Thyroid hormones (THs) exert a multiplicity of effects. Among these are crucial effects on development, differentiation and metabolism. The first two are particularly relevant in the early stages of development, and a deficit in THs in the neonatal period has serious consequences, such as mental retardation and growth disturbance. However, notwithstanding the knowledge of this wide spectrum of activity, the publication of an enormous number of reports and a long list of hypotheses, the mechanism by which THs exert their diverse actions has not finally been established. In particular, with regard to the stimulatory effects of THs on metabolism we can distinguish three historical periods during which some major hypotheses regarding their mechanisms of action have been developed and have attracted the attention of investigators in the field. The first period was from the early 1950s to the middle 1960s. At this time, the most intriguing hypothesis put forward was "the uncoupling hypothesis", which suggested that THs stimulated metabolic rate by acting at the mitochondrial level by uncoupling the electron transport chain from ATP synthesis (1,2). Mainly because of the large doses employed and also because of the non-reproducibility of the observations "in vivo", this hypothesis was subsequently discarded on the grounds that it was not physiologically relevant.
Then, at the beginning of the 1960s the results obtained by Tata et al. (3-5) provided a basis for the most widely recognized action of THs. They showed that the stimulation of metabolic rate induced by a single injection of T₃ took about 2-3 days to achieve a maximal effect and that the effect was blocked by simultaneous administration of the DNA-transcription suppressor actinomycin D (AD). These results for the first time clearly pointed to the nucleus as the cellular site of action of THs. At the beginning of the 1970s, Oppenheimer et al. (6) were the first to describe the presence of specific nuclear binding sites with a high affinity and low capacity for T₃ in rat liver and kidney. Such sites were subsequently found in other tissues and cell cultures (7). In 1986, two groups (8,9) reported the identification of the cellular proto-oncogene c-erbA, which encodes the high-affinity thyroid hormone receptor (TR). Now, it is well established that most of the physiological effects of T₃ within cells are exerted at the level of transcription, via interactions with specific TRs belonging to the superfamily of nuclear hormone receptors (10-12). TRs are transcription factors that modulate transcription by binding to thyroid-hormone response-elements (TREs). The genes for TRs each express alternative receptor isoforms, including TRb1 (widely expressed), TRb2 (expressed in cochlea, retina and pituitary), TRb3 (expressed in lung, kidney and in an osteosarcoma cell-line), TRa1 (widely expressed) and TRa2 (a C-terminal splice variant that does not bind T₃) (for further details, see Refs. 11-13).
 
Non-nuclear actions of THs
 
For a long time, the predominant view was that the actions of THs are initiated exclusively by an interaction of T₃ with TRs. In recent years (end 1980s-beginning 1990s), however, an increasing number of effects have been described for which a nuclear genomic-dependence can be excluded, leading to the possibility of distinguishing between nuclear and extranuclear effects (due to the presence of a mitochondrial genome, the terms "nuclear" and "extranuclear" are preferable to "genomic" and "non-genomic"). Because of the dogmatic nature of the oft-repeated statement that thyroid hormones act via nuclear receptors, it is difficult to convince many people that they may not necessarily always do so (14). In fact, several extranuclear effects have been described at the level of: 1) the plasma membrane; 2) the cytoskeleton; 3) the sarcoplasmic reticulum and the endoplasmic reticulum; 4) the cytoplasm; 5) mitochondria; and 6) contractile elements in vascular smooth muscle cells. The mechanisms responsible for these extranuclear effects may involve: a) a direct interaction of THs with effector-cell proteins; b) cell-surface receptors coupled to G proteins; c) activation of protein kinase; d) promotion of protein trafficking; e) protein polymerization; f) a change in intracellular calcium concentration; g) ionic exchange. All these aspects have been extensively covered in two reviews (15,16) and will not be further discussed here.
What are the characteristics that should allow us to distinguish between the nuclear and extranuclear effects of THs? Unlike the nuclear effects, the extranuclear ones: I) are independent both of the nuclear receptors for THs and of protein synthesis; ii) may be mediated by signal-transducing pathways; iii) have a short latency to onset (minutes or few hours, even if some nuclear effects have a short latency: see Spot 14 (17); iiii) may involve various iodothyronines. Concerning the last point, until some years ago it was a common assumption that T₃ was the active hormone (following its formation by deiodination of the precursor T₄). A growing body of evidence, however, has led to a revision of that opinion: it seems that at least four iodothyronines exist, and that these all have significant (although not identical) biological activities. These are: L-thyroxine (T₄), triiodo-L-thyronine (T₃), reverse T₃ (rT₃) and 3,5-diiodo-L-thyronine (T₂).
  
The case of T₂
 
T₂ is particularly intriguing because its effects on metabolism seem to be mostly extranuclear. In recent years, in fact, a growing volume of evidence has accumulated to indicate that T₂, a putative product of the inactivating deiodination pathway in T₃ metabolism, could be of biological relevance. In 1989, Horst et al. (18), who studied the effects of several iodothyronines on the oxygen consumption of perfused livers, showed that T₂, like T₃ and T₄, was able to rapidly stimulate hepatic oxygen uptake. While the effects induced by T₃ and T₄ were abolished by inhibiting hepatic deiodinase activity, those induced by T₂ were unaffected by such inhibition. These results stimulated our group and others to focus more deeply on a putative physiological role for T₂. Several investigators, indeed, have demonstrated a rapid effect of T₂ on mitochondrial respiration both in vitro and after its in vivo administration (18-27). The clearest demonstration of an effect of T₂ on energy metabolism comes from "in vivo" studies (28-33). In one of these (29), the effects of a single injection of T₂ were measured on resting metabolic rate (RMR; oxygen consumption at rest, in thermoneutrality and in the post-absorbitive state). This study employed an animal model in which all three known types of deiodinase enzymes were inhibited and the rats made hypothyroid, effects produced by administration of propylthiouracil (PTU) and iopanoic acid (IOP) ("P+I" in Fig.1). Under such conditions it was shown that T₂, as well as T₃, is able to enhance the RMR of hypothyroid rats, although their effects differed in terms of both time course and dependency on protein synthesis. Injection of T₃ stimulates RMR through a nuclear-mediated pathway: indeed, its latency to maximal effect is 2-3 days and the effect is completely blocked by simultaneous administration of actinomycin D. In contrast, T₂ stimulates RMR more rapidly, the effect reaching peak on day one after the injection and being insensitive to actinomycin D (see Fig.1).
 

 
Fig.1 For explanations see text

The effect of T₂ is most likely due to a direct interaction of this iodothyronine with mitochondria. The major difficulty encountered in studying the effect of T₂ in vivo is that injection of T₂ into euthyroid rats results in a slight, insignificant change in RMR. A rapid metabolic degradation of T₂, differences in the metabolic status of the animals or the need to be formed from a precursor such as T₃ could be some of the reasons for this inefficacy of T₂ when it is injected into euthyroid rats. How is T₂ formed? Is T₃ a possible precursor from which T₂ may be formed intracellularly (even if an enzyme that converts T₃ into 3,5-T₂ has not yet been discovered, at least from in vitro studies)? The best way to answer these questions is to inject T₃ into euthyroid rats and to compare the time course of the variations in RMR (either in the presence of or absence of actinomycin D) with the time course of the changes in the serum and hepatic levels of T₂. If some of the effects on RMR that follow the administration of T₃ rely on its transformation into T₂, then in the curves expressing the time course of the change in RMR and the time course of the increases in the serum and tissue levels of T₂, the initial rising phases should be of similar steepness (only if T₂ acts instantaneously). In a recent study, designed to test this hypothesis, was observed that an acute injection of T₃ had an evident effect on RMR earlier in euthyroid rats ("N" in Fig.2) than in rats made hypothyroid by administration of PTU plus IOP (see above) thus indicating that the effects observed following the administration of T₃ to euthyroid rats are not entirely due to T₃ itself (34). In fact, following administration of T₃ the maximal increase in RMR occurred 2-3 days after the injection in hypothyroid rats but after only 25 h from the injection in euthyroid ones. The patterns of response induced by T₃ in euthyroid rats and by T₂ in hypothyroid rats were temporally similar (compare Fig.1 with Fig.2) suggesting that at least part of the early effect of T₃ in euthyroid animals might be due to T₂. In addition, the peak observed on day one after T₃ injection into euthyroid rats was markedly reduced when inhibitors of deiodinases were simultaneously injected (see Fig.2).
 


Fig.2 For explanations see text
 

Moreover, the serum and hepatic levels of T₂ were increased at 12-24 h after T₃ injection and peaked on day one, coincident with the peak in the actinomycin D-insensitive metabolism. Collectively, these data promote confidence in the physiological nature of the effects exerted by T₂, even if further studies are needed to confirm this idea.
As stated before, T₃ is also able to stimulate metabolism, although its effect occurs through a different (nuclear-mediated) mechanism. The recently discovered uncoupling-protein homologues may play a major role in the T₃ -mediated effect (for review, see 35) as their expressions and uncoupling activities in skeletal muscle are regulated by T₃ and increase in line with the increase in RMR (36).
 

On the basis of the available evidence, we believe that the coexistence of nuclear and extranuclear mechanisms of action for THs action may adequately explain the multiplicity of effects exerted by iodothyronines (including the short-term and the long-term ones).
As a last consideration, we should like to outline some problems related to studies of the effects of THs. Some of the controversies surrounding the mechanism of action of THs may be, at least to some extent, a consequence both of the actual TH used and of the thyroid state of the animals. Indeed, discrepant results and opinions may derive from the use of :1) acute vs. chronic treatment, 2) smaller vs. larger doses of the hormones, 3) different iodothyronines, and/or 4) different animal models (created using different ways of inducing hypothyroidism).
Among the points cited above, number 4 is of particular relevance. In fact, the clarification of the biochemical properties of deiodinase enzymes has revealed that surgical and chemical thyroidectomy result in very different animal models of hypothyroidism, characterized by different TH serum levels and different effect on deiodinase enzymes. In addition, different animal models of hypothyroidism are obtained when different drugs are used to induce chemical thyroidectomy. When methimazole is used, there is an inhibition of the synthesis of TH, while the activities of the three types of deiodinase enzymes respond in the same way as they do to surgical thyroidectomy [type I deiodinase (present in liver and kidney) decreases; type II deiodinase (present in brain and brown adipose tissue) increases; type III deiodinase (present in brain, skin and placenta) is differently affected depending on the tissue examined). When PTU is used alone, there is an inhibition of the synthesis of TH and a concomitant strong inhibition of type I deiodinase. When IOP is used alone there is no influence on the synthesis of TH but a significant change in the peripheral metabolism of TH resulting from the inhibition of all three types of deiodinase enzymes. All this suggests that considerable caution needs to be exercised when comparing results obtained from different animal models.
 

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