Hibernation has fascinated scientists for centuries (1–3). Obligate hibernators, e.g., the 13-lined ground squirrel (Ictidomys tridecemlineatus) can survive many months under harsh conditions (no food and very low ambient temperatures) in winter. They recover in spring, apparently without any functional or organ damage. The squirrel enters this state, termed torpor, by rapidly decreasing its metabolism and lowering the core temperature down to 3 or 4°C. After a few weeks in torpor, brief periods (12–24 h) of “interbout euthermia”, which are essential for survival, are observed (4). The processes of hibernation (entry and arousal) are of interest, e.g., for organ preservation in transplantation (5), ischemia–reperfusion damage and cardio protection in the context of cardiac surgery (6, 7), organ protection after hemorrhagic shock or global ischemia after cardiac arrest (8), and protection of brain from ischemic injury (9). Likewise, understanding the conservation of bone and skeletal muscle mass and performance (10) despite many months of inactivity may be of interest for NASA, planning a Mars flight, perhaps with a “torpid” crew (11). A torpor-like state, but not multiday hibernation, can be induced in mice by fasting and a cage temperature below their thermoneutral zone. The search for endogenous signals that trigger hibernation or a torpor-like state started in 1969 (12) and remains topical, with the aim of developing drugs for therapeutic hypothermia. Among the endogenous compounds mentioned in reviews (13, 14) is 3-monoiodothyronamine (T1AM). This paper shall serve as a focused review on torpor induction by T1AM in the context of its pharmacology and the mysteries of its biological origin.

“These unique molecules [i.e. T1AM] have developmental potential as cryogens for the treatment of stroke, in which rapid and prolonged cooling offers outstanding therapeutic benefit to patients” (15). “Such potent actions of 3-T1AM, its metabolites, and synthetic congeners are of eminent interest in emergency and critical care medicine, surgery, tissue transplantation, metabolic and eye clinics, as well as space science. Application of an endogenous biogenic cryogen derived from a hormone provides a rather safe and valuable “lead compound” to be tested and developed by the pharmaceutical industry for various medical applications…” (16). “From the current body of literature, potential therapeutic applications with T1AM are quite apparent, ranging from sleep/torpidity induction, conferring protection against ischemic injury, and anti-obesogenic by inducing increased metabolic reliance on lipid oxidation” (17). “The major endogenous thyroid hormone metabolite 3-iodothyronamine (3-T1AM) exerts marked cryogenic, metabolic, cardiac and central nervous system actions. It is bound to apolipoprotein B-100 (ApoB-100), possibly facilitating its cellular uptake via interaction with the low density lipoprotein-receptor” (18).

T1AM, after its isolation from rat brain, was tested as a putative endogenous ligand (19) for activation of trace amine-associated receptors (TAARs) (20, 21). Trace amines (e.g., octopamine, tyramine, β-phenylethylamine) received their name from their two to three orders of magnitude lower abundance in brain tissue compared to classical amine neurotransmitters such as noradrenaline, serotonin, or dopamine.

Trace amines are formed enzymatically from aromatic amino acids by decarboxylation and were, for a long time, regarded as curiosities. The situation changed when high-affinity binding sites were identified in brain membranes by classical grinding and binding experiments with radiolabeled trace amines. The sensitivity of these agonist binding sites to guanylyl nucleotide inhibition indicated their relationship to the family of Gαs G protein-coupled receptors, which finally led to cloning of the first prototype TAAR1 of a larger family in 2001. For a review of its discovery and properties, we recommend the review by Grandy (22). Except TAAR1, all other TAARs function as odorant receptors expressed on olfactory neurons. Mice possess 14 of such receptors in their nose epithelium (23) compared to five TAARs in humans. Among them is human TAAR5, which is activated by trimethylamine, occurring in rotten fish (24) and TAAR2, which also occurs in human white blood cells (25) and in mucosal layers of the gastrointestinal tract of mice (26). TAAR1 protein is expressed in brain but also in the periphery [e.g., heart, T-lymphocytes, stomach (27), duodenum, and pancreatic β-cells (28, 29)]. Human TAAR1 is implicated in drug addiction, eating behavior, sleep-wake balance, and neuropsychiatric disorders (30, 31). This explains the initial excitement for T1AM that was postulated to be a metabolite of T₄ and an endogenous physiological signal acting rapidly via cell surface receptors similar to the actions of T₄ and T₃ on α_Vβ₃ integrins (32).

Injecting T1AM into mice induces a “torpor-like” state. This immediately fascinated scientists and even convinced a National Space Lab program in South Korea to fund research on the newly found “hibernating” drug (33).

Rodents such as mice and rats have a large surface area compared to their volume, consequently suffering from much greater heat loss compared to larger animals (34). At cage temperatures below thermoneutrality (about 28°C for rats and 30°C for mice), the sympathetic nervous system and brown adipose tissue (BAT) are always activated (35), and a considerable fraction of the total energy expenditure is spent for cold-induced thermogenesis via BAT. Many small mammals have a natural defense mechanism during the colder season, upon a decline in food supply (36). The set point is lowered in the hypothalamus, and the core temperature approaches ambient temperatures. This “torpor-like” state may not be confused with hibernation (4) but is nevertheless often used as a readout for drug candidates investigated for therapeutic hypothermia. Among them are adenosine agonists (37–39) and α₂ adrenergic agonists (40).

Upon intraperitoneal injection of T1AM, the rectal temperature of mice dropped in a dose-dependent manner with an ED50 of around 25 mg/kg. Mice injected with 100 or 200 mg/kg died, suggesting a very small or even absent therapeutic window for torpor induction (19). Moreover, the heart rate dropped significantly, and a strong negative inotropic effect was observed in the isolated perfused rat heart preparation. The T1AM-treated mice are sedated, have a hunched back, closed eyes, and the tail rolled around the body. This “hibernated” state could be reproduced several times by multidose application, provided the animals were always warmed up between the applications (33). Despite their sleep-like state, T1AM-receiving rodents are under extreme metabolic stress: plasma levels of corticosterone, glucagon, and glucose increase several-fold in rats, but insulin is not responding to the raised glucose levels (41). The combination of sedation, bradycardia, and hypothermia in rodents is typical for centrally acting α₂ adrenoceptor agonists such as the approved drugs clonidine, guanabenz, the sedo-analgesic dexmedetomidine, or the Servier experimental compound S18116, which is one of the most potent and selective drugs from this class (42).

These agonists decrease sympathetic outflow from the brainstem and inhibit BAT thermogenesis in rodents (40). Guanabenz was able to maintain the torpid state in rats for up to 7 days without warming-up periods (43). Subcutaneous S18116 injection lowers the body temperature of mice by up to 10°C in the dose range from 0.1 to 40 µg/kg, with an EC50 value of 2.5 µg/kg (42). None of the aforementioned drugs killed the animals at acutely effective hypothermic doses, indicating a sufficiently high therapeutic index for this effect. Unfortunately, quantal dose–effect curves for T1AM are not available but exist for all of the drugs mentioned. T1AM is a highly potent agonist for the receptor subtype α₂A (29). This can explain the inhibition of insulin release despite hyperglycemia (41) and provides the most likely mechanism by which the rapid drop in core temperature occurs. Blockade of the sympathetic outflow from the brain by T1AM inhibits heat generation by BAT, which is further enhanced by low cardiac output. All of the mentioned α₂ adrenoceptor agonists had much wider hypothermic windows than T1AM. The large number of metabolites (see Section 6) and already demonstrated additional targets possibly contribute to the pronounced toxicity. An excellent study with NMR-based metabolomics in obese mice (44) supported earlier findings in rats (41). Upon chronic application of T1AM (10 mg/kg/day) for 7 days, the compound initially increased lipolysis and β-hydroxybutyrate concentrations in plasma, indicating acute inhibition of insulin secretion. On days 5–7, a shift from lipid oxidation to either carbohydrate or protein metabolism as macronutrients was observed. After 7 days, the T1AM-treated mice, in contrast to the vehicle-treated animals, were still not gaining weight for additional 14 days and had increased valine and glycine concentrations in plasma. The authors commented on these toxic posttreatment effects as follows: “The discovery that protein catabolism induction can occur after chronic application of T1AM at low concentration is important and demonstrates the power of combined analyses for anti-obesity drug evaluations to identify unexpected side effects” (44).

Contrary to earlier expectations, TAAR1 is not responsible for the T1AM-induced torpor-like state. TAAR1 knockout mice still respond to the compound by lowering their body temperature. Classical activators of these receptors even increase the core temperature (45). Peripheral signals for the adaptive behavior of rodents upon fasting in a cold environment have been identified: lower leptin and insulin, higher ghrelin and uridine (46, 47) signal to the hypothalamus for a decrease of the temperature set point. Compared to α₂A receptor agonists (either approved or experimental), adenosine, its analogs or uridine (48), the low potency of T1AM to induce a torpor-like state in comparison to its toxic effects disqualify it as an “endogenous biogenic cryogen” (16).

Synthetic T1AM is a potent activator of rat (EC50: 14 nM) and mouse (EC50: 112 nM) TAAR1, stably expressed in HEK-293 cells (19). In another cell line, the EC50 was determined as 22.4 nM for the rat and 1,510 nM for human TAAR1. These values may be compared to those of β-phenylethylamine, which activates human TAAR1 with an EC50 of 106 nM and rat TAAR1 with 206 nM (49). The low potency of T1AM for human TAAR1 was confirmed by others (EC50: 1,690 nM) (50). It is therefore unlikely that there exist any significant effects in humans via TAAR1. A useful comparison of these values may be drawn with data for a selective ligand, RO5166017 [(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine]. RO5166017 has EC50 values in HEK-293 cells of 3.3 nM (mouse), 2.7 nM (rat), and 31 nM (human) (51). In addition to a favorable pharmacokinetic profile, a radioligand screening of RO5166017 against 123 target proteins revealed little or no interaction with other receptors, transporters, or enzymes. To prove that the effects of such selective drugs on, e.g., animal behavior (51) or metabolism (28) are indeed mediated by TAAR1 and not via “off-targets”, the TAAR1 knockout mouse is employed as a negative control. Alternatively, actions could be blocked by a selective TAAR1 antagonist (52).

With respect to the target profile of T1AM, α₂ adrenoceptors are activated with a similar potency as noradrenaline. Neuronal membrane as well as vesicular transporters for dopamine and noradrenaline (45, 53) and all subtypes of muscarinic acetylcholine receptors (54) are functionally blocked in the micromolar or sub-micromolar range. Other identified “targets” are cited in review articles (16, 17, 55). One very high-affinity binding site, apolipoprotein B-100 (ApoB-100), is mentioned here for two reasons. First, ApoB-100 is suggested to be relevant for delivery of the novel hormone to tissues (18) and second, its seemingly problematic nature in the context of accurate quantification of T1AM via MS/MS technology (56) deserves mention. ApoB-100 is a component of circulating VLDL and LDL lipoproteins and binds to T1AM in a 1:1 stoichiometry with a K_d of 17 nM (57). The concentration of serum ApoB-100 in healthy adults ranges from 500 to 1,500 nM but is considerably lowered in patients undergoing statin therapy.

In sum, T1AM may be genuinely termed as a “multi-target” compound, or in plain words: it is a “dirty” drug.

Unfortunately, the pharmacological profile is worsened upon a review of T1AM’s pharmacokinetics and metabolism in rodents. A great deal of effort was spent with MS/MS technology to elucidate the fate of injected T1AM in rodents (58). After intraperitoneal injection, the parent drug is rapidly cleared from plasma with an apparent half-life of 7–8 min during the first hour. Thereafter, a slower elimination with a half-life of about 50 min takes place (59). Oxidative deamidation by monoamine oxidase to 3-iodothyroacetic acid (TA1) (60, 61), glucuronidation, sulfation (62), acetylation, and deiodination are observed. Within 3 h after a single intraperitoneal injection, the sum of the three main metabolites in the serum of mice is approximately 3 µM. At this time, the concentration of the parent compound (T1AM) is reduced by approximately two orders of magnitude from 16.6 to 0.19 µM. Moreover, the concentration of TA1 (17.7 µM) is equal to that of T1AM (16.6 µM) after 10 min. The authors were surprised about the extent and speed of T1AM breakdown: “[A] rich, diverse metabolism such as this is not generally seen with synthetic drugs or xenobiotics” (58).

In conclusion, suggestions regarding the value of such properties as a lead compound for further cryogenic drug development will probably not convince the pharmaceutical industry.

The discovery of T1AM as an endogenous novel metabolite of T₄ exemplifies the great analytical power of MS/MS technology to identify and quantify molecules occurring in various matrices, including human plasma or animal tissues at incredibly low concentrations. The exciting finding that T1AM activates rodent TAARs with nanomolar EC50 values (19) stimulated further research. A breakthrough in the mysterious biological pathway regarding the formation of T1AM was that in addition to deiodinases such as DIO1, ODC is involved (74). One may conclude that the rise of fame of T1AM started with a rat brain extract and ended in the gut, but the question of a route from gut to brain remains (see Figure 1). T1AM shares an analogy with another hormone, abscisic acid (ABA). This phytohormone was isolated from pig and rat brain in 1986, guided by a highly specific antibody. The identity of ABA was proven by MS/MS and the purified compound was shown to be functionally active in a conventional ABA bioassay (90). The authors were surprised about the presence of a phytohormone in mammalian brain and kept rats on an ABA-deficient diet for a long time. To their surprise, the ABA diet-deficient rats almost doubled the content in the brain, suggesting that ABA is possibly synthesized in the absence of external supplies. Some years later, others discovered greatly reduced ABA concentrations in brain samples of ruminants but confirmed the high concentrations in rodents (91). As an explanation, the authors pointed out that ruminants had bacteria in the upper intestine whereas rats have them in the distal part. Indeed, ABA is produced by gut bacteria (92, 93). Before discarding these citations as completely irrelevant to T1AM, one should take notice that ABA is circulating in human plasma in nanomolar concentrations. ABA is not an inert contaminant from plant-derived sources. Instead, it is a powerful regulator of glucose metabolism in humans in doses of a single microgram per kilogram (94–97). One can agree with Hoefig et al. (16), that it will take much less time today compared to earlier discoveries, to unravel the mysteries of the novel T₄ metabolite.

HG collected the data; HG and OL wrote the paper; OL provided assistance during production of the final format of the manuscript for readers not acquainted with the subject.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.