| The Thyroid and its Diseases | ||||
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| The Thyroid and its Diseases | ||||
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Chapter
3
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In healthy humans the thyroid gland produces predominantly the prohormone T4 together with a small amount of the bioactive hormone T3. Most T3 is produced by enzymatic outer ring deiodination (ORD) of T4 in peripheral tissues. Alternative, inner ring deiodination (IRD) of T4 yields the metabolite rT3, the thyroidal secretion of which is negligible. Normally about one-third of T4 is converted by ORD to T3 and about one-third to rT3. The remainder of T4 is metabolized by different pathways, in particular glucuronidation and sulfation. T3 is further metabolized largely by IRD and rT3 largely by ORD, yielding in both cases the metabolite 3,3'-T2. Thus, ORD is regarded as an activating pathway and IRD as an inactivating pathway.
Three enzymes catalyzing these deiodinations have been identified, called type I (D1), type II (D2) and type III (D3) iodothyronine deiodinases. In the last decade all three deiodinases have been cloned and characterized in different species. This established that the deiodinases are a family of homologous selenoproteins consisting of "250-280 amino acids, containing an essential selenocysteine residue in the active center. It is remarkable, therefore, that production and metabolism of thyroid hormone are dependent on two trace elements, namely iodine and selenium.
D1 is expressed mainly in the liver, the kidneys and the thyroid. In particular the hepatic enzyme is thought to be responsible for the major part of peripheral T3 production as well as for the clearance of plasma rT3. These processes are mediated by the ORD activity of D1, but this enzyme has also IRD activity especially towards sulfated T4 and T3. Therefore, in addition to the bioactivation of T4 to T3, D1 also catalyzes the degradation of thyroid hormone. An important property distinguishing D1 from the other deiodinases is its sensitivity to inhibition by the anti-thyroid drug propylthiouracil (PTU). The important role of D1 in the peripheral production of plasma T3 has been demonstrated by the marked decrease in plasma T3 levels in T4-substituted athyreotic subjects treated with PTU.
D2 has been studied extensively in the central nervous system, the pituitary and brown adipose tissue of experimental animals. D2 has only ORD activity and its expression shows adaptive changes in reponse to alterations in thyroid state, which serves to maintain tissue T3 levels in the face of varying plasma T4 and T3 concentrations. These findings have led to the general opinion that D2 is important for the generation of local T3 in these tissues but does not contribute much to the production of plasma T3. However, the recent identification of D2 in human skeletal muscle has provided support for the suspicion that a significant part of plasma T3 may be generated by an extra-hepatic, PTU-insensitive mechanism, in particular in subjects with lowered plasma T4 levels. D2 has also been localized in the human thyroid gland.
D3 mediates the degradation of thyroid hormone since it has only IRD activity. The brain is the predominant D3-expressing tissue in adult animals, and may thus be the main site for the clearance of plasma T3 and for the production of plasma rT3. However, high D3 activities have been demonstrated in the placenta and the pregnant uterus as well as in different fetal tissues. The high D3 activities at these sites appear to prevent exposure of fetal tissues to high T3 levels, allowing the growth of these tissues. T3 is only required at the differentiation stage of tissue development.
The bioavailability of thyroid hormone is determined by various factors, including a) thyroidal secretion of T4 and T3, b) activation of the prohormone T4 by ORD to T3, and c) inactivation of T4 and T3 by IRD to rT3 and 3,3'-T2, respectively. Since the deiodinases and the T3 receptor are located intracellularly, transport of T4 and T3 across the plasma membrane plays an important role in the regulation of tissue thyroid hormone bioavailability. Clinically, the importance of the deiodinases in the regulation of thyroid hormone bioactivity is apparent when their activity is affected by pathophysiological conditions. Examples of such conditions are thyroidal and non-thyroidal illness and malnutrition.
Expression of D1 and D3 is under positive control and that of D2 is under negative control by thyroid hormone. Therefore, the relative contribution of D1 and D2 to peripheral T3 production varies with thyroid state, with D1 prevailing in the hyperthyroid and D2 in the hypothyroid state. The proportions of T3 being produced via D1 or D2 in euthyroid subjects remains to be established. In non-thyroidal illness (NTI) plasma T3 is often decreased and plasma rT3 increased; plasma FT4 is usually (in some reports) still in the normal range. The changes in plasma T3 and rT3 are explained by a diminished conversion of T4 to T3 and of rT3 to 3,3-T2 by D1 in the liver. Although this may be caused to some extent by decreased D1 expression or cofactor levels, a diminished activity of transporter(s) mediating hepatic uptake of T4 and rT3 appears to be another important mechanism. This also holds for the generation of the low T3 syndrome in malnutrition. A special situation is selenium deficiency. In rats chronically fed a Se-deficient diet, hepatic and renal D1 activities are markedly decreased. This is associated with a relatively small decrease in plasma T3 and significant increase in plasma T4. Deiodinase activities in other tissues are less affected by Se deficiency for reasons which are not fully understood. Low Se intake may also result in decreased peripheral T4 to T3 conversion in humans. It has been demonstrated recently that subclinical or even overt hypothyroidism may be induced by expression of high D3 activities in hemanigiomas, a condition which has been termed consumptive hypothyroidism. These findings support the idea that the low T3 syndrome of NTI may also result in part from increased expression of D3, for instance in liver where it is also expressed in the fetal stage.
Finally, peripheral production of T3 can be inhibited by a variety of drugs, including PTU, dexamethasone, propranolol, and iodinated compounds such as the radiographic agents iopanoic acid and ipodate and the anti-arrhythmic drug amiodarone. PTU is a specific uncompetitive inhibitor of D1, while iopanoic acid and ipodate are competitive inhibitors not only of D1 but also of D2. In addition, the radiographic agents inhibit hepatic uptake of thyroid hormone. Amiodarone and its metabolite desethylamidarone may also interfere with peripheral thyroid hormone levels by inhibition of deiodinase activities and tissue thyroid hormone transport. Little is known about the mechanisms by which propranolol and dexamethasone inhibit peripheral T3 production. Combinations of these drugs (e.g. PTU, ipodate, dexamethasone and/or propranolol) may be used to acutely decrease plasma T3 levels in patients with severe hyperthyroidism.
Thyroid hormone metabolism in humans
In healthy human subjects with an adequate iodine intake, the thyroid gland produces predominantly the prohormone T4 and a small amount of the bioactive thyroid hormone T3. Roughly 80% of T3 is produced by outer ring deiodination (ORD) of T4 in peripheral tissues (1-4). The relative contribution of T3 secretion increases in iodine deficiency and other conditions where the thyroid gland is stimulated by TSH or TSH receptor antibodies, since this is associated with increased de novo T3 synthesis and thyroidal expression of both D1 and D2, and thus increased intrathyroidal T4 to T3 conversion (see below). Nevertheless, there is good agreement that about 1/3 of T4 daily produced (~130 nmol) in normal humans is converted to T3, which corresponds to about 40 nmol and thus 80% of the estimated total daily T3 production of 50 nmol. For a recent comprehensive review of thyroid hormone metabolism and the role of the iodothyronine deiodinases therein, the reader is referred to Ref. 4.
That most plasma T3 is derived from peripheral conversion of T4 is supported by the fact that normal plasma T3 levels are obtained in athyreotic patients treated with sufficient T4 to achieve high normal plasma T4 levels. A recent study has shown that administration of T4 replacement doses to hypothyroid rats achieving normal plasma T4 levels results in subnormal plasma T3 levels not only because of the lack of T3 secretion but also because of a decreased T3 production by D1 in peripheral tissues, since this enzyme is under positive control by T3 itself (5). This study as well as a recent clinical study (6) suggest that thyroid hormone replacement of hypothyroid subjects is done best by treatment with a combination of T4 and T3 (preferably as a slow-release formulation) in proportion to their normal thyroidal secretion.
Besides ORD to T3, T4 is converted by inner ring deiodination (IRD) to the metabolite rT3 (Fig. 1), which accounts for about 40% of T4 turnover, while thyroidal secretion of rT3 is negligible (1-4). T3 and rT3 undergo further deiodination, predominantly to the common metabolite 3,3'-diiodothyronine (3,3'-T2), which is generated by IRD of T3 and by ORD of rT3 (1-4). Thus, ORD is an activating pathway by which the prohormone T4 is converted to active T3, whereas IRD is an inactivating pathway by which T4 and T3 are converted to the metabolites rT3 and 3,3'-T2, respectively.
In addition to deiodination, iodothyronines are metabolized by conjugation of the phenolic hydroxyl group with sulfate or glucuronic acid (Fig. 1) (1,7,9). Sulfation and glucuronidation are so-called phase II detoxication reactions, the general purpose of which is to increase the water-solubility of the substrates and, thus, to facilitate their biliary and/or urinary clearance. However, iodothyronine sulfate levels are normally very low in plasma, bile and urine, because these conjugates are rapidly degraded by D1, suggesting that sulfate conjugation is a primary step leading to the irreversible inactivation of thyroid hormone (9). Plasma levels and, if investigated, biliary excretion of iodothyronine sulfates are increased by inhibition of D1 activity with PTU or iopanoic acid (IOP), and during fetal development, NTI and fasting (9-11). Under these conditions, T3 sulfate (T3S) may function as a reservoir of inactive hormone from which active T3 may be recovered by action of tissue sulfatases and bacterial sulfatases in the intestine (9-11).
In contrast to the sulfates, iodothyronine glucuronides are rapidly excreted in the bile. However, this is not an irreversible pathway of hormone disposal, since after hydrolysis of the glucuronides by bacterial ß-glucuronidases in the intestine part of the liberated iodothyronines is reabsorbed, constituting an enterohepatic cycle (1,7). Nevertheless, about 20% of daily T4 production appears in the feces, probably through biliary excretion of glucuronide conjugates (7). Cleavage of the ether bond connecting the inner and outer ring as well as oxidative deamination of the alanine side chain, producing the acetic acid metabolites Tetrac and Triac, represent minor pathways of iodothyronine metabolism (2) which will not be further discussed here. In the following sections especially the biochemical aspects of the deiodination and sulfation pathways will be reviewed.
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| Figure 3c-1. Pathways of thyroid hormone metabolism. |
Three iodothyronine deiodinases have been identified, with distinct tissue distributions, catalytic specificities, physiological functions, and regulations (Fig. 2) (1-4). D1 is expressed predominantly by liver parenchymal cells, kidney proximal tubular cells, and thyroid follicular cells (1-4,12). Evidence suggests that the enzyme is associated in rat liver with the endoplasmic reticulum and in rat kidney with the plasma membranes (2). In cells transfected with D1 cDNA constructs, the expressed FLAG-tagged proteins appear to be located in the plasma membrane (13). D1 catalyzes the ORD and/or IRD of a variety of iodothyronine derivatives, although it is most effective in catalyzing the ORD of rT3, while the IRD of both T4 and T3 is strongly facilitated by sulfation of these iodothyronines (1,9). In the presence of dithiothreitol (DTT) as the cofactor, D1 displays high Km and Vmax values (1-4). Hepatic D1 is probably the primary site for the clearance of plasma rT3 and a major source of circulating T3 (1-4).
Figure 3c-2. Characteristics of the Three Types of Iodothyronine Deiodinases
Type |
D1 |
D2 |
D3 |
T4 |
T4 |
T4 |
|
T3 , rT3 |
rT3 , T3 |
rT3 |
|
T2 |
T2 |
T2 |
|
Tissues, e.g. |
liver, kidney, thyroid |
brain, pituitary, skeletal muscle, heart (?) |
brain, placenta fetal tissues |
Substrates |
rT3 > T4 > T3 |
T4 > rT3 |
T3 > T4 |
Km Values |
0.1 – 10 m M |
1 nM |
10 nM |
Function |
plasma T3 production |
local T3 production |
T3 degradation |
Inhibitors (IC50, m M) |
|||
PTU |
5 |
> 1000 |
> 1000 |
IAc |
2 |
1000 |
1000 |
GTG |
0.05 |
1 |
5 |
Hypothyroidism |
decrease |
increase |
decrease |
Hyperthyroidism |
increase |
decrease |
Increase |
D1 activity in liver and kidney is stimulated in hyperthyroidism and decreased in hypothyroidism, representing the regulation of D1 activity by T3 at the transcriptional level (14). T3 reponse elements (TREs) have been identified in the upstream region of the D1 gene (15,16). Studies in T3 receptor (TR) knockout mice have indicated that D1 expression in liver is primarily controlled by the TRb isoform (17). This agrees well with the colocalization of TRb and D1 in the pericentral zone of rat liver (18). In thyroid, D1 expression is stimulated by T3, TSH and TSH receptor antibodies (19,20). The effects of TSH and TSH receptor antibodies on D1 expression are mediated by cAMP.
D1, D2 and D3 have been cloned in different species, including man, rat, mouse, dog, frog, chicken and fish (21-38). The deduced amino acid sequences of human D1, D2 and D3 are presented in Fig. 3. The deiodinases appear to be homologous proteins, consisting of 249-278 amino acids. Although they are all hydrophobic proteins, a particulary lipophilic sequence is present at the N terminus of all three deiodinases, which probably represents a membrane-spanning region. Studies of the topography of rat D1 have suggested that the major part of the protein is exposed on the cytoplasmic surface of the membrane (39). Analysis of detergent extracts of rat liver or kidney membranes have indicated an apparent molecular weight of 50-60 kDa for native D1 (2), which is about twice the molecular weight of the protein encoded by the cDNA, suggesting that D1 is a dimer of two identical subunits. This has recently been confirmed in experiments using cells coexpressing endogenous pig D1 and exogenous rat D1 constructs (40).
The most remarkable feature of all three iodothyronine deiodinase is that they are selenoproteins, i.e. they contain a selenocysteine (Sec) residue in the center of the amino acid sequence. In all selenoproteins, Sec is encoded by a UGA triplet which is an opal stop codon because it usually signals termination of translation. However, if the 3' untranslated region (3'UTR) of the mRNA contains a particular stem loop structure, termed selenocysteine-insertion sequence (SECIS) element, the UGA codon specifies the insertion of Sec (41).
The D1 gene is located on human chromosome 1p32-33. It consists of four exons, with exon 1 coding for the 5'UTR and amino acids 1-112, exon 2 for amino acids 113-160, exon 3 for amino acids 161-227, and exon 4 for amino acids 228-249 and the 3'UTR, including the SECIS element. Before D1 was identified as a selenoprotein, this was suspected from findings that D1 activities are strongly reduced in liver and kidney, but not in thyroid, of rats fed on a selenium (Se)-deficient diet (42). This is associated with a small decrease in serum T3 and a marked increase in serum T4 (42). The Sec residue in D1 is essential for deiodinase activity since replacement of Sec by Cys results in a 100-fold decrease in catalytic activity, while substitution of Sec by Leu produces an enzymatically inactive protein (43). In addition, D1 is extremely sensitive to inactivation by iodoacetate due to carboxymethylation of a highly reactive residue, probably Sec, in the enzyme active center which is prevented in the presence of substrate (1-4). Moreover, D1 activity is inhibited by very low concentrations ("10-8 M) of goldthioglucose (GTG), which is known to form very stable complexes with Sec residues, and this inhibition is also competitive with substrate (44). Therefore, Sec is probably the catalytic center of D1.
Two other observations have provided important clues about the possible catalytic mechanism of D1. Firstly, D1 shows ping-pong type reaction kinetics in catalyzing the deiodination of iodothyronines by DTT (1,2), suggesting that reaction of iodothyronine substrate with D1 produces an enzyme intermediate, from which native enzyme is regenerated by reaction with thiol cofactor (DTT). Secondly, D1 is potently inhibited by PTU, and this inhibition is uncompetitive with substrate and competitive with cofactor, suggesting that PTU and cofactor react with the same enzyme intermediate. Thiouracil derivatives are particularly reactive towards protein sulfenyl iodide (SI) groups, and presumably even more reactive towards selenenyl iodide (SeI) groups, suggesting that such an intermediate is generated in the catalytic cycle of D1. Therefore, the selenolate (Se-) group of the native enzyme is thought to act as an acceptor of the iodonium (I+) ion which is substituted in the substrate by a proton, and the SeI intermediate thus generated is reduced back to native enzyme by thiols such as DTT or converted into a dead-end complex by PTU (Fig. 4). Unlike the mammalian enzyme, D1 from tilapia is insensitive to PTU inhibition (26). In contrast to expectation, cloning of tilapia D1 has demonstrated the presence of a Sec residue in a position corresponding to that in other, PTU-sensitive D1s (26). The reason for the PTU-insensitivity of tilapia D1 remains to be elucidated.
 |
| Figure 3c-4. Putative catalytic
mechanism of D1 and inhibition by PTU, IAc and GTG. *) Selenocysteine (Sec) |
D2 is expressed primarily in the brain, the anterior pituitary gland and (rodent) brown adipose tissue (BAT) (1-4). D2 activity has recently also been shown in rat skin (45), human thyroid (46-48) and human skeletal muscle (30,49), while D2 mRNA is also expressed in human heart (29,30). The enzyme is induced in cultured glial cells by a variety of factors (50), and localization of D2 mRNA in rat brain by in situ hybridization has recently indicated that the enzyme is expressed in astrocytes, in particular in tanycytes lining the third ventricles (51). In cells transfected with D2 cDNA constructs, FLAG-tagged D2 proteins were localized to the endoplasmic reticulum (13).
D2 has only ORD activity, exhibiting low Km and Vmax values, and a slight preference for T4 over rT3 as the substrate (1-4). In contrast to D1, it does not catalyze the deiodination of sulfated iodothyronines. The amount of T3 in tissues expressing D2 is derived to a large extent from local conversion of T4 by this enzyme and to a minor extent from plasma T3 (1-4). In general, D2 activity is increased in hypothyroidism and decreased in hyperthyroidism (1-4). Part of this negative control is explained by substrate-induced inactivation of the enzyme by T4 and rT3 (1-4,50,52). Both substrate-induced endocytosis of plasma membrane-associated enzyme as well as ubiquitination of the protein followed by its degradation in the proteasomes have been suggested as mechanisms for substrate-induced inactivation of D2 (52-55). In addition, presumably receptor-mediated inhibition of D2 activity by T3 has been demonstrated in pituitary tumor cells (56), and D2 mRNA levels in brain, pituitary and BAT are up-regulated in hypothyroid rats and down-regulated in hyperthyroid animals (57-59). The central Sec residue plays an important role in the catalysis and turnover of D2. Replacement of this Sec with Cys results in a 1000-fold increase in the Km value of the substrates T4 and rT3, and a 10-fold decrease in turnover number (60,61). Substitution of Sec by Ala completely inactivates the enzyme. Also the mechanism of substrate-induced D2 degradation is strongly or completely impaired by replacement of Sec by Cys or Ala, respectively (55), suggesting that modification of this Sec residue during catalysis may be an essential step in the inactivation of the enzyme. Interestingly, mammalian and avian D2 also have a second Sec residue near the C-terminus which, however, is not important for catalytic activity (62).
The D2 gene is located on human chromosome 14q24.2-q24.3. It consists of 2 exons of 0.7 kb and 6.6 kb, seperated by a 7.4 kb intron (63-67). The SECIS element in the 3'ÚTR is separated by ~5 kb from the UGA triplet coding for the catalytic Sec residue, resulting in a poor translation efficiency of the D2 mRNA (67). This is even further hampered by the presence of multiple short open reading frames in the 5'UTR of human D2 mRNA (67). Analysis of detergent extracts of brain cell membranes have indicated an apparent molecular weigth of "200 kDa, suggesting a multimeric complex of different subunits (68). Supposedly, one of these is the "30 kDa protein encoded by the D2 cDNA, while another "29 kDa 'substrate-binding' subunit (p29) appears to be involved in the substrate-induced internalization and inactivation of D2 in brain cells (50,52). This p29 has recently been cloned and characterized (69). Interestingly, transient expression of p29 induces a dramatic increase in D2 activity in cAMP-stimulated glial cells (69).
D2 is expressed in human thyroid but not in rat thyroid. Both D2 mRNA and D2 activity in human thyroid are greatly stimulated by TSH and TSH receptor antibodies circulating in patients with Graves' disease (47,48). The expression of D2 in human thyroid has been associated with functional TTF-1 binding sites in the 5' flanking region of the human D2 gene which are lacking in the 5' flanking region of the rat D2 gene (65). The stimulatory effects of TSH and TSH receptor antibodies on D2 expression in human thyroid are mediated by cAMP, which has been associated with the presence of a cAMP response element (CRE) in the 5' flanking region of the D2 gene (63,64). Recently, a D2 knockout mouse has been generated, showing modest phenotypic changes (70) The homozygous D2-/- knockouts have increased serum T4, normal T3, and increased TSH levels (70). Thermogenesis in BAT of D2 knockout mice shows a modest impairment, resulting in hypothermia in cold-exposed animals, confirming the role of D2 in local T3 generation in BAT (71).
D3 activity has been detected in a variety of tissues, i.e. brain (especially fetal brain) of different animals, rat skin (especially neonatal skin), fetal human liver, fetal rat intestine, the embryonic chicken liver, placenta, and the pregnant rat uterus (1-4,72-74). D3 activity has also been demonstrated in monkey hepatocarcinoma cell lines (75). Because of its expression in fetal liver and liver tumors, D3 has been named an oncofetal protein. The subcellular localization of the enzyme has not been assessed, but highest activities are associated with the microsomal fractions of the tissues (1-4). D3 has only IRD activity, catalyzing the inactivation of T4 and T3 with intermediate Km and Vmax values. The expression of D3 in placenta, pregnant uterus, embryonic and fetal tissues may protect developing organs against undue exposure to active thyroid hormone. Also in adult subjects, D3 appears to be an important site for clearance of plasma T3 and production of plasma rT3 (1-4). In brain and skin, but not in placenta, D3 activity is increased in hyperthyroidism and decreased in hypothyroidism (72), which in brain is associated with parallel changes in D3 mRNA levels (76).
The D3 gene is located on human chromosome 14q32 and contains no intron. In all species, D3 cDNA codes for a selenoprotein which is homologous with the amino acid sequences of D1 and D3, including the essential Sec residue positioned in a strongly conserved region (Fig. 2). Recent evidence suggests that D3 plays a much more important role in the regulation of thyroid hormone bioactivity than previously assumed. It has been shown that region-specific expression of D3 in fetal and adult human brain is negatively associated with local tissue T3 levels (77,78). Recent reports show that high expression of D3 in vascular tumors may result in subclinical and even overt hypothyroidism in patients with such tumors, which conditions are referred to as consumptive hypothyroidism (79,80). Recent findings have also demonstrated significant expression of D3 in liver and skeletal muscle in patients who died after severe illness (81). Finally, D3 knockout (D3-/-) mice which have been generated recently show remarkable neonatal mortalitity and growth retardation (82). The heterozygous D3+/- mice showed either almost normal or strongly decreased D3 expression, depending on whether the defective allele was inherited from the mother or the father, respectively, indicating paternal imprinting of the D3 gene (82).
In contrast to the marked decrease in hepatic and renal (but not thyroidal) D1 activities, the unexpectedly small effects of Se deficiency on tissue D2 and D3 activities in rats, despite that they all appear to be Sec-containing proteins, may be explained by findings that the selenium state of different tissues varies greatly in Se-deficient animals (83). In addition, the efficiency of the SECIS element to complex with protein factors necessary for the read-through of the UGA codon may vary between different selenoproteins, which could result in the preferred incorporation of Sec into some selenoproteins, e.g. deiodinases, over others, e.g. glutathione peroxidase (84).
The presence of Sec in a strongly conserved region of the proteins strongly suggests the same mechanism of deiodination for the different deiodinases. This seems to be contradicted by the widely different susceptibilities of D1 versus D2 and D3 to the different mechanism-based inhibitors PTU, IAc and GTG (Fig. 2). It also seems to be in conflict with previous findings that, in contrast to the ping-pong kinetics of D1, the other two enzymes appear to follow sequential-type kinetics, suggesting the formation of a ternary enzyme-substrate-cofactor complex during catalysis (1,2). A possible explanation for the low susceptibility of D2 and D3 to IAc and GTG inhibition is that the reactivity of the selenol group in these enzymes may be much lower than that in D1. This would imply that turnover numbers are much lower for D2 and D3 than for D1. It has been shown that the 100-fold lower turnover number for the Sec126Cys mutant versus wild-type rat D1 is associated with a dramatic decrease in its sensitivity for inhibition by GTG and PTU (44).
hD1 MGLPQP GLWLKRLWVL LEVAVHVVVG KVLLILFPDR VKRNILAMGE KTGMTRNP.. hD2 MGILSVDLLI TLQILPVFFS NCLFLALYDS VILLKHVVLL LSRSKSTRGE WRRMLTSEGL hD3 MLHSLLLH SLRLCAQTAS CLVLFPRFLG TAFMLWLLDF LCIRKHFLGR RRRGKPEPEV hD1 .......... ......HFSH DNWIPTFFST QYFWFVLKVR WQRLEDTTEL GGLAPNCPVV hD2 R......... ......CVWK SFLLDAYKQV KLGEDAPNSS VVHVSSTEGG DNSGNGTQEK hD3 ELNSEGEEVP PDDPPICVSD DNRLCTLASL KAVWHGQKLD FFKQAHE... GGPAPNSEVV hD1 RLSG.QRCNI WEFMQGNRPL VLNFGSCTUP SFMFKFDQFK RLIEDFSSIA DFLVIYIEEA hD2 IAEG.ATCHL LDFASPERPL VVNFGSATUP PFTSQLPAFR KLVEEFSSVA DFLLVYIDEA hD3 LPDGFQSQHI LDYAQGNRPL VLNFGSCTUP PFMARMSAFQ RLVTKYQRDV DFLIIYIEEA hD1 HASDGWAFKN N....MDIRN HQNLQDRLQA AHLLLARS.. .PQCPVVVDT MQNQSSQLYA hD2 HPSDGWAIPG DSSLSFEVKK HQNQEDRCAA AQQLLERFSL PPQCRVVADR MDNNANIAYG hD3 HPSDGWVTTD SP...YIIPQ HRSLEDRVSA ARVLQQGA.. .PGCALVLDT MANSSSSAYG hD1 ALPERLYIIQ EGRILYKGKS GPWNYNPEEV RAVLEKLHS 249 hD2 VAFERVCIVQ RQKIAYLGGK GPFSYNLQEV RHWLEKNFSK RUKKTRLAG 273 hD3 AYFERLYVIQ SGTIMYQGGR GPDGYQVSEL RTWLERYDEQ LHGARPRRV 278 |
| Figure 3c-3. Alignment of the amino acid sequences of human D1, D2,
and D3 U = Selenocysteine (Sec) |
Importance of D3 during fetal development
The important role of hepatic D3 in the regulation of circulating thyroid hormone during development has been investigated in detail in the embryonic chicken (25,85). These studies have demonstrated that during the last (third) week of incubation there is a gradual increase in plasma T4 levels paralleled by a steady increase in hepatic D1 activity although hepatic D1 mRNA levels do not change much (25). D3 mRNA and D3 activity show a parallel increase to maximum levels at day 17 of embryonic development, followed by a steep decrease in both parameters in particular immediately before hatching (25). This is associated with an equally steep increase in plasma T3, strongly suggesting that the latter is importantly and negatively regulated by hepatic D3 activity (25).
A study of the ontogeny of hepatic D1 and D3 during human development has indicated similar profiles of deiodinase expression, with substantial and relatively constant D1 activities from mid-gestation onwards, and high D3 activities at mid-gestation declining to very low levels around term (86). Since in rat liver D1 is not expressed until the last days of gestation, while hepatic D3 expression is low at all stages of rat development (73), these results indicate that the embryonic chicken is a better model than the fetal rat for the regulation of hepatic deiodinases during human development. Injection of the chicken embryo with growth hormone or glucocorticoids induces an acute down-regulation of hepatic D3 mRNA levels and D3 activities, suggesting that the D3 mRNA in the embryonic chicken has a very short half-life, and that transcription of the D3 gene is acutely blocked by these treatments (85). If D3 expression in the fetal human liver is also rapidly down-regulated by GH and glucocorticoids remains to be determined. It is likely that the high D3 activities expressed in the fetal liver, in addition to the high D3 activities in the placenta (87,88) and perhaps the uterus (74), plays an important role in the regulation of fetal circulating T3 levels.
Iodothyronine sulfotransferases
Relatively little is known about the sulfotransferases involved in the sulfation of thyroid hormone in humans. Sulfotransferases represent a family of soluble enzymes with a molecular weight of "34 kDa, located in the cytoplasmic fraction of different tissues, e.g. liver, kidney, intestine and brain (89). They catalyze the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to usually a hydroxyl group of the substrate (89). On the basis of substrate specificity and aminoacid sequence homology, two sulfotransferase subfamilies have been recognized in human tissues, i.e. phenol sulfotransferases, including estrogen sulfotransferase, and hydroxysteroid sulfotransferases (89). Different phenol sulfotransferases have been identified with significant activity towards iodothyronines. These include human SULT1A1, SULT1A2, SULT1A3, and SULT1B1 (90,91). Strange enough, these studies have indicated a large substrate preference of the recombinant enzymes as well as the native enzymes in human liver and kidney for 3,3'-T2, the sulfation of which is catalyzed orders of magnitude faster than that of T3 or rT3, while sulfation of T4 is hardly detectable (91). Surprisingly, recent studies have also demonstrated that human estrogen sulfotransferase (SULT1E1) is an important isoenzyme for sulfation of thyroid hormone. Although human SULT1E1 shows much higher affinities for estrogens (Km "nM) than for iodothyronines (Km "µM), it is about as efficient as other isoenzymes in sulfating 3,3'-T2 and T3, and much more efficient in sulfating rT3 and T4 (92). Human tissues known to express SULT1E1 include liver, uterus, and mammary gland (93). In particular the enzyme expressed in the endometrium may be a significant source for the high levels of iodothyronine sulfates in human fetal plasma (see below).
Deiodination of iodothyronine sulfates
Although D1 is capable of converting T4 with similar efficiency by ORD to T3 and by IRD to rT3, this is changed dramatically after sulfate conjugation, i.e. IRD of T4S by rat D1 is accelerated "200-fold, while ORD of T4S becomes undetectable (Fig. 5) (9). IRD of T3 by rat and human D1 is also markedly stimulated ("40-fold) by sulfation (Fig. 5) (9). As mentioned before, rT3 is by far the preferred D1 substrate; its ORD is not influenced by sulfation, suggesting that the catalytic efficiency of D1 is already optimal with nonsulfated rT3 (9). While sulfation inhibits ORD of T4 and is without effect on ORD of rT3, it markedly stimulates ORD of 3,3'-T2 (Fig. 5). Thus, sulfation facilitates the IRD of T4 and T3, while it either inhibits (T4), does not affect (rT3) or markedly stimulates (3,3'-T2) the ORD of other substrates (9). The mechanism by which sulfation stimulates especially the IRD of different substrates remains unclear. In some cases sulfation primarily effects an increase in Vmax, while in others there is a predominant decrease in apparent Km value (9). The facilitated deiodination of sulfated iodothyronines by rat liver D1 may be due to interaction of the negatively charged sulfate group with protonated residues in the active center of this basic protein (9). The effect of sulfation on deiodination of iodothyronines is both conjugation type and deiodinase type-specific since D1 does not catalyze the deiodination of T3G, while D2 and D3 do not accept T4S and/or T3S as substrates.
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| Figure 3c-5. Efficiency of
deiodination of iodothyronines and their sulfates by rat liver D1. *) Vmax (pmol/min/mg protein)/Km (µM) ratio. |
Importance of thyroid hormone sulfation
Serum concentrations of T4S, T3S, rT3S and 3,3'-T2S are low in normal human subjects but they are high in fetal and cord blood, in patients with NTI, and in patients treated with PTU or IOP, both inhibitors of D1 (94-100). The serum T3S/T3 ratio is also increased in hypothyroid patients (95). High iodothyronine sulfate levels have also been detected in serum, bile, allantoic fluid and amniotic fluid of fetal sheep (101-104). The high serum iodothyronine sulfate levels during NTI, hypothyroidism and fetal development have been ascribed to a low peripheral D1 activity in these conditions (9). These results are in accordance with experimental findings in rats, showing marked increases in the serum concentration and biliary excretion of iodothyronine sulfates in animals with impaired hepatic and renal D1 activities due to administration of D1 inhibitors or selenium deficiency (9). These changes are not caused by an increased sulfation of iodothyronines but by a decreased clearance of the sulfated iodothyronines by D1 (9). Thus, sulfation is a primary step leading to the irreversible degradation of T4 and T3 by D1. However, if D1 activity is low, inactivation of thyroid hormone by sulfation is reversible due to expression of sulfatases in different tissues and by intestinal bacteria (105-107). It has been speculated that especially in the fetus T3S has an important function as a reservoir from which active T3 may be released in a tissue-specific and time-dependent manner (9,106).
Since thyroid hormone action is initiated, by and large, by binding of T3 to its nuclear receptors, it is important to consider the role of the processes discussed above in the regulation of nuclear T3. There are two sources of intracellular T3, i.e. T3 derived from plasma T3, and T3 produced locally from T4, and the degree to which they contribute to the occupied receptors varies among the different tissues (4,108, 109). The kidney and liver are typical of most tissues in the body in which most of the T3 specifically bound to the T3 receptor is derived directly from plasma. In the cerebral cortex, BAT, and anterior pituitary there is a substantial contribution to nuclear T3 from locally produced T3.
Under normal conditions, plasma T3 is largely derived from deiodination of T4 by D1 in liver and kidneys (1-4). Most tissues depend on plasma T3, but some critical tissues, such as the central nervous system (CNS), have acquired a second deiodinase, D2, for local T4 to T3 conversion which allows them to regulate intracellular T3 levels independent of variations in plasma T3 (1-4). D3 plays an additional important role in maintaining intracellular T3 concentrations in these tissues by catalyzing the degradation of T3 in case of excess or by diverting the metabolism of T4 to rT3. Indeed, the adaptations of deiodinase activities in response to changes in thyroid state are thought to serve the purpose of keeping intracellular T3 in the CNS constant. Thus, when T4 supply is decreased in hypothyroidism both D1 and D3 activities are down-regulated, so that relatively more T4 is available for conversion to T3 by D2 in the CNS, the activity of which is up-regulated. Opposite changes occur in hyperthyroidism. These adaptations are not only important for the optimal function of the CNS in adult life, they are also essential for the development of the CNS which is critically dependent on thyroid hormone (110). Although the adaptations in deiodinase activities during hypo- or hyperthyroidism go a long way in securing T3 availability in the CNS, in severe iodine deficiency they may not fully compensate for the extreme decrease in T4 supply (101). This may result in severe impairment of neurological development in the child even when plasma T3 levels in the mother are sufficient to maintain a euthyroid state (101).
The view that D1 in liver and kidney is largely responsible for plasma T3 production and that D2 is responsible for local T3 production in tissues such as brain has been changed by recent findings that D2 is also expressed in human thyroid and skeletal muscle. The expression of both D1 and D2 in the human thyroid implicates that in certain pathophysiological conditions, such as Graves' disease, hypothyroidism, and iodine deficiency, the ratio of T3 and T4 secretion may be much higher than their content in thyroglobulin due to intrathyroidal T4 to T3 conversion. The presence of D2 in skeletal muscle suggests that it does not only produce T3 'locally' but because of the size of this tissue it may also contribute significantly to plasma T3 production. This would imply a dual source for plasma T3: a) D1 conversion of T4 in liver and kidneys, and b) D2 conversion of T4 in skeletal muscle. Because of the positive control of D1 and negative control of D2 by thyroid hormone, their relative contributions to plasma T3 production may depend on thyroid state, with D2 being more important in hypothyroidism and D1 in hyperthyroidism. This is supported by findings of a non-linear relationship between the plasma T3/T4 ratio and plasma T4 in patients substituted with various T4 doses, suggesting two mechanisms of peripheral T3 production (111). It is also supported by findings that the efficacy of PTU in the inhibition of peripheral T3 production is greater in hyper- than in euthyroid subjects (4), supporting an increased contribution of hepatic and renal D1 to plasma T3 production in hyperthyroid subjects. The low T3 syndrome in patients with NTI may thus result not only from decreased T3 production in D1 tissues such as liver but also from decreased T3 production in D2 tissues such as skeletal muscle, and in addition from increased T3 breakdown in D3 tissues such as skeletal muscle.
