T₁AM or T₀AM (0.5 mg/kg body weight) produced even more pronounced effects on peripheral glucose metabolism in male Wistar rats (28). No leakage of the icv applied substances into peripheral compartments was found. These observations support a central action of 3-T₁AM and T₀AM on hepatic glucose output and metabolism. The authors suggested an increased sympathetic tone on pancreatic islets via central TAM effects as the underlying mechanism. In addition, sympathetic and parasympathetic projections might activate hepatic glucose production. Furthermore, a direct TAM effect, stronger for 3-T₁AM than for T₀AM, on the activation of the hypothalamic-pituitary-adrenal axis has to be postulated to account for the increase in corticosterone serum concentration induced by 3-T₁AM and T₀AM, which was also observed in these studies. Although systemic administration of T₁AM and T₀AM decreased plasma TSH, T₄, and T₃, no such changes were found after icv injection of the low TAM doses.

A recent independent study demonstrated that direct 3-T₁AM injection into the arcuate nucleus caused a 3-fold increase in food intake in rodents, suggesting that the orexigenic effect of 3-T₁AM is not caused by energy expenditure but rather is regulated on the hypothalamic level (21). Intraperitoneal or icv injection of low doses of 3-T₁AM (4 and 1.2 μmol/kg body weight, respectively) into rats or mice caused a significant increase in food intake without affecting oxygen consumption and locomotor activity. However, at high 3-T₁AM doses (50 mg/kg body weight, ∼127 μmol), the authors confirmed the previously reported reduction of oxygen consumption and locomotor activity (3). Stimulation of primary hypothalamic explants with 3-T₁AM led to a significant increase of neuropeptide Y release, which might account for the increased food intake observed in the in vivo experiments.

J. Inhibition of monoamine transporters

In an attempt to elucidate the mechanisms associated with the in vivo effects of TAM, the interactions between TAMs and neuronal monoamine transporters were studied. These investigations were motivated by the presence of TAMs in brain and by their structural similarity to monoamine transmitters and trace amines (31). In hetero-logous expression systems, all TAMs except rT₃AM inhibited the activity of the plasma membrane human and rat dopamine transporter (DAT). Similarly, all TAMs except T₄AM inhibited the rat vesicular monoamine transporter 2 (VMAT2), which is not localized to the plasma membrane but to the intracellular vesicular membrane. By contrast, the plasma membrane-bound human and rat serotonin transporter was inhibited exclusively by 3,3′-T₂AM, but it was not affected by the remaining TAMs. Furthermore, the plasma membrane-bound human norepinephrine transporter (NET) exhibited a mixed behavior becoming activated by several TAMs and inhibited by others, including 3-T₁AM. In these heterologous expression systems, 3-T₁AM inhibited DAT, VMAT2, and NET at IC₅₀ values in the lower micromolar range and displaying either a competitive or a mixed mode of inhibition. In both the heterologous expression system and primary rat brain synaptosome or synaptic vesicle preparations, 3-T₁AM was confirmed as an inhibitor and excluded as a substrate of DAT, VMAT2, and NET. Therefore, 3-T₁AM is the only known endogenous phenethylamine identified to date that inhibits VMAT2 but fails to be recognized as a substrate. Taken together, the potencies of TAMs to inhibit monoamine transporters were comparable to the potencies of the biogenic trace amines, which also display IC₅₀ values in the lower micromolar range (31). Accordingly, the inhibition of DAT and NET by TAMs, which might lead to extracellular monoamine accumulation, and the inhibition of VMAT-2 by TAMs, which is likely to cause a depletion of neurotransmitter stores and subsequent reduction of further monoamine signaling, might have a physiological relevance (31).

V. Receptors

To this end, the physiological receptor(s) of TAMs has not been identified unambiguously. Although some receptors have been excluded as candidates, some experimental evidence has been gathered for others to qualify as potential receptors mediating TAM signaling (1).

A. Early descriptions of thyronamine binding sites

T₄AM displaced ¹²⁵I-labeled T₃ or T₄ from binding to cytosolic and nuclear protein fractions obtained from human leukocytes at apparent K_i values in the lower nanomolar range, i.e., comparable to unlabeled T₃ and T₄, respectively (32, 33). T₃AM, 3,5-T₂AM, and T₀AM did not only interfere with ligand binding to β-adrenergic receptors expressed at the plasma membrane of turkey erythrocytes but also inhibited the downstream activation of cAMP synthesis (4, 8). Notably, these TAMs exhibited apparent K_i values comparable to the K_i of isoproterenol, which is a well-established β-adrenergic receptor ligand. By contrast T₄, T₃, rT₃, and 3,3-T₂ failed to show any effect (4, 8). From these studies, it was concluded that T₃AM, 3,5-T₂AM, and T₀AM bind to β-adrenergic receptors but do not activate cAMP signaling. Instead, they prevented cAMP signaling by isoproterenol. However, in a more recent study, the application of 1 μm isoproterenol to isolated, denervated ex vivo rat hearts pretreated with 38 μm 3-T₁AM caused a reversal of heart rate, cardiac output, and systolic aortic pressure to baseline, within 30 sec to 1 min. Accordingly, it was concluded that signaling via β₁ adrenergic receptors is preserved in the presence of 3-T₁AM (3). The reasons for the discrepancy between these studies remain elusive.

B. Receptors for thyroid hormones and biogenic amines

The structural similarities of TAMs with THs and biogenic amines suggested that TAMs might signal via nuclear thyroid hormone receptors or receptors of biogenic amines. On the other hand, the rapid onset of TAM effects had not only led to their early classification as “non-genomic effects” but also motivated the proposal that TAMs might not signal via slowly acting nuclear thyroid hormone receptors (34).

In an early study, T₃AM did not displace ¹²⁵I-T₃ from isolated rat liver nuclei (4). In line with this, T₀AM and 3-T₁AM were reported to neither bind to nor activate nuclear thyroid hormone receptors using nuclear receptor binding and reporter gene assays (3). Furthermore, neither T₀AM nor 3-T₁AM activated the heterologously expressed G_αs-coupled dopamine D₁ and β₂ adrenergic receptor (3).

Thus, despite their structural similarities, TAMs, THs, and certain biogenic amines appear to signal via different receptors.

C. Trace amine-associated receptor 1

The first receptor suggested to mediate TAM effects was the membrane-bound G protein-coupled TAAR-1 (3). This suggestion was based on the finding that 3-T₁AM, 3,3′-T₂AM, 3,5-T₂AM, T₃AM, and T₀AM stimulated the intracellular accumulation of cAMP in HEK-293 cells stably transfected with rat TAAR-1, with EC₅₀ values ranging from 14 to 131 nm with respect to the indicated order (3). Analysis of structure-activity relationships of single and double mutant rat and mouse TAAR-1 stably expressed in HEK-293 cells revealed surprisingly distinct, species-specific ligand preferences for a series of TAMs in comparison to 3-T₁AM. Residues critical for the distinct, high-affinity ligand binding were identified in the transmembrane helices 4 and 7 of TAAR-1 of both species (35). Given the structural similarity between TAMs and biogenic amines, the expression of TAAR-1 transcript in TAM target tissues such as heart (11), brain (2), and pancreas (27), as well as the specific binding of [3-¹²⁵I]T₁AM to cardiac membrane preparations, TAAR-1 has been considered an excellent candidate receptor for T₀AM and 3-T₁AM (20). This notion was further supported by the observation that synthetic analogs of 3-T₁AM exhibiting comparable or even higher potencies than 3-T₁AM at activating heterologously expressed TAAR-1 also induced hypothermia after ip injection into mice (36). These synthetic derivatives included a 3-T₁AM-like compound lacking the 4′-OH group, an N-methylated 3-T₁AM derivative containing a fluorine atom instead of the 4′-OH group, and a derivative with a methyl group at the 3-position of the TAM scaffold.

However, the concept of TAM signaling via a G_αsPCR, such as TAAR-1, has been challenged. In parallel experiments of the study by Scanlan et al. (3) using murine TAAR-1, only 3-T₁AM and 3,5-T₂AM activated cAMP accumulation at EC₅₀ values in the nanomolar range, whereas all other TAMs were unable to trigger cAMP synthesis at concentrations of 1 μm or less. Likewise, perfusion of isolated rat hearts with 3-T₁AM did not increase intracardial cAMP level, although transcripts of several TAAR isoforms including TAAR-1 were detected in rat heart and specific binding of [3-¹²⁵I]-T₁AM to cardiac membrane preparations was observed (11). Moreover, the β-adrenergic receptor agonist isoproterenol, which increases the intracardial cAMP concentration upon activation of the cardiac β₁ adrenergic receptor, reversed the negative chronotropic and inotropic effects of 3-T₁AM in the perfused rat heart (3). In line with this, T₃AM, 3,5-T₂AM, and T₀AM inhibited the cAMP synthesis in turkey erythrocytes, which had been prestimulated with isoproterenol (4). Moreover, 3-T₁AM did not have any effect on blood glucose and insulin levels in a mouse model carrying a G_αiPCR knockout specific to pancreatic β-cells (27). Bordetella pertussis toxin, which ADP-ribosylates and uncouples all G_αi proteins except G_z from their upstream GPCRs, prevented the negative effect of 3-T₁AM on the glucose-stimulated insulin release from primary murine and human pancreatic β-cells (27).

In conclusion, the hypothermic, negative chronotropic, negative inotropic, and hyperglycemic effects of 3-T₁AM were more consistent with an inhibition of cAMP accumulation through G_αi activation than with an effect on G_αs. Yet, TAAR-1 has not been withdrawn as a candidate TAM receptor for several reasons. The observed pharmacological TAM effects may be secondary to the direct activation of TAAR-1 (3). Furthermore, although TAAR-1 couples to G_αs in stably transfected HEK293 cells, it might couple differently in different tissues in vivo (3). However, in a Xenopus oocyte expression system in which the M2 muscarinic receptor readily coupled to G_αi, no coupling of TAAR-1 to G_αi was detected (27).

However, a recent report rejects the hypothesis that TAAR-1 mediates the thermoregulatory response to 3-T₁AM and other trace amines (37). TAAR-1 knockout and wild-type male mice were used to prepare synaptosomes employed for binding and uptake assays, behavioral, and pharmacological studies with 3-T₁AM in comparison to other trace amines and amphetamine-like psychostimulants. Experiments using cells transfected with rhesus monkey TAAR-1 and human DAT complemented these studies. Although 3-T₁AM competed for DAT uptake as well as for serotonin uptake in these models, 3-T₁AM-induced TAAR-1 signaling was not increased in DAT-transfected cells. Surprisingly, the previously demonstrated impressive hypothermic cryogenic response to 3-T₁AM administration was maintained in TAAR-1 knockout mice to a similar extent and with the same dose response at 25 and 50 mg/kg (∼70 and 140 μmol 3-T₁AM/kg) body weight. Whether other members of the TAAR family or other plasma membrane receptors mediate this impressive cryogenic 3-T₁AM response, therapeutically relevant for treatment of stroke, cardiovascular complications, and intensive care medicine, remains to be studied.

D. Alpha_2A adrenergic receptor

A second receptor proposed to mediate the effects of 3-T₁AMs was the α_2A adrenergic receptor (Adra_2A), which is an established G_αi coupled receptor expressed in many cell types including pancreatic β-cells (27, 38). In vitro studies demonstrated a higher affinity of human and murine Adra_2A for 3-T₁AM compared with epinephrine, an accepted endogenous ligand of Adra_2A (27). Coadministration of the Adra_2A antagonist yohimbine with 3-T₁AM to wild-type mice inhibited the hyperglycemic effects of 3-T₁AM. Moreover, 3-T₁AM had no effect on blood glucose and insulin levels in mice carrying a G_αiPCR knockout specific to pancreatic β-cells and failed to induce hyperglycemia in Adra_2A-null mice (27).

E. Concerted function of TAAR-1 and Adra_2A?

So far, several lines of evidence indicate that Adra_2A and TAAR-1 might play a concerted role in mediating the effects of 3-T₁AM on carbohydrate metabolism. As described above, 3-T₁AM inhibited the glucose-stimulated insulin release from primary murine and human pancreatic β-cells in a Bordetella pertussis toxin-sensitive fashion (27). However, 3-T₁AM stimulated insulin secretion from the MIN6 insulinoma cell line, which expresses TAAR-1 and Adra2 in a reversed ratio compared with murine islets. This effect was potentiated by Bordetella pertussis toxin or the Adra2A antagonist yohimbine. Moreover, 3-T₁AM did not only fail to induce hyperglycemia in Adra2A-null mice, but rather induced hypoglycemia (27). Taken together, 3-T₁AM might activate insulin secretion in pancreatic β-cells via G_αs-coupled TAAR-1 and inhibit it via G_αi-coupled Adra2a. Consistent with this model, the Adra2 effect dominates in primary pancreatic β-cells, which express higher concentrations of Adra2A than TAAR-1. Likewise, the TAAR-1 effect dominates in MIN6 cells in which the TAAR-1 expression is higher relative to that of Adra2.

VI. Signal Transduction Pathways Activated by Thyronamines

To assess the signaling pathways activated by TAMs more systematically, ex vivo adult rat working heart preparations, which had been pretreated with various inhibitors of signaling cascades, were perfused with T₀AM and 3-T₁AM (11). Remarkably, Bordetella pertussis toxin, the aforementioned inhibitor of G_αi signaling, did not interfere with the negative inotropic effects of T₀AM and 3-T₁AM, thus excluding a role of G_αi proteins for TAM effects on the heart. Likewise, inhibitors of various kinases, directed against protein kinase A, protein kinase C, Ca²⁺-calmodulin kinase 2, phosphoinositide 3-kinase (PI3K), and MAPK2 did not show any effect. By contrast, the negative inotropic effects of 3-T₁AM were potentiated by genistein, a tyrosine kinase inhibitor, and attenuated by vanadate, a phosphotyrosine phosphatase inhibitor (11). Because vanadate also acts as a P-type ATPase inhibitor and thus possesses intrinsic inotropic effects, it could not be concluded directly that 3-T₁AM might signal by activating phosphotyrosine phosphatases. However, the detection of tyrosine phosphorylations and dephosphorylations in the cytosolic and microsomal fraction of rat ventricles perfused with 3-T₁AM strengthened the hypothesis that 3-T₁AM might signal via tyrosine phosphorylation and dephosphorylation pathways (11). In the same study, Ca²⁺ depletion or inhibition of phospholipase C also potentiated the negative inotropic effects of 3-T₁AM, indicating a more complex signaling by TAMs (11).

VII. Biosynthesis and Deiodination Reactions

The pharmacological effects reported for TAMs identified an interesting discrepancy between TAMs and classical THs. On the one hand, TAMs and THs are highly similar in physicochemical characteristics and structure (Fig. 1), but on the other hand the short-term hypothermic, negative chronotropic and negative inotropic effects of TAMs are opposite in direction to the actions of the classical active thyroid hormone T₃ (1). Thus, TAMs were suggested to be derivatives of thyroid hormones that might serve to fine-tune or even antagonize thyroid hormone effects (39).

Yet, the pathways of TAM biosynthesis are still unknown. If TAMs were derivatives of TH (e.g., T₄ or T₃), decarboxylation of the alanine side chain would be required for their biosynthesis (1). So far, no TH decarboxylating enzyme has been identified, although some preliminary evidence that T₄ might be a substrate of aromatic L-amino acid decarboxylase has been reported since 1974 (4, 19).

This hypothesis could not yet be supported using human recombinant amino acid decarboxylase preparations and all THs as substrates, whereas its substrate L-DOPA (L-Dihydroxyphenylalanine) was readily decarboxylated to yield dopamine under comparable reaction conditions (Ref. 40 and our unpublished data).

If the putative decarboxylating enzyme was converting only THs with higher iodine content, deiodinases (Dio1, Dio2, and Dio3) would be directly required to complete 3-T₁AM and T₀AM biosynthesis by removing at least one to four iodine atoms. Intracellular Dios catalyze the reductive removal of iodine from their substrates (41). Because a role of Dios in TAM biosynthesis requires their ability to accept TAMs as substrates, it was investigated whether TAMs are converted by Dios (14, 42). TAMs were incubated with isozyme-specific Dio preparations. Deiodination products were analyzed using a newly established method applying LC-MS/MS. Phenolic ring deiodinations of rT₃AM, 3′,5′-T₂AM, 3,3′-T₂AM, as well as tyrosyl ring deiodinations of T₃AM and 3,5-T₂AM were observed with Dio1. These reactions were completely inhibited by the Dio1-specific inhibitor 6n-propyl-2-thiouracil. Dio2-containing preparations also deiodinated rT₃AM and 3′,5′-T₂AM at the phenolic rings, but in a 6n-propyl-2-thiouracil-insensitive fashion. All TAMs with tyrosyl ring iodine atoms were deiodinated at the tyrosyl ring by Dio3-containing preparations. In functional competition assays, the newly identified TAM substrates inhibited an established TH deiodination reaction. By contrast, TAMs, which had been excluded as Dio substrates in LC-MS/MS experiments, failed to show any effect in the competition assays, thus excluding them as substrates and verifying the former results.

These data support a role for Dios in TAM biosynthesis and contribute to confining the biosynthetic pathways for 3-T₁AM and T₀AM (Fig. 3) (14).

Fig. 3

Pathways suggested for TAM biosynthesis. Diagonal arrows, Phenolic ring deiodination reactions catalyzed by Dio1 or Dio2 and tyrosyl ring deiodination reactions catalyzed by Dio1 or Dio3. Horizontal arrows, Putative decarboxylation reactions that might represent the first step of TAM biosynthesis. Dashed lines, TAMs that have been excluded as precursors of the biosynthesis of 3-T₁AM and T₀AM.

VIII. Metabolism

A. First findings

The first study analyzing potential metabolizing reactions of TAMs (43) was motivated by previous experiments demonstrating that ip injection of a synthetic 3-T₁AM derivative lacking the 4′-OH group into C57BL/6 wild-type mice caused a rapid and pronounced decrease in body temperature (from 36.7 C to ∼23.6 C within 120 min) but also toxic effects, such as stiffening of hind limbs, leading to death 24 h after injection (36). Similarly, an N-methylated 3-T₁AM derivative containing a fluorine atom instead of the 4′-OH group caused hypothermia comparably with 3-T₁AM but concomitant toxic effects within 1 wk. Other synthetic 3-T₁AM derivatives that were even as potent as 3-T₁AM at activating heterologously expressed TAAR-1 caused similar hypothermic effects but no toxicity. Taken together, among the compounds studied, those lacking the 4′-OH group exhibited toxicity. Considering that the 4′-OH group represents a target site of sulfation and glucuronidation, it was hypothesized that the lack of 4′-OH might prevent sulfation and glucuronidation of the compounds and thus impair their clearance, which may be a factor in the observed toxicity (36).

B. Sulfation

T₀AM and 3-T₁AM, which are present in vivo, but also T₃AM were found to be readily sulfated by distinct human liver sulfotransferase (SULT) isoforms (43). Moreover, 3-T₁AM was sulfated by homogenates of human brain and cardiac tissue, i.e., target tissues of TAM action. These SULT actions might serve to attenuate and thus regulate TAM action (43).

C. Oxidative deamination

3-T₁AM and T₃AM were also found to be subject to oxidative deamination of the ethylamine side chain yielding thyroacetic acids (7). This represents yet another alternate pathway of TH metabolism (44, 45). 3-T₁AM and T₃AM were converted to their respective thyroacetic acids (TA₁ and TA₃) by cultured cells, tissue homogenates, and in vivo. These reactions were catalyzed by an amine oxidase activity and were inhibited by the monoamine oxidase and semicarbazide-sensitive amine oxidase inhibitor iproniazid (44).

The biological activity of TA₁ is unknown, so far. Taken together, there is evidence suggesting that sulfation and oxidative deamination are relevant reactions of TAM metabolism. So far, no evidence has become available for 4′-O-methylation or glucuronidation of TAMs.

IX. Cellular Uptake and Transport

3-T₁AM has recently been found to be transported into a variety of cell types via a specific transport mechanism that can be saturated and inhibited (46). To identify the putative 3-T₁AM transporter(s), a systematic large-scale screening analysis of the solute carrier transporter family was performed. No single specific TAM transporter was identified from this screen; however, the sodium- and chloride-independent, pH-dependent, TAM-specific intracellular transport may involve multiple transporters, eight of which were characterized in more detail (46).

The hypothesized receptors, biosynthesizing and/or metabolizing enzymes, and transporters of TAMs are summarized in Table 2.

Table 2

Summary of hypothesized receptors, biosynthesizing and/or metabolizing enzymes, and transporters of TAMs

	Experimental model	Mechanism and/or interpretation	Refs.
Receptors
TAAR-1	Transfected cell lines, isolated rat hearts	Intracellular cAMP accumulation	3, 11, 27, 35
Adra2A	Cell lines, Adra2A-null mice	May modulate effects on metabolism	27, 38
Signal transduction	Ex vivo adult rat working heart	Tyrosine phosphorylation and dephosphorylation pathways	11
Biosynthesis and metabolism
Deiodination(Dio1, Dio2, Dio3)	Cell lines	May be involved in TAM biosynthesis, provided that TAMs are formed by decarboxylation of THs with ≥2 iodine atoms per molecule	14
Oxidative deamination (MAO, SSAO)	Cell lines, homogenates of human thyroids	May serve as precursors in the biosynthesis of thyroacetic acids (e.g., TA1, triac)	44
Sulfation (SULT1A1, SULT1A3, SULT1E1)	Transfected cell lines, homogenates of human liver, brain, and heart	May deactivate TAMs and thus attenuate TAM effects	43
Transporters
Hypothesized transporters (SLC7A1, SLC16A7, SLC17A5, SLCO3A1, SLCO4A1, SLC29A2, SLC31A1, SLC43A3)	Transfected cell lines, siRNA screening	Uptake of 3-T1AM occurs in multiple cell types, uptake properties identified, so far: specific, saturable, inhibitable, pH-dependent, sodium- and chloride- independent	46

MAO, Monoamine oxidase; SSAO, semicarbazide-sensitive amine oxidase; SLC, solute carrier family; siRNA, small interfering RNA.

X. Therapeutic Applications

Although their physiological function remains elusive, 3-T₁AM and T₀AM have already revealed promising therapeutic potential. In one study published so far, 3-T₁AM and T₀AM were used successfully to treat experimentally induced stroke (Fig. 4) (20). In adult C57BL/6 mice injected ip with 50 mg/kg 3-T₁AM or T₀AM either 1 h after or 2 d before the experimental induction of stroke, the infarct volumes were reduced compared with vehicle-treated control mice. The effect was abolished when the TAM-treated mice were placed on a heat pad, indicating that the neuroprotective effect of TAMs required the induction of hypothermia. This study was the first to apply endogenous compounds to induce hypothermia as a treatment of stroke that might be expected to cause fewer side effects than synthetic compounds (20). Moreover, it reported the first evidence that a cryogen, namely 3-T₁AM, may be prophylactically administered in situations of an anticipated ischemic injury (20).

Fig. 4

Infarct volume (percentage of ipsilateral hemisphere) after treatment with 3-T₁AM and T₀AM following middle cerebral artery occlusion (left) and under conditions of hypothermia blocked (n = 8 per group). Error bars represent sem. *, P < 0.05 compared with vehicle-injected. [Reprinted with permission from K. P. Doyle et al.: Stroke 38:2569–2576, 2007 (20). Copyright American Heart Association, Inc.]

XI. Thyronamines in Comparison to Other Recently Characterized TH Metabolites and Their Targets

During the last few years, the classical concept of mechanism of TH action has experienced significant expansion and modifications. Apart of the well-documented and most prevailing mechanism of action of the thyromimetic ligand T₃, which acts by modulating the activities of nuclear T₃ receptors (TRs) TRα1, TRβ1, and TRβ2, several other TH-related effects were described and were supported by convincing experimental evidence (Fig. 5) (for recent reviews, see Refs. 47–50).

Fig. 5

Schematic overview of effects and target molecules involved in mechanisms of action of thyroid hormones T₄ and T₃ and their metabolites, Tetrac, rT₃, 3,5-T₂, and 3-T₁AM. DIO1,2, Type 1 and type 2 Dio; LAT2, L-type amino acid transporter 2; MCT8, monocarboxylate transporter 8; OATP14, organic anion transporter 14; PKC, protein kinase C; PLC, phospholipase C; RXR, retinoic acid X receptor; TRα43, mitochondrial form of TRα.

A. Mitochondrial TRα1 and effects of 3,5,-T₂

A TRα1 variant lacking the N-terminal A/B domains and thus the nuclear location signal is found in mitochondria (Table 3), acting there as a bona fide TR transcription factor for mitochondrial circular DNA, which contains T₃-responsive elements (47, 51). Apart from transcription modulation, this TR form is also involved in regulation of the mitochondrial proton gradient and Ca-²⁺ mediated signaling. Mitochondrial biogenesis, ATP generation, and energy metabolism have been known for decades to be regulated by TH status. T₃-mediated effects on various mitochondrial targets and reactions have been established, and strong evidence has been presented that 3,5-T₂ is a powerful TH metabolite targeting mitochondrial function (for reviews, see Refs. 48 and 52). 3,5-T₂ in rather high concentrations (25 μg/100 g body weight) rapidly stimulates basal metabolic rate and oxidative capacity in hypothyroid rats, and these effects neither require de novo protein biosynthesis nor lead to thyrotoxic cardiac effects, well known for T₃ administration in high concentration. In rodents fed on high-fat diets, 3,5-T₂ also prevents obesity by activating AMP-kinase, which mediates this remarkable stimulation of thermogenesis and mitochondrial fatty acid oxidation in skeletal muscle (52). 3,5-T₂ stimulates flavin-adenin-dinucleotide, reduced form (FADH₂)-linked mitochondrial respiratory pathways and alters lipid partitioning by enhancing mitochondrial import and oxidation of long-chain fatty acids. Whether such effects are also observed in euthyroid animals or obese humans remains to be studied. The 3,5-T₂ effects resembling mitochondrial uncoupling in rat muscle and leading to decreased efficiency of energy utilization appear not to be mediated by altered expression of the uncoupling proteins (UCPs) UCP2 or UCP3 (53). Partial uncoupling and decreased H⁺/electron stoichiometry had been assigned to binding of 3,5-T₂ to cytochrome c-oxidase subunits, which alters the function of this element of the respiratory chain (48). These effects are distinct from the well-known TRβ-mediated stimulatory effect of T₃ on UCP1 in brown adipose tissue (54), which involves induction of the gene by synergistic action of β3-adrenergic stimulation of cAMP production and induction of Dio2. Modulation of this interaction occurs by additional stimulation of Dio2 by bile acid-mediated activation of the TGR5 (Gpbar-1) plasma membrane GPCR in brown adipocytes and myocytes (55).

Table 3

Overview of targets and effects of natural endogenous and thyroid hormone metabolites as well as synthetic analogs

Tissue/serum concentration	Target structure, receptor, protein	Relevant target organs/cells	Major biological or pharmacological effects	Refs.
3,5,3′,5′-Levo-tetraiodothyronine (l-T4) (nm to pm)	Plasma membrane ion pores (Na+/K+-ATPase,Na+/H+-exchanger)	Erythrocytes, hepatocytes, several cell types	Rapidly altered signal transduction; control of proliferation and differentiation	49, 56
	ανβ3 integrin	Several cell types (endothelium, vascular smooth muscle cells, cancer cells, osteoclasts)	Activation of MAPK, angiogenesis cell proliferation, bone metabolism	49, 50
	ERK1/2
	PI3K
	F-actin	Developing cerebellum, astrocytes	Neuronal migration under glial guidance	59
3,5,3′-Levo-triiodothyronine (l-T3) (nm to pm)	TRα, TRβ	Whole body	Permissive and direct effects, basal metabolic rate, growth, differentiation	47
	Plasma membrane receptors (ανβ3 integrin)	Several cell types including tumor cells	PI3K activation, TRα1 translocation, HIF1α induction; rapidly altered signal transduction; control of proliferation and differentiation	49, 74
3,3′,5′-Levo-triiodothyronine (reverse-T3, l-rT3) (pm)	Dio1, Dio3	Many cells	Competing for extrathyroidal T3 production, Dio inhibitor	50, 75
	Plasma membrane receptors (ανβ3 integrin)	Several cell types	Activation of several cytosolic kinase pathways	59
	TRΔα1	Astrocytes	F-actin polymerization
3,5-Diiodo-l-thyronine(3,5-T2) (μm to nm)		Liver	Enhanced fatty acid oxidation and carnitine palmitoyl transferase activity	53
	Cytochrome c oxidase subunits	Liver	Mitochondrial uncoupling	48, 53
	AMP-kinase	Skeletal muscle	Enhanced thermogenesis and basal metabolic rate in hypothyroid rats	52
Tetraiodothyroacetic acid (Tetrac)	ανβ3 integrin (inhibitor)		Angiogenesis	50
Triiodothyroacetic acid (Triac)	TRβ2	Anterior pituitary	TSH suppression	48, 76
		Liver	Cholesterol lowering	66
		Bone	Bone loss
Triiodothyronamine (Triam)	β-adrenergic receptor	Turkey erythrocytes	Antagonist	4
	Dopamine receptor		Reduced dopamine requirement in intensive care patients	8
TAMs (3-T1AM/T0AM)(pmol/g or nm)	See Table 2	See Table 1	See Table 1
GC-1	TRβ		Partially similiar to T3	77
	ανβ3 integrin		Angiogenesis	78
Eprotirome (KB2115)	Hepatic TRβ1	Liver	Decreases atherogenic lipoproteins	71, 72
3,5-Diiodothyropropionic acid (DITPA)	TR	Several cell types	Partially similiar to T3
	ανβ3 integrin		Angiogenesis	50, 61, 79
	TR	Heart	Congestive heart failure
d-Thyroxine/d-triiodthyronine (d-T4/d-T3)	TR	Liver, adipocytes	Lipolysis	68

B. Plasma membrane-mediated effects of T₄, T₃, and Tetrac

The TH effects described above are obviously opposite to the rapid cryogenic action of 3-T₁AM (37). Rapid plasma membrane-initiated effects of THs and their metabolites have also been observed during development in chicken hepatocytes and various rodent and human cells, including cancer cells (49). Rapid modulation of activity of Na+,K⁺-ATPase, both activation and inhibition depending on cell type and developmental stage (56), has been reported for T₃. However, T₄ and 3,5,-T₂ also exert such effects. Evidence presented so far indicates that this fast activation involves cytosolic kinase signaling cascades such as ERK1/2 and PI3K. Whether these direct effects on Na⁺,K⁺-ATPase are mediated by TH binding to the plasma membrane ανβ3 integrin receptor or depend on a membrane-associated or cytosolic TR form that is known to interact with PI3K (57) requires further study. Apart from Na⁺,K⁺-ATPase, the Na⁺/H⁺ exchanger and the Na⁺-dependent amino acid transport system A are rapidly modulated in their activity by T₄, T₃, and to some extent also by rT₃ and 3,5-T₂ (49). The major cellular target initiating these effects appears to be the ανβ3 integrin receptor, which exhibits distinct binding sites for T₄, T₃, and Tetrac. This membrane-initiated signaling requires propagation of the signal by phospholipase C, protein kinase C, Src, or MAPK pathways. The T₄ binding site on the ανβ3 integrin receptor, which is located at the RGD peptide recognition site, shows high T₄ affinity in the range of free T₄ serum concentrations, whereas that of T₃ requires supraphysiological serum concentrations. Surprisingly, Tetrac, the deaminated T₄ metabolite circulating in serum in concentrations comparable to those of T₃, antagonized these effects of T₄ and T₃. Beyond that, Tetrac also blocks integrin-signaling cross talk with other cellular pathways. One final endpoint of action is enhanced angiogenesis, also in the absence of T₄ or T₃ stimulation (49, 50, 58). T₄ stimulation of integrin signaling also increases proliferation of various tumor cells, and again this effect can be efficiently blocked by Tetrac on Tetrac-nanoparticles, which do not enter cells.

Neuronal migration, guided by laminin on glial cells, is affected by rT₃ involving a mechanism associated with polymerization of F-actin, and again this rapid effect exerted by T₄ and rT₃ but not by T₃ involves ανβ3 receptor signaling sensitive to RGD interference (47, 59). Rapid ανβ3 integrin receptor-initiated and RGD sensitive T₄ and T₃ effects have also been reported for bone cells (60) and were related to osteoblast proliferation and T₃-sensitive demineralization of the bone.

The remarkable rapid effects of Tetrac (inhibitory) and T₄ and to a lesser extent T₃ (stimulatory) on angiogenesis during development and in tumors open novel routes toward targeted therapies involving innovative formulations of TH-derived metabolites, e.g., nanoparticular Tetrac (50). Whether endogenous Tetrac is also involved in the regulation of angiogenesis remains to be studied. Currently, no data are available to suggest a contribution of T₄AM or T₃AM in this context, either as potential intermediates formed from T₄ by decarboxylation and further metabolized to Tetrac or Triac (tiratricol) by oxidative deamination or as TH-derived ligands rapidly activating ανβ3 integrin receptors. It might well be that various metabolites generated from the amino acid-derived T₄ and T₃, the main secretory products of the thyroid gland, are specifically formed by enzymatic processes in particular tissues during developmental, physiological, or pathophysiological conditions. They may thus expand the repertoire of possibilities to modulate and fine-tune metabolic processes and pathways sensitive to and dependent on thyroid hormone-derived signaling compounds such Tetrac, rT₃, 3,5-T₂, and 3-T₁AM.

XII. Novel Synthetic Analogs of Thyroid Hormones, Specific Ligands, DITPA, and Eprotirome, Specific Ligands for T₃ Receptor Isoforms

Although currently no clinically relevant synthetic analogs of T₄, Tetrac, rT₃, 3,5,-T₂, or 3-T₁AM are administered in (pre-)clinical trials, several T₃ analogs selective for the TR isoforms TRα1, TRβ1, or TRβ2 have been tested at various stages (Table 3). 3,5-Diiodothyroproionic acid (DITPA) has recently received high interest as a potent TR ligand that does not require cellular uptake by the plasma membrane T₃ transporter MCT8 and thus might reach relevant neuronal targets during development in patients with X-linked Allan-Herndon-Dudley syndrome via other TH transporters. Apart from its classical TR agonistic metabolic effects, DITPA has also been used experimentally as a powerful ligand for the rapid plasma membrane-initiated TH effects on angiogenesis, on cardiac remodeling after infarction, and in ischemia models (58, 61, 62).

The successful administration of Triac has been reported for patients with pituitary and general resistance to thyroid hormone due to mutations of TRβ (63–65). Triac and similarly D-T₄ have also been used as cholesterol- and lipid-lowering drugs (66), but long-term administration resulted in adverse effects on bone metabolism and density (Triac) (67) or cardiac problems (D-T₄) (68). Several TRα- or TRβ-selective ligands, both agonists and antagonists, have been developed and tested in cellular and animal experimental models (69, 70). A number of x-ray structures and detailed structural models of the TR ligand binding sites with various ligands have been published, and some compounds such as the T₃ mimetic compound eprotirome (KB2115) have been reported to effectively decrease plasma low-density lipoprotein cholesterol and to stimulate bile acid synthesis in humans (71). Remarkably, these beneficial effects of this TRβ-selective ligand occur without cardiac effects under dosages administered. Even more promising are recent reports on this novel drug demonstrating significant dose-dependent reduction in several atherogenic lipoproteins [low-density lipoprotein cholesterol, apolipoprotein B, Lp(a) lipoprotein] and triglycerides in patients after 12 wk of treatment that was administered in addition to the state-of-the-art statin therapy (72). These alterations occurred without change in serum TSH and T₃ levels and with only minor decreases in total and free T₄ levels, and they did not result in a compensatory increase in serum TSH during the intervention period. No adverse eprotirome effects on cardiac or bone parameters were observed, and free fatty acid levels were not increased. Obviously, it might be very interesting to know whether circulating serum levels of rT₃, Tetrac, 3,5-T₂, or 3-T₁AM are affected by eprotirome administration. More details of eprotirome effects on respiratory coefficient, lipid compartmentation, and subcellular partitioning as well as fatty acid vs. glucose oxidation might especially be required to compare the action of this T₃-agonist targeting hepatic TRβ1 with the unique metabolic and cryogenic impact of administration of 3-T₁AM in various animal models.

XIII. Conclusions

3-T₁AM and T₀AM have recently been identified as endogenous signaling molecules exhibiting remarkable in vivo effects, such as rapid and profound hypothermia, hyperglycemia, reduction of the RQ, negative chronotropy, and negative inotropy (3, 6). So far, basically all fields of TAM biology appear insufficiently characterized and are in need of further research. It is not known whether further representatives of TAMs apart from 3-T₁AM and T₀AM are present in vivo and, if so, which tissue concentrations and half-lives they display. It remains equally elusive whether the aforementioned in vivo effects represent the physiological TAM actions or are merely pharmacological in nature. Moreover, the physiological receptor(s) of TAMs remain to be identified unequivocally.

Recent findings, which have not been published in peer-reviewed journals yet, suggest that TAM total serum concentrations are orders of magnitude higher than previously reported, depending on how the sample is processed before analysis (12, 73). Possibly, the strong binding of 3-T₁AM to apolipoprotein B100 has to be taken into account for systematic blood sample workup.

Additional unsettled issues include the sites, pathways, and regulation of TAM biosynthesis, their transport in blood and across biological membranes, as well as possible pathophysiological and therapeutic implications. Yet, their presence in tissues and serum is established, and so is their interesting and promising pattern of biological effects.

Acknowledgments

We thank Prof. Dr. Lutz Schomburg for his critical evaluation of the manuscript.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Graduiertenkolleg 1208, TP3; to J.K.) and the Charité Universitätsmedizin Berlin (to J.K.). This review contributes in part to the Ph.D. thesis of C.S.H.

Disclosure Summary: S.P., C.S.H., and J.K. have nothing to disclose. T.S.S. is an inventor on United States Patents 6,979,750 B1; 7,339,079 B2; and 7,355,079 B2.

Abbreviations

• Adra_2A

  α_2A Adrenergic receptor

• DAT

  dopamine transporter

• Dio

  deiodinase

• DITPA

  3,5-diiodothyroproionic acid

• GPCR

  G protein-coupled receptor

• icv

  intracerebroventricular

• LC-MS/MS

  liquid chromatography-tandem mass spectrometry

• LP

  long photoperiod

• NET

  norepinephrine transporter

• PI3K

  phosphoinositide 3-kinase

• RQ

  respiratory quotient

• SP

  short photoperiod

• SULT

  sulfotransferase

• TA

  thyroacetic acid

• TAAR

  trace-amine-associated receptor

• TAM (or T₀AM)

  thyronamine

• 3-T₁AM

  3-iodothyronamine

• TH

  thyronine

• TR

  T₃ receptor

• UCP

  uncoupling protein

• VMAT2

  vesicular monoamine transporter 2.

Author notes

^*

S.P. and C.S.H. contributed equally to this work.