The Thyroid and its Diseases
	Chapter 3 3A-Thyroid Hormone serum Transport Proteins 3B--Cellular Uptake of Thyroid Hormone 3C--Thyroid Hormone Metabolism 3D--Cellular Action of Thyroid Hormone	Downloads

Chapter 3B
Cellular Uptake of Thyroid Hormones
Revised by Georg Hennemann, MD, and Theo Visser, PhD. -- 22 February 2005

During the last two decades it has become apparent that thyroid hormones are transported into cells by specific carrier-mediated uptake mechanisms. Before that time, it was envisaged that crossing the plasma membrane of tissue cells was a matter of simple diffusion, because thyroid hormones are lipophilic and thought to smoothly pass the lipid bilayer of the plasma membrane. There is now a vast literature showing that this is apparently not the case. In fact, diffusion probably plays a minor, if any, role in crossing the plasma membrane.

CLINICAL SUMMARY

About 80% of circulating T3 is produced outside the thyroid gland by peripheral conversion of T4 into T3 and 20% is directly secreted by the thyroid gland. The liver plays an important role in this extra-thyroidal production of T3 (1). Other organs that may be involved are the kidneys and skeletal muscle that contain deiodinases that catalyze the conversion process (2) (see section on metabolism, this chapter). T3 is considered the bioactive moiety of thyroid hormone, while T4 is mainly a prohormone that becomes activated upon its conversion to T3. To exert its biologic action, T3 has first to bind its nuclear receptor in target cells. Nuclear bound T3 is partly derived from plasma and partly from local generation from T4. For biological action of thyroid hormone, both T3 and T4 have to cross the plasma membrane of target cells.

Thyroid hormones cross the plasma membrane by specific processes that may be energy- and/or sodium-dependent. It is unlikely that significant plasma membrane crossing takes place by diffusion (3). The potential clinical importance of the uptake process of thyroid hormones is that it may play a part in the regulation of thyroid hormone activity in the different organs. It has been shown in vitro in human and rat liver, rat pituitary and other cell types, that inhibition of transport of T3 into the cell leads to a proportionate decrease of nuclear T3 binding. Furthermore it appears that cellular uptake of thyroid hormone determines subsequent metabolism (e.g. T3 production from T4), and not vice versa (3). Studies in the intact perfused rat liver, and also in humans, show that when hepatic ATP is decreased by administration of fructose, uptake of thyroid hormone into the liver is diminished (4). Starvation in humans and in rats results in an attenuation of liver ATP content and a proportional decrease of uptake of T4. Caloric deprivation in humans also results in decreased uptake of T4 and T3 in other organs than the liver (5). It is known that in starvation in the rat and in man, circulating T3 drops some times to 50% of normal. This "low T3" syndrome in the rat is caused mainly by a decrease of thyroid function as serum TSH decreases substantially, followed subsequently by a decrease in T4 and T3 production (6). In humans, however, serum TSH is normal or slightly decreased while T4 production rate and serum T4 levels remain normal (7). Despite this, serum T3 may drop substantially under these circumstances, apparently by a decrease of peripheral T4 to T3 conversion. There is evidence that this decrease is caused both by a decrease in deiodinase activity in the liver (see section on metabolism, this chapter) but also that inhibition of T4 transport into the liver plays a role in the generation of this biochemical "syndrome" (see also Chapter 5).

A patient has been described in whom plasma T3 production was found to be low, while T4 production was normal (8). T4 transport into the liver was decreased, but it was normal into non-hepatic tissues. No abnormalities were found with regard to T3 transport into tissues. Deiodinase activity in the liver, measured using thyroid hormone tracer kinetics, was normal. From these data it was concluded that the low T3 production rate was caused by the inhibition of T4 transport into the liver. In other words, uptake of T4 into the liver may have a regulatory role in plasma T3 production and thus the regulation of thyroid hormone activity at the tissue level (8).

The lowering of T3 production in NTI is probably much more complicated, than in caloric deprivation. In both situations a negative energy balance exists, leading to decreased liver ATP and thus inhibition of T4 uptake into the liver. In both situations there is an increase in serum nonesterified fatty acids to concentrations that inhibit T4 uptake in hepatocytes. However, in NTI other compounds, that inhibit hepatic T4 uptake in vitro, circulate in increased amounts in the serum, depending on the type of disease. In patients with uremia, these substances are indoxyl sulfate and a furan fatty acid (CMPF, see basic section below). Bilirubin is such a compound that is increased in patients with liver disease, and in patients with critical illness (for review see ref (3)). Furthermore, drugs may also have an effect on thyroid hormone tissue uptake (see also Chapter 5). Inhibitory activity on hepatic T4 transport in serum of patients with NTI, was found to be negatively correlated with serum T3 concentration of the same serum sample, underlining the importance of the liver in the determination of serum T3, and the regulatory role of T4 hepatocyte plasma membrane transport in this process (9).

It is not known at the moment to what extent changes in tissue uptake, and the T4 to T3 conversion process per se, contribute to lowered plasma T3 production in NTI. Presumably this depends in part on the type of the disease, the medications used and other unknown factors. Both in the low T3 syndrome caused by starvation and in NTI, serum rT3 may be elevated (except in patients with renal or brain disease). This elevation is explained by a normal production rate, while plasma clearance is decreased. The latter is caused by decreased uptake and deiodination in the liver (3).

Although not established yet, many thyroidologists consider lowered plasma T3 production during caloric deprivation and during illness (see below) as an adaptation mechanism, since energy and protein (i.e. organ function) are being conserved (for review see ref (10)). Lowered plasma T3 production occurs during both acute and chronic non-thyroidal illness. Because the patients are not clearly hypothyroid, the term "euthyroid sick syndrome" is used synonymously. (See also the section on Non-thyroidal Illness for a different interpretation.) As mentioned above, lowered T3 production in NTI, is considered by many as a beneficial mechanism in stress, although there is no substantiation for this standpoint. Other thyroidologists believe that the patients are hypothyroid and advocate replacement therapy with thyroid hormone. Whether treatment is beneficial or harmful is uncertain. It is the personal opinion of this author that, at present, administration of T3 in NTI is generally not indicated and possibly even harmful.

Recently, two thyroid hormone-specific transporters have been identified. OATP1C1 shows a preference for T4 and rT3 above T3 and is expressed almost exclusively in brain, in particular in capillaries (11). OATP1C1 is probably important for transport of T4 across the blood-brain barrier. MCT8 is another specific iodothyronine transporter which is expressed in different tissues (12). Mutations in the MCT8 transporter have been identified in young boys with a novel syndrome of severe X-linked psychomotor retardation and elevated serum T3 levels (13, 14)

CELLULAR UPTAKE AND RELEASE OF THYROID HORMONE IN VITRO
Cellular uptake of thyroid hormones

Most of the early studies, showing carrier-mediated uptake were performed with rat liver cells. In the late 1970s, Rao et al. (15) and Krenning et al. (16) independently showed that L-T3 was taken up by rat liver cells by a carrier-mediated process that was energy and sodium dependent. Pliam and Goldfine identified high-affinity binding sites for T3 on purified plasma membranes of rat liver cells (17). In subsequent years carrier-mediated transport was identified in many cell types from different species. Thus, saturable specific transport of thyroid hormones, often found to be temperature-, energy- and sodium-dependent, has been found in normal human and rat liver cells, human and rat hepatoma cells, and trout hepatocytes. Such transport was identified In cells from the central nervous system of humans, mice and rats. Carrier-mediated uptake has been reported in the normal anterior pituitary of the rat and in rat pituitary tumor cell lines and in human and rat red blood cells. Specific transport was detected in a variety of other cells e.g. in a human choriocarcinoma cell line, rat myoblasts, human white blood cells, mouse thymocytes, rat thymus and human and mouse fibroblasts.(for review see (18). Energy dependence has been confirmed with regard to transport of T3 in most studies, except for erythrocytes where reports conflict. Transport of T4 has been studied much less extensively than for T3. Both energy dependence (1) and independency (19) of T4 translocation was noted. However it was argued that less than optimal conditions for restoration of optimal ATP concentration after isolation of cells could explain the lack of finding energy dependence of T4 transport. Transport of T4 is greatly affected by a slight decrease in ATP concentration, which affects T3 transport much less (Fig. 3b-1) (3, 20).

Figure 3b-1.  Percent uptake (of control) of T3, rT3 and T4 versus ATP concentration in rat hepatocytes (from ref.7, with permission).

In different cell types like hepatocytes and fibroblasts two specific binding sites were detected. One, of high capacity and low affinity, with a Km value in the micromolar range, appears to represent binding to the outer surface of the cell membrane. A second, with high-affinity, low-capacity characteristics, with a K_m value in the nanomolar range, appears to be involved in the transport process (1). As the K_m of transport is one order of magnitude higher than the concentration of free hormone (in the picomolar range), no regulation of transport can be exerted by the phenomenon of saturation. However, regulation by the energy charge (ATP) of the cell has been shown in vitro (Fig 3b-2), in the isolated intact rat liver and in the human liver in vivo (see below). Inhibition of transport of thyroid hormones by structurally related compounds is also seen. The best known are amiodarone, aromatic amino acids, benzodiazepine, and iodine-containing X ray contrast agents (18, 21, 22). Furthermore, uptake of thyroid hormones into the liver, but not the pituitary, is also inhibited by substances like fatty acids, indoxyl sulfate and bilirubin, that circulate in increased amounts in patients with nonthyroidal illness (see Chapter 5 and below). In hepatocytes T4 and T3 are transported through different pathways, although they inhibit each other’s active translocation over the plasma membrane. rT3 seems to share the T4 system in the rat liver (20) but in humans evidence has been presented that rT3 also has its own specific liver transport system (23). In contrast, in the rat pituitary T4 and T3 are reported to share the same transport site (24).

Figure 3b-2 Dose dependant inhibition of the specific 1-min uptake of T3 (circles) or T4 (triangles), by cultured rat hepatocytes, in the presence of increasing dilutions of monoclonal antibodies ER-22 or ER-15. Er-22 is a specific antibody directed against the putative thyroid hormone membrane transporter(s). ER-15 is a high affinity antibody directed against a rat hepatocyte membrane protein not involved in transport of thyroid hormones and is used as a positive control. (from ref. 13, with permission)

Studies using a monoclonal antibody against the putative carrier protein(s), demonstrated that diffusion does not contribute to the uptake process in human and in rat liver (Fig 3b-3) (25, 26). It was also shown that T3 sulfate (T3S) was minimally, if at all, taken up by uninjected oocytes of Xenopus laevis, while uptake was stimulated when injected with rat liver mRNA (Fig. 3b-4, below) (27). Uptake of T3S was not detectable in rat anterior pituitary cells (28). These results underline the absence of significant diffusion. It was shown using electron spin resonance stop-flow technique that T3 does not cross the lipid bilayer but gets stuck in the outer layer (29).

Figure 3b-3 Initial uptake of T3 sulphate (T3S) in Xenopus Laevis oocytes injected with water (control), fractionated rat liver mRNA, cRNA of rat liver type I deiodinase (G2I) or both (G2I +mRNA). (from ref.8, with permission)

Figure 3b-4. Uptake of T4 in the isolated perfused rat liver, in the absence (circles) or presence (triangles) of fructose. (from ref. 28, with permission).

Of potential physiological importance is the fact that the process of transport of thyroid hormone over the plasma membrane is rate-limiting for subsequent metabolism. Thus, inhibitors of uptake of T4, T3 and rT3 reduced iodide production in the incubation medium of cultured rat hepatocytes. This decrease reflected a decrease in hormone availability for deiodination, as it was demonstrated that the inhibitory substances had no effect on the deiodination process per se (Table 3b-1) (30). As hepatic conversion of T4 plays an important role in total plasma T3 production (see below), this means that transport of T4 into the liver is co-determinant for plasma production of bioactive hormone. The biological importance of the translocation process is underlined by results of studies showing occupancy of T3 in the cell nucleus is dependent on the uptake rate of the plasma membrane transport process (31-33). Increasing the metabolic activity of oocytes of Xenopus laevis, by injecting the cRNA of type 1 deiodinase, has no influence on the uptake rate of thyroid hormone (Fig. 3b-3, above). This demonstrates that the transport process is independent of the deiodinase activity of the cell, that controls about 80% of total cellular T4 metabolism (28).

Cellular release of thyroid hormones

Cellular efflux of thyroid hormones has been studied only on a limited basis. When rat liver cells in culture were loaded with radioactive T4 and T3 and, a half-life of efflux of 7 to 8 min was detected and efflux was independent of cellular ATP (Fig. 3b-4, shown above) (32). Saturation of efflux of T3 by about 40% was observed when cells were loaded with 5 µM free T3 (Hennemann et al. unpublished). In rat erythrocytes, saturable uptake of T3 was seen, and effllux appeared saturable as well (34). Saturable efflux of thyroid hormones was inhibited by verapamil in primary cultures of rat hepatocytes, cardiocytes and fibroblasts (35). As inhibition of efflux (as also true for the uptake mechanism) occurs only at unphysiologically high concentrations of free hormone (10^-4 M) and is an energy-independent process (see above), it probably does not play a role in the regulation of the intracellular hormone concentration. However the expression of the exporter may be operative in efflux regulation.

UPTAKE OF THYROID HORMONE IN THE PERFUSED LIVER

Experiments using rat liver perfusion to study uptake of thyroid hormone have been reported. Using a two pool model of thyroid hormone distribution and metabolism, uptake of T3 into the cellular compartment of liver was inhibited by 40% by 2 days of fasting (36). Transport was normalized within 0.5 h when insulin, cortisol and/or glucose were added to the perfusion medium. This short period of time suggest that restoration of energy stores was responsible for normalization (36). That the cellular energy charge is involved in the regulation of thyroid hormone uptake was further evidenced by the fact that fructose added to the perfusion medium decreased uptake of T4 entry into the isolated perfused rat liver (Fig. 3b-5). This was paralleled by a decrease in cellular ATP (4). Like the in vitro data, results with liver perfusion show that T4 and T3 are transported into the liver by different uptake systems. When rats were treated with amiodarone, uptake of T4 was inhibited whereas that of T3 was unaffected (37). When livers of hypothyroid rats were perfused, uptake of T3 was not different from normal but metabolism was decreased (38). In livers of hyperthyroid rats uptake of T3 was decreased, but metabolism was increased. These data point to an adaptation mechanism, at the cellular level to maintain tissue euthyroidism when the supply of T3 is abnormal (38). As energy is involved in the uptake process it is to be expected that a gradient of the free hormone is present over the plasma membrane. Indeed, using again the isolated perfused rat liver, a gradient of ~18 was found both for T4 and for T3 (39).

Figure 3b-5. T4 uptake into (a) the rapidly equilibrating compartment (REC) and (b) the slowly equilibrating compartment (SEC) of the subject (closed circles) and the controls (open circles).(from ref. 32, with permission)

TISSUE UPTAKE OF THYROID HORMONES IN-VIVO.

Studies to evaluate saturability and specificity of tissue uptake of thyroid hormones in vivo, in experimental animals or in humans, have not been reported in the literature. Furthermore, there is, as far as we know, only one report dealing with energy dependence of initial uptake in the liver in humans (4). In this study, initial liver uptake was measured, using T4 tracer kinetics and non-compartmental analysis of data, before and after intravenously administered fructose, to four healthy subjects. Initial liver uptake after fructose was depressed compared to the control situation, but uptake normalized subsequently when fructose was metabolized. Obviously, liver ATP could not be measured in the liver in these studies. Comparable experiments in the perfused rat liver showed a decrease in liver ATP with a concomitant decrease in uptake of T4 (4). In the human study, serum lactate and uric acid levels increased after fructose, indirectly reflecting a decrease in liver ATP (4). Another report showed the rate-limiting potential of liver uptake of T4 in determining the amount of circulating plasma T3 (8). Thyroid hormone kinetic studies were performed in a 60 years old T4-substituted woman, who showed a decreased serum T3/T4 ratio (as compared to a T4-substituted control group) in the presence of normal serum thyroid hormone-binding proteins and normal serum TSH. The results indicated decreased transport of T4 into the rapidly equilibrating compartment (predominantly the liver), but a normal transport to the other tissues (slowly equilibrating compartment) (Fig. 3b-6, below). Transport kinetics of T3 were normal. Deiodination properties effecting T4 to T3 conversion in the liver were indirectly found to be normal. It was concluded that the inhibition of T4 transport into the liver leads to low normal serum T3 and an elevated serum T4, resulting in a decreased T3/T4 ratio. This study was originally reported together with similar serum findings in an eight year old boy (40). This boy had in addition an elevated serum TBG that substantially decreased upon treatment with small doses of T3. As TBG is synthesized in the liver and elevated in serum in hypothyroidism, these observations indicate the potential importance of the transport process of T4 into the liver with regard to the regulation of biological activity of thyroid hormone at the tissue level. Genomic DNA of another patient with a similar serum thyroid hormone phenotype revealed no structural abnormalities of the coding and 5’-flanking region of the type 1 iodothyronine deiodinase gene (41, 42)

Figure 3b-6. Relationship of iodide production from T4 (as a reflection of net hepatocyte uptake of T4, corrected for differences in free T4) in the presence of 10% NTI serum, expressed as percentage of production in the presence of 10% serum of healthy controls and serum T3.(from ref. 42, with permission)

THE ROLE OF PLASMA MEMBRANE TRANSPORT IN THE REGULATION OF BIOAVAILABILITY OF THYROID HORMONE

Certain requirements have to be fulfilled before the process of transport of thyroid hormones over the plasma membrane of target cells can be considered as being of (patho)physiological significance (Table 3b-2, and for review ref (3)). In the sections above several of these aspects have been discussed with emphasis on transport into liver cells, because many studies related to these requirements have been done with these cells, and because the liver plays a dominant role in plasma T3 production. Specificity has been shown by the K_m values of the translocation process and by the competitive activity on transport by structurally related compounds. No significant diffusion is apparent ((3) and above). Regulation of the process is effected by intracellular ATP and by certain compounds that circulate in increased amounts in patients during starvation and nonthyroidal illness (see below). Evidence has been presented that the uptake process is rate-limiting for subsequent metabolism of thyroid hormone.

To what extent are changes in transport of thyroid hormone appropriate from the (patho)physiological point of view? Although not established yet, many authors consider the lowered T3 production in starvation and in nonthyroidal illness as an energy and protein (i.e. organ function)-saving mechanism in situations of stress, thus an adaptive effect. If this is indeed the case, then inhibition of uptake of T4 into the liver (leading to decreased plasma T3 production) and of T4 and T3 uptake into tissues in general would be an appropriate change in transport from the pathophysiological point of view. It should be noted however that the pathophysiology of lowered plasma T3 production in NTI is probably much more complex (see also clinical summary) than in starvation and consequently may have a different interpretation.

When obese, but otherwise healthy, volunteers were subjected to a calorie restricted diet, thyroid hormone kinetics revealed an inhibition of T4 and T3 uptake into rapidly and slowly equilibrating tissues (5). In starvation and in non-thyroidal illness, nonesterified fatty acids circulate in increased amounts that have been shown to inhibit T4 uptake into liver cells in-vitro. Bilirubin is often increased in patients with liver disease, and in critical illness in general, and also inhibits T4 transport in vitro into liver cells in concentrations that are found in patients. Finally in patients with renal disease, increased amounts of indoxyl sulphate and of a furan fatty acid (CMPF) are present that inhibit this process (21, 43, 44). Also these changes in transport seem to be appropriate. Serum TSH is generally normal in starvation and in non-thyroidal illness despite the low serum T3 and sometimes low T4 concentration. It would be inappropriate, from the teleological point of view, for TSH to be elevated (normally expected when thyroid hormone parameters are low in serum), because this would result in increased thyroidal T4 secretion that could result in augmented T3 synthesis by the liver, which would counteract the supposedly beneficial lowered T3 production. It would therefore be inappropriate if the above mentioned serum compounds that inhibit T4 uptake in the liver, would have the same action on the pituitary, in this way promoting an increase in serum TSH. So far three out four tested substances, CMPF, indoxyl sulphate and bilirubin, had no inhibitory activity on pituitary T4 uptake (45-47).

That transport inhibition of T4 into the liver may have a significant impact on the concentration of circulating T3 has also been shown in the following study (9). When rat hepatocytes were incubated with serum from patients with nonthyroidal illness of varying severity, a correlation was found between the degree of transport inhibition of T4 into the hepatocytes (effected by the inhibitory compounds in the serum) and the degree of plasma T3 decrease in that particular patient. In other words the more inhibition of T4 transport, the lower serum T3 (Fig. 3b-8). From this study it also appeared that the more severe the illness of the patients, the more depressed was serum T3 and the higher was mortality.

TRANSPORTERS

Organic anion transporters

Cellular thyroid hormone uptake by organic anion transporters was considered when it was found that transporters expressed in rat liver mediated uptake not only of different iodothyronines but also of their sulfonated derivatives (48). This was confirmed by expression studies in Xenopus laevis oocytes, demonstrating that the multispecific Na-taurocholate cotransporting polypeptide (NTCP) and Na-independent organic anion transporting polypeptide 1 (OATP1) facilitated uptake of the different iodothyronines as well as their sulfate and sulfamate derivatives (49). NTCP is expressed exclusively in liver, and is a member of the solute carrier 10 (SLC10) family (50).

OATPs represent a large family of homologous proteins, many of which have been shown to transport different iodothyronines (Table 1) (51). The genes coding for these transporters are now referred as the SLCO family. The OATPs accept a wide range of ligands, not only anionic but also neutral and sometimes even cationic compounds. Some members are expressed in a single tissue, whereas others have a wider tissue distribution. Interestingly, the SLCO1A2, 1B1, 1B3 and 1C1 genes are clustered together with a related pseudogene on human chromosome 12p12. The encoded OATPs have all been shown to transport iodothyronines (Table 1). Of these, OATP1B1 and 1B3 are expressed liver-specifically, OATP1C1 is expressed only in brain and testis, while OATP1A2 is expressed in brain, liver and kidney. In terms of thyroid hormone transport, OATP1C1 is the most intriguing as it shows a high specificity and affinity towards T4 and rT3. In brain, it is localized preferentially in capillaries, suggesting that OATP1C1 is particularly important for transport of T4 across the blood-brain barrier (11, 52, 53).

Amino acid transporters

Iodothyronines are a particular class of amino acids built from two tyrosine residues. Therefore, it is no surprise that amino acid transporters, in particular the L and T type amino acid transporters, have been shown to be involved in thyroid hormone uptake into several tissues (54-58). L type transporters mediate uptake of large neutral, branched-chain and aromatic amino acids, whereas T type transporters are specific for the aromatic amino acids Phe, Tyr and Trp.

Recently, two L type transporters (LAT1 and LAT2) have been identified among the members of the heterodimeric amino acid transporter (HAT) family. HATs consist of a heavy chain and a light chain, linked through a disulfide bond (59). There are 2 possible heavy chains (4F2hc and rBAT) belonging to the SLC3 gene family, and 7 possible light chains belonging to the SLC7 gene family. The 4F2 or CD98 cell surface antigen is expressed in many tissues, especially on activated lymphocytes and tumor cells. 4F2hc is glycosylated protein with a single transmembrane domain, whereas the light chains are not glycosylated and have 12 transmembrane domains (59). The LAT1 and LAT2 light chains form a functional transporter with the 4F2hc heavy chain.

Significant Na-independent transport of iodothyronines has been observed in Xenopus oocytes expressing heterodimeric transporters consisting of human 4F2hc and either human LAT1 or mouse LAT2 (60). The rate of iodothyronine uptake by the 4F2hc/LAT1 transporter decreased in the order 3,3’-T2 > T3 ~ rT3 > T4. Apparent K_m values were found to be in the micromolar range, being lowest for T3 (1.5 µM) (60).

Ritchie et al. have reported on the stimulation of T3 transport in oocytes injected with mRNA for 4F2hc and for the IU12 Xenopus LAT1 homolog (61). They have also showed that overexpression of the heterodimeric L type transporter in cells results in increased intracellular T3 availability and, thus, augmented T3 action (62). Furthermore, they demonstrated T3 uptake via the 4F2hc/LAT1 transporter into the human BeWo placental choriocarcinoma cell line, suggesting that this transporter plays an important role in the transplacental transfer of maternal thyroid hormone to the fetus (63).

A T type amino acid transporter (TAT1) has recently been cloned from rats and humans, and shown to transport Phe, Tyr and Trp but not iodothyronines (64, 65). This protein is a member of the monocarboxylate transporter (MCT) family, and is also called MCT10. The MCT family consists of 14 members, and MCT10 shows the highest homology with MCT8. Friesema et al. have recently identified MCT8 as an active and specific iodothyronine transporter (12). MCT1-4 have been characterized as monocarboxylate transporters but the compounds transported by most other MCTs, in spite of their name, have not been identified. MCT8 and MCT10 are now also referred to as SLC16A2 and SLC16A10, respectively.

MCT8

The human MCT8 gene is located on chromosome Xq13.2, consists of 6 exons, and codes for a protein of 539 or 613 amino acids, depending on which of the two alternative translation start sites (TLSs) is used (66). Both forms of the protein contain 12 predicted transmembrane domains, characteristic of a transporter protein. The N-terminal end of the protein contains a so-called PEST domain , rich in Pro (P), Glu (E), Ser (S) and Thr (T) residues. This is why MCT8 was initially called X-linked PEST containing transporter (XPCT) (66). MCT8 is expressed in many tissues, including human liver, kidney, heart, brain, placenta, lung and skeletal muscle.

After the cloning of MCT8 in 1994, no reports on the biological function or the transported ligands were published until Friesema et al. identified rat MCT8 as a specific thyroid hormone transporter (12). Expression of MCT8 in Xenopus oocytes induced a ~10-fold increase in iodothyronine uptake, much greater than that induced by any other transporter, including rat NTCP, rat OATP1 and human LAT1 (12). Although rat MCT8 does not discriminate between T4, T3, rT3 and 3,3’-T2, it does not transport iodothyronine sulfates and sulfamates, the amino acids Phe, Tyr, Trp and Leu, or the monocarboxylates lactate and pyruvate. Apparent Km values amount to 2-5 µM for the different iodothyronines in the absence of protein in the medium. T4 and T3 transport are largely Na-independent (12).

To date it is unknown if either the long (613 amino acids) or the short (539 amino acids) isoform of human MCT8 or both are expressed in vivo, and if there are any functional differences between them. In rats and mice, only the short form of MCT8 exists due to the lack of the first TLS. The short form of human MCT8 is an active and specific thyroid hormone transporter, showing perhaps a slight preference for T3 as the ligand.

Dramatic evidence for the pathophysiological importance of thyroid hormone transport by MCT8 was obtained in studies of 5 young boys with a novel syndrome of severe psychomotor retardation and strongly elevated serum T3 levels (67). The boys vary in age between 1.5 and 6 years, and in none of them is there any development of speech. Communication skills are limited to smiling and crying. None of the boys can sit or stand independently. Poor head control due to truncal hypotonia is observed in all subjects. All patients were found to have a mutation in MCT8 (Fig. 2). One patient shows a deletion of 24.5 kb in size, encompassing exon 1, and another patient has a deletion of 2.4 kb in size, including part of exon 3 and entire exon 4. The other patients show single nucleotide mutations, associated with the introduction of a premature stop in one boy, and with amino acid substitutions in two boys, i.e. an Ala to Val substitution in the second transmembrane domain, and a Leu to Pro substitution in the nineth transmembrane domain. The mothers of all patients proved to be carriers of the mutations. None of the mothers show psychomotor retardation, and their serum thyroid hormone levels are mostly within the normal range.

Mutations in MCT8 have also been reported by Dumitrescu et al., who reported on two unrelated patients with similar severe neurological abnormalities and increased serum T3 levels (14). In one patient, a point mutation in exon 5 was detected, resulting in a Leu to Pro substitution in the fifth intracellular loop. In the other patient, a single nucleotide deletion in exon 3 results in a frameshift and the generation of a premature stop in the seventh transmembrane domain.

The observations in these patients strongly suggest that MCT8 plays an essential role in T3 supply to neurons in the central nervous system, which are the primary targets of T3 action, in particular during brain development. These neurons also express D3, involved in the termination of T3 action in these cells. However, neurons do not express D2, that is required for local production of T3 from T4. For this, neurons depend on the neighboring astrocytes. Indeed, in rodent brain expression of D2 has been demonstrated in astrocytes, whereas MCT8 and D3 are almost exclusively expressed in neurons (68, 69). Inactivating mutations in MCT8 will result in an impaired supply of T3 to the neuron, which will have detrimental effects on neuronal migration, differentiation and myelination, causing the psychomotor phenotype in our patients. In addition, inactivation of MCT8 will block the access of T3 to neuronal D3, resulting in a decreased T3 clearance and, thus, in the increased serum T3 levels in our patients. However, it is not excluded that MCT8 is involved in the transport of other, still unidentified ligands, whose reduced bioavailability may contribute to the phenotype.

Mutations in MCT8 appear to cause tissue-specific hypothyroidism in the brain. Since the gene is located on the X chromosome, mutations in MCT8 represent a novel cause of X-linked psychomotor retardation. These findings demonstrate the dramatic consequences of a mutation in a thyroid hormone transporter, resulting in an impaired tissue thyroid hormone supply, and representing a novel mechanism for thyroid hormone resistance.