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Iodothyronine deiodinase
Iodothyronine Deiodinases Donald L. St. Germain
[reviewed by Leonard (1991)]. Recent affinity-labeling studies have confirmed this impression (Safran and Leonard 1991). These three processes have been designated the type I, II, and III iodothyronine deiodinases, and a summary of their tissue distribution and catalytic activity in the rat is shown in Figure 2. Type I Deiodinase
The iodothyronine deiodinases constitute a family of enzymes that catalyze the removal of iodine atoms from various thyroid hormones (THs) in the thyroid gland and extrathyroidal tissues. As such, they are responsible for both the activation and inactivation of these compounds, and are thus important regulators of TH action. Recently, new insights have been gained into the biochemical characteristics of these proteins and their physiologic roles in TH metabolism. In particular, the availability of affinity-labeling techniques, molecular probes, and specific antisera for these enzymes, and the recent identification of the type I deiodinase as a selenoprotein, have ushered in a new era in the study of thyroid hormone deiodination. (Trends Endocrinol Metab 1994;5:3642)
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Overview of Thyroid Hormone Metabolism
The metabolism of thyroid hormones (THs) in extrathyroidal tissues involves a complex series of enzymatic reactions that result in the deiodination, conjugation, andlor deamination and oxidative decarboxylation of these iodothyronine compounds (Figure 1). The extent to which these reactions occur within a given cell or organ has profound effects upon the circulating and tissue levels of THs and thus their metabolic effects (Wu 1991). Of importance, these metabolic reactions are not mutually exclusive, but may occur sequentially. For example, sulfoconjugation appears to enhance the rate of certain deiodination reactions selectively in some tissues (see the following). Deiodination is the predominant mechanism of TH metabolism and involves the removal of an iodine atom from either the tyrosyl ring (at the 3 or biochemically equivalent 5 position) or the phenolic ring (at the 3’ or 5’ position). Although thyroxine (T,) is the principal secretory product of the thyroid gland, it has a relatively low affinity Donald L. St. Get-main is at the Departments of Medicine and Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA. 36
for the nuclear TH receptors when compared with 3,5,3’-triiodothyronine (T,), and functions primarily as a prohormone. Thus, the 5’-deiodination of T, to form T, represents a key activation step in the action of THs. In contrast, deiodination of T, or T, on the tyrosyl ring results in the formation of the inactive iodothyronines 3,3’,5’-triiodothyronine (reverse T,, rT,) or 3,3’-diiodothyronine (T,), respectively. The physiologic importance of deiodination to overall TH economy is highlighted by the finding that -80% of the secreted T, is deiodinated to form T, and rT, in approximately equimolar amounts (Engler and Burger 1984).
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Classification and Characteristics of Iodothyronine Deiodinases
Until recently, the methods available for studying the 5’- and 5-deiodinase enzymes (5’-D, 5-D, respectively) that catalyze these reactions have been quite limited, in large part because reliable purification methods for these relatively low-abundance microsomal proteins have not yet been developed. Based on activity measurements in tissue homogenates, three major patterns of deiodination have been identified and, because of their diverse characteristics, have been assumed to result from the catalytic properties of three separate enzymes
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The type I deiodinase is found predominantly in the liver and kidney and can catalyze either 5’- or 5-deiodination (Visser 1988). Both processes are extremely sensitive to inhibition by propylthiouracil (PTU). In euthyroid rats, 70% of the circulating T, appears to be derived from the catalysis of T, at the 5’ position by this enzyme (Silva et al. 1984). In certain species such as humans and rats, type I deiodinase is also present in the thyroid gland and appears to contribute significantly to thyroidal T, production (Beech et al. 1993). The sulfation state of the iodothyronine substrates appears to have a major influence on the type I catalytic reaction; nonsulfated iodothyronines, such as T, and rTT,, undergo predominantly 5’deiodination, whereas T, sulfate and T, sulfate are deiodinated at the 5 position (Visser 1988). As investigated recently in HepG2 cells, sulfation appears to be an obligate step in the 5-deiodination process by the type I enzyme (Van Stralen et al. 1993). Thus, the sulfation pathway(s) in tissues expressing type I activity may play an important role in regulating TH metabolism by dictating the relative extent of tyrosyl versus phenolic ring deiodination. Of potential physiologic importance, the catalytic efficiency with which various substrates are deiodinated by the type I deiodinase is markedly different, as judged by the V,,,/K, ratios of the individual compounds (Visser 1988). T, is a relatively poor substrate for 5’- and 5-deiodination by this enzyme; the V,,,/ K,,, ratio for the 5’-deiodination of rTJ is at least 500 times greater than that of T,, and both T, sulfate and T, sulfate are 5-deiodinated with considerably greater efficiency than unconjugated T,. Such data, derived with dithiothreitol (DTT) used as a cofactor in tissue homogenates, where free hormone concentrations are uncertain, permits only cautious extrapolation to the in vivo setting, but suggests that in tissues such as the
TEM Vol. 5,No.1,1994
liver and kidney, where tissue T, levels are only modest,
the predominant
cata-
lytic processes carried out by the type I enzyme are the 5’-deiodination of rTs and the 5-deiodination
of the sulfated
1 DEIODINATION
conjugates of T, and T,. In the thyroid, however, where intracellular concentrations of T, are high, the 5’-deiodination of T, to T, may be the principal reaction process. Recent studies demonstrating little change in serum T, levels in rats with impaired extmthyroidal type I deiodinase activity are consistent with such an analysis and implicate the thyroid gland as the principal source of circulating T, in this species (Chanoine et al. 1993). Similarly, in the C,H/He mouse, which manifests an impairment in type I deiodinase activity, serum T, levels are no different from those in control mice, but circulating rT, levels are increased -3fold, demonstrating the importance of this enzyme in rTJ clearance (Schoenmakers et al. 1993). Type II Deiodinase The central nervous system, pituitary gland, and brown adipose tissue (BAT) express type II deiodinase activity, which catalyzes deiodination exclusively at the 5’ position (Leonard 1991). Both T, and rT, are efficiently metabolized by this pathway, with the product of T, deiodination being the active TH T,. Unlike the type I deiodinase, this process is relatively resistant to the inhibitory effects of PTU and aurothioglucose. The type II enzyme appears to be broadly distributed in the rat brain, with relatively high levels present in the cerebral cortex, cerebellum, and hypothalamus (Kaplan et al. 1981). Although the cell types expressing this enzyme have not yet been defined in vivo, type II activity is present in cultured astroglial cells (Leonard and Larsen 1985, Courtin et al. 1991) and in the NB41A3 neuroblastoma cultured cell line (St. Germain 1986). Studies in fetal and neonatal rats have demonstrated that this deiodinase is of considerable importance in ensuring the adequate intracellular concentration of T, in the brain during critical periods of development (Silva and Matthews 1984, Ruiz de Oiia et al. 1988). During cold adaptation, a rapid increase in type II deiodinase activity in brown fat contributes significantly to T, levels in that tissue, and thus the THinduced increase in thermogenesis (Bi-
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I
1
I
m I
I
I T3
I T4
rever.se T3
1 CONJUGATION *
I
I T4
(
1
SO4&-& 1 I T4 sulfate
DEAMINATION I DECARBOXYLATION
1
*-* T4
Tetrac
Figure 1. Enzymatic processes involved in the metabolism of thyroid hormones. anco
and Silva
1988).
In the anterior
pituitary lobe, the local generation of T, by the type II deiodinase appears to contribute significantly to nuclear receptor T, occupancy, suggesting a mechanism whereby pituitary function can be modulated by circulating T4 as well as T, levels (Van Door-n et al. 1983). Of note, transplantation of the anterior pituitary to the kidney capsule results in a marked decrease in the level of type II activity and an increase in type I activity, suggesting that hypothalamic releasing factors may play a role in regulating deiodination in this tissue (St. Germain et al. 1985). Type II deiodinase has also been described in lower species, such as in fish liver (MacLatchy and Eales 1992) and in several tissues of the metamorphosing tadpole (Galton and Hiebert 1988). Type III Deiodinase An exclusively tyrosyl-ring-deiodinating enzyme, the type III deiodinase, is found primarily in the cerebral cortex and skin of adult nonpregnant rats (Kaplan et al. 1983, Huang et al. 1985), but is also expressed in large amounts in the placenta and in several fetal rat tissues,
little affinity
for the nuclear
tors and are metabolically type III deiodinase may
TH recep-
inactive, the serve during
development to protect the fetal brain and other tissues against excessively high levels of active THs. The function of this enzyme in the skin is at present uncertain. Skin contains the highest levels of rT, of any tissue in the adult rat (Huang et al. 1985>, however, suggesting that the high levels of type III activity noted in skin homogenates accurately reflect the activity of this enzyme in vivo. Other Deiodinases A recent report suggests that the classification of deiodinases into the three types described above may need to be broadened. In the kidney of the fish species tilapia (Oreochromis niloticus), a s’Figure 2. Classification and tissue distxibution of the iodothyronine deiodinases as identified in the adult rat. BAT, brown adipose tissue.
such as skeletal muscle, intestine, liver, and cerebellum (Huang et al. 1988). An analogous deiodinase has been demonstrated in human placenta (Roti et al. 1981) and brain (Kodding et al. 1988). The exact physiologic role of this deiodinating pathway is uncertain. However, since the products of 5-deiodination (rT3 converted from T,, and T, from T,) have
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37
been
it parallels the decrease in activity of the
controlled
described that displays properties of both the type I and the type II deiodinases (Mol et al. 1993). Similar to the type I enzyme, the preferred substrate for the fish deiodinase is rT,, and the K, value in the presence of high concentrations of
type III 5-deiodinase in the brain and skin. In contrast, the activity of the type II 5’-deiodinase in brain, pituitary, and brown fat increases markedly in response to decreases in circulating TH levels, and thus serves to maintain relatively normal T, levels in these tissues.
1993). In the latter conditions, the decreased mRNA levels may reflect tissue hypothyroidism induced by the altered nutritional status of the animal. Because cDNAs for the type II and type III deiodinases have not yet been isolated,
deiodinating
process
has recently
DTT is in the micromolar range. The reaction process, however, is quite resistant to inhibition by PTU and aurothioglucose, which are properties more consistent with a type II deiodinase.
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Coordinate Regulation of Deiodinases in Response to Alterations in Thyroid Hormone Status
Dramatic changes in deiodinase activity accompany alterations in TH status (Kaplan 1986). From a physiologic viewpoint, these changes appear to be coordinated in such a fashion as to maintain T, levels as normal as possible in the circulation and selected tissues such as the brain, and may have developed as a protective mechanism in response to iodine deficiency. As illustrated in Figure 3, the response in the rat to hypothyroidism includes a marked decrease in the activity of the type I enzyme in liver and kidney. To the extent that this enzyme is involved in 5-deiodinating reactions as noted above, this decrease in activity of an “inactivating” process may serve to protect circulating T, and T, levels, and
Figure 3. Alterations in iodothymnine deiodinase activities in response to hypothyroidism. Changes in the relative size of the boxes and weight of the arrows relative to Figure 2 represent the alterations in the various deiociinase processes and reactions that occur in the hypothyroid state.
TYPE II Brain, Pituitary,
38
T4
TYPE III
T3
m.el”. Sk,”
T3 7.2
BAT
rT3 T2
An exception to this pattern is the type I deiodinase in the thyroid gland, which increases in the hypothyroid state under the influence of TSH stimulation (Erickson et al. 1982). Such a response likely serves to increase the proportion of T, secreted by the thyroid in states of impaired thyroid function such as iodine deficiency and may explain in part the increased T,iT, ratio observed in the hypothyroid state. The administration of T, to experimental animals to induce a hyperthyroid state results in opposite changes in activity of all three deiodinating pathways (Kaplan 1986). In addition to being regulated by TSH, the type I activity in the thyroid gland is stimulated by the thyroid-stimulating immunoglobulins that circulate in Graves’ disease, an effect that likely contributes to the increased thyroidal secretion of T, in this condition (Ishii et al. 1981).
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Cellular Mechanisms Deiodinase Activity
Pretranslational
Regulating
Regulation
The complex pattern of altered deiodinase activity resulting from changes in TH status involves both pre- and posttranslational regulatory mechanisms. The regulation of type I activity in response to altered TH levels results from changes in the mENA level for this enzyme (Berry et al. 1990, St. Get-main et al. 1990). We have quantified this response by using an RNA solution hybridization assay and have demonstrated in hypothyroid rats an approximate 50-fold increase, over a 72-h time period, in type I deiodinase mRNA level in both liver and kidney in response to T, administration (O’Mara et al. 1993). This increase in mRNA level is accompanied, after an 8- to 24-h delay, by an increase in type I activity of similar magnitude. Alterations in mRNA level are also responsible for the stimulation of enzyme activity by TSH in the thyroid (Toyoda et al. 1992) and the decreases in activity observed in fasting and the poorly
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diabetic
state (O’Mara
et al.
the pretranslational regulation of these enzymes has not been well defined. Posttranslational
Regulation
In addition to this pretranslational
mode
of regulation, the type I deiodinase, as well as the type II enzyme, are subject to rapid posttranslational inactivation, induced by THs and competitive inhibitors such as sodium ipodate (Leonard et al. 1984, St. Germain 1988a and b). This in vivo loss of catalytic activity is manifested by a decrease in maximal enzyme velocity, and we have termed this process ligand-induced inactivation. The cellular mechanisms mediating this loss of activity have not yet been fully delineated and may vary depending on the enzyme and tissue involved. For the type I deiodinase in liver and kidney, inactivation appears to be closely linked to the sulfhydryl state of the enzyme and may require direct interaction between the ligand and the active site as a triggering mechanism (St. Germain 1988a). PTU, by binding to the active site of the enzyme and inhibiting catalytic cycling, protects the type I deiodinase from inactivation (St. Germain and Croteau 1989). These concepts have recently been confirmed and extended by Santini and Chopra (1992). Using a specific RIA for this enzyme, these investigators have demonstrated that treatment of rats with sodium ipodate is associated with a decreased mass of type I deiodinase in rat liver and kidney, whereas PTU treatment results in an increase in the liver content of this protein. Studies conducted on the GH, rat pituitary tumor cell line suggest that the sulfhydryl status of the cell is also important in the inactivation of the type II deiodinase; treatment of intact cells with the sulfhydryl-oxidizing agent diamide causes a rapid loss of type II catalytic activity, whereas treatment with DTT protects against ligand-induced inactivation (St. Germain 1988b). Leonard et al. (1990) have investigated the inactivation of the type II deiodinase by THs in cultured astroglial cells and have found that this loss of activity is
TEM Vol.5,No. I.1994
inhibited by cytochalasins, implicates
a finding that
the actin cytoskeleton in this In subsequent studies using
process. affinity-labeling
techniques,
it has been
demonstrated that T, rapidly promotes actin polymerization and internalization of the type II deiodinase catalytic subunit to an endosomal pool resulting in its inactivation (Farwell et al. 1990 and 1993). Whether this unique and important extranuclear mechanism of TH action demonstrated in cultured glial cells is applicable to the inactivation of deiodinases in other tissues remains uncertain. For example, in GH, cells, the structural requirements of the iodothyronines for inducing inactivation of the type II deiodinase differ from those observed in astroglial cells (St. Germain 1988b, Safran et al. 1993) and cytochalasins do not influence the inactivation process (St. Germain unpublished observation 1990). Cultured astroglial cells, which express type III deiodinase activity as well as type II activity, have also proved to be an excellent model system for studying the effects of other regulatory mechanisms on deiodinase enzymes. In this cell system, CAMP, phorbol esters, and fibroblast growth factor a have marked stimulatory effects on deiodinase activity (Courtin et al. 1991).
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Thiol Cofactors
Thiol cofactors are essential components of the deiodination reaction process and interact with the deiodinase enzymes and substrates in either a ping-pong or a sequential kinetic pattern (Leonard 1991). In tissue homogenates, dithiols such as DTT are the most potent in supporting deiodinase activity. Of note, the kinetic characteristics of 5’-deiodination in rat liver and kidney homogenates differ significantly, depending on the cofactor system used in the in vitro assay system. When DTT is used in the reaction mixture, the I$,, values for the 5’-deiodination of T4 and rT, are in the micromolar range. Such concentrations far exceed the intracellular levels of these substrates in liver and kidney, and thus called into question the physiologic significance of this process. This was brought into further relief by the demonstration that physiologic cofactors such as reduced glutathione (GSH) or a reconstituted thioredoxin-NADPH system supported 5’-deiodination in homogenates
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of these tissues nanomolar
with K,
values in the and Rosen-
1988). Indirect evidence suggests a close
1988, Bhat et al. 1989). This sug-
association between PDI and deiodinase enzymes. Initial attempts to isolate a
gested that, in addition to the DTTsupported process, one or more “low K,”
cDNA for the type I enzyme made use of a polyclonal antiserum that was demon-
5’-deiodinases might be present in these tissues. This uncertainty as to the number of 5’-deiodinases in the liver and the kidney was recently resolved by the
strated in vitro to precipitate 5’-deiodinase activity from solubilized liver microsomes. Of potential significance, the use of this antiserum to screen a hgtl 1 cDNA expression library resulted in the isolation of a cDNA for PDI (Boado et al. 1988). More recently, Leonard and colleagues have demonstrated that PDI is
berg
range (Goswami
demonstration that the type I deiodinase, when expressed in Xenopus laevis oocytes by the injection of mRNA prepared in vitro with use of the full-length cDNA for this enzyme as template, manifests both high K, and low K, activity, depending on the thiol cofactor system utilized in the in vitro reaction assay (Sharifi and St. Germain 1992). Of importance, in the absence of added thiols, significant levels of low K, 5’-deiodinase activity are demonstrable in this system, suggesting that this is the kinetic pattern of the enzyme when endogenous cofactors are utilized. Thus, the liver and kidney express only a single 5’-deiodinase, the type I enzyme, whose reaction kinetics differ markedly depending on the available thiol cofactor. Irrespective of the cofactor utilized, however, rTT, is a much more efficient substrate than T, for 5’-deiodination by this enzyme (Sharifi and St. Germain 1992). Of the three deiodinase processes described to date, only type 1 activity has been demonstrated in the presence of a “physiologic” cofactor system such as GSH or thioredoxin; types II and III deiodinase activity have been demonstrated only with relatively high concentrations of DTT, and GSH does not efficiently support deiodination by these latter enzymes in vitro. Thus, the identity of the cofactor system(s) that supports deiodination in vivo remains uncertain.
??
Deiodination Isomerase
and Protein
Disulfide
The study of the deiodinases has recently become intertwined with that of another microsomal enzyme, protein disulfide isomerase (PDI). This multifunctional, 55-kD protein was first identified because of its ability to catalyze the isomerization of protein disulfide bonds in vitro [reviewed in Noiva and Lennarz (1992)]. Of note, PDI binds T, with relatively high affinity and is downregulated by posttranslational mechanisms in response to T, treatment in GH, cells (Obata et al.
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translocated in glial cells by the actin cytoskeleton in response to T, treatment in a manner analogous to the type II deiodinase (Safran et al. 1992). Although the significance of these observations remains uncertain, they suggest that PDI could be involved in either the posttranslational regulation of the deiodinases as described here, or function as part of the thiol cofactor system necessary for deiodinase catalytic activity. Of note regarding the latter possibility is that PDI contains two thioredoxinlike active sites (Noiva and Lennarz 1992).
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The Role of Selenium Deiodination
in
The characterization of full-length cDNAs for the rat and human type I deiodinases has confirmed earlier evidence that these enzymes are selenoproteins that, like the glutathione peroxidases, contain a selenocysteine at their active site (Berry et al. 1991, Mandel et al. 1992). Additional studies by Berry and Larsen (1992) have provided important insights into the molecular mechanisms controlling selenocysteine incorporation into proteins during the translational process. Using mutation analysis, these investigators have also demonstrated that the presence of the selenocysteine is responsible for several of the unique properties of the type I enzyme, such as the marked sensitivity to inhibition by PTU and the gold compound aurothioglucose. Indeed, the lack of sensitivity of the type II and III deiodinases to these inhibitors suggests that they may not be selenoproteins or may lack a selenocysteine at the active center of the enzyme (Berry et al. 1991, Safran et al. 1991, Santini et al. 1992). In experimental animals, selenium deficiency leads to a marked reduction in type I deiodinase activity and protein content in the liver and kidney, but,
39
surprisingly, not in the thyroid gland, which appears to be markedly resistant to selenium depletion (Behne et al. 1990, Vadhanavikit and Ganther 1993, Chanoine et al. 1993). The cellular basis for this conservation of selenium within the thyroid gland is uncertain, but it appears to be responsible for the observation that moderate selenium deficiency in the rat does not lower serum T, levels in spite of markedly decreased extrathyroidal type I deiodinase activity. Severe selenium deficiency, however, can impair thyroidal function as well as peripheral TH metabolism, and evidence from both human and animal studies suggests that concurrent selenium deficiency may be an important determinant of the severity of the clinical manifestations of iodine deficiency (Arthur et al. 1992, Berry and Larsen 1992).
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Clinical Implications
The complexity of the deiodinating pathways has important implications for human thyroid physiology. In Graves’ disease, for example, T, to T, conversion is increased in the thyroid gland and other tissues containing the type I deiodinase, a process that likely contributes to the hyperthyroid state. Thus, high doses of the type I deiodinase inhibitor PTU, or radiographic contrast agents, such as sodium ipodate, which are potent inhibitors of all deiodinase processes, are of therapeutic benefit (Croxson et al. 1977, Wu et al. 1982). To date, very few patients with defects in deiodination have been described, a situation that contrasts with the many case reports of the TH resistance syndromes caused by defective T, receptors (Refetoff et al. 1993). Kleinhaus et al. (1988) described a clinically euthyroid patient with elevated T, and rTs levels accompanied by normal T, and TSH concentrations, findings similar to those found in the C,H/I-Ie mouse with deficient type I deiodinase activity (Berry et al. 1993, Schoenmakers et al. 1993). Thus, mild-to-moderate degrees of type I deficiency may be manifest only by hyperthyroxinemia, with little or no impact on clinical thyroid status (Maxon et al. 1982). The clinical phenotypes of deficient type II or type III deiodinase activity are uncertain, because patients with these defects have not been recognized. Furthermore, selective inhibitors of these
40
processes are not yet available, and animal models with defective activity have not been described. Given the distribution of the these pathways, however, one might expect deficiencies of type II or type III activity to have an adverse effect on fetal development in general and brain maturation in particular.
techniques,
and
biochemical
the investigation
methods
of selenium
in
metabolism.
Biol Trace Elem Res 2627:439447. Berry
MJ,
Larsen
selenium
PR:
in thyroid
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The
hormone
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of En-
doer Rev 13:207-219. Berry MJ, Kates A, Larsen PR: 1990. Thyroid hormone
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RNA in rat liver.
mes-
Mol Endocrinol
4:743-748. ??
Future Studies
Berry
In spite of important
new insights
into
the mechanisms of TH deiodination, considerable work remains to be accomplished before we have a complete understanding of these important metabolic processes. Our knowledge of the physiologic role of these enzymes in individual tissues and in overall TH economy remains incomplete. This is due in part to the complexity of these enzymatic processes, each of which can utilize multiple substrates, and the lack of specific inhibitors for the different deiodinases in various tissues. In this regard, the availability in the future of experimental animal models produced by recombinant DNA techniques with impaired deiodinase activity of specific enzymes in specific tissues should provide important information, particularly regarding the role of deiodination in TH action in the brain and during development. In addition, little is currently known of the thiol cofactor system(s) that supports deiodination in vivo. The interplay of deiodination with other pathways of TH metabolism is likely also to be an important area of future study.
MJ,
Kieffer
Evidence
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TEA4 Vol. 5, No. 1, 1994
Farwell AP, DiBendedetto DJ, Leonard JL: 1993. Thyroxine targets different pathways of internalization of type II iodothyronine 5’deiodinase in astrocytes. J Biol Chem 2685055-5062. Galton VA, Hiebert A: 1988. The ontogeny of iodothyronine 5’-monodeiodinase activity in Rana catesbeiam tadpoles. Endocrinology 122:640-645. Goswami A, Rosenberg IN: 1988. Effects of glutathione on iodothyronine 5’deiodinase activity. Endocrinology 123:192-202. Huang T, Chopra IJ, Beredo A, Solomon DH, Chua Teco GN: 1985. Skin is an active site of inner ring monodeiodination of thyroxine to 3,3’,5’-triiodothyronine. Endocrinology 117:2106-2113. Huang T, Chopra IJ, Boado R, Solomon DH, Chua Teco GN: 1988. Thyroxine inner ring monodeiodinating activity in fetal tissues of the rat. Pediatr Res 23: 196-199. Ishii H, Inada M, Tanaka K, et al.: 1981. Triiodothyronine generation from thyroxine in human thyroid: enhanced conversion of Graves’thyroid tissue. J Clin Endocrinol Metab 52:1211-1217. Kaplan MM. 1986. Regulatory influences on iodothyronine deiodination in animal tissues. In Hennemann G, ed. Thyroid Hormone Metabolism. New York, Marcel Dekker, pp 23 l-253. Kaplan MM, McCann UD, Yaskoski KA, Larsen PR, Leonard JL: 1981. Anatomical distribution of phenolic and tyrosyl ring iodothyronine deiodinases in the nervous system of normal and hypothyroid rats. Endocrinology 109:397-402. Kaplan MM, Visser TJ, Yaskoski KA, Leonard JL: 1983. Characteristics of iodothyronine tyrosyl ring deiodination by rat cerebral cortical microsomes. Endocrinology 112:35-42.
deiodinases by thyroid hormone. Endocrinology 114:998-1004. Leonard JL, Siegrist-Kaiser CA, Zuckerman CJ: 1990. Regulation of type II iodothyronine 5’-deiodinase by thyroid hormone: inhibition of actin polymerization blocks enzyme inactivation in CAMP-stimulated glial cells. J Biol Chem 265940-946. MacLatchy DL, Eales JG: 1992. Properties of T, 5’-deiodinating systems in various tissues of the rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 86~313-322.
Safran M, Farwell AP, Leonard JL: 1992. Thyroid hormone-dependent redistribution of the 55kilodalton monomer of protein disulfide isomerase in cultured glial cells. Endocrinology 131:2413-2418. Safran M, Fat-well AP, Rokos H, Leonard JL: 1993. Structural requirements of iodothyronines for the rapid inactivation and internalization of type II iodothyronine 5’-deiodinase in glial cells. J Biol Chem 268:14,224-14,229.
Maxon HR, But-man KD, Premachandra BN, et al.: 1982. Familial elevations of total and free thyroxine in healthy, euthyroid subjects without detectable binding protein abnormalities. Acta Endocrinol (Copenh) 100:224-230.
St. Germain DL: 1986. Hormonal control of a low K, (type II) iodothyronine 5’deiodin ase in cultured NB41A3 mouse neuroblastoma cells. Endocrinology 119:840-846.
Mol K, Kaptein E, Darras VM, de Greef WJ, Kuhn ER, Visser TJ: 1993. Different thyroid hormone-deiodinating enzymes in tilapia (Oreochromis niloticus) liver and kidney. FEBS Lett 321:140-144. Noiva R, Lennarz WJ: 1992. Protein disulfide isomerase: a multifunctional protein resident in the lumen of the endoplasmic reticulum. J Biol Chem 267:3553-3556. Obata T, Kitagawa S, Gong Q, Pastan I, Cheng S: 1988. Thyroid hormone down-regulates ~55, a thyroid hormone-binding protein that is homologous to protein disulfide isomerase and the l&subunit of prolyl-4hydroxylase. J Biol Chem 263:782-785. O’Mara BA, Dittrich W, Lauterio TJ, St. Germain DL: 1993. Pretranslational regulation of type I 5’-deiodinase by thyroid hormones and in fasted and diabetic rats. Endocrinology 133:1715-1723
K&lding R, Fuhrmann H, Ranella RR: 1988. Elevated T, and T, 5-deiodinase activity in different human brain tumors [abst]. Ann Endocrlnol49:240.
Refetoff S, Weiss RE, Usala S: 1993. The syndromes of resistance to thyroid hormones. Endocr Rev 14:348-399.
Leonard JL: 1991. Biochemical basis of thyroid hormone deiodination. In Wu S, ed. Thyroid Hormone Metabolism, Regulation and Clinical Implications. Boston, Blackwell, pp l-28.
Roti E, Fang SL, Green K, Emerson CH, Braverman LE: 198 1. Human placenta is an active site of thyroxine and 3,3’,5-triiodothyronine tyrosyl ring deiodination. J Clin Endocrinol Metab 53:498-50 1.
Leonard JL, Larsen PR: 1985. Thyroid hormone metabolism in primary cultures of fetal rat brain cells. Brain Res 327:1-13.
Ruiz de Oiia C, Obregon MJ, Escobar de1 Rey F, Morreale de Escobar G: 1988. Developmental changes in rat brain 5’-deiodinase and thyroid hormones during the fetal period: the effects of fetal hypothyroidism and maternal thyroid hormone. Pediatr Res 24:588-594.
TEM Vol. 5, No. 1, 1994
Safran M, Farwell AP, Leonard JL: 199 1. Evidence that type II 5’-deiodinase is not a selenoprotein. J Biol Chem 266:13,47713,480.
Mandel SJ, Berry MJ, Kieffer JD, Hamey Jw, Wame RL, Larsen PR: 1992. Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase. J Clin Endocrinol Metab 75: 1133-l 139.
Kleinhaus N, Faber J, Kahana L, Schneer J, Scheinfeld M: 1988. Euthyroid hyperthyroxinemia due to generalized 5’deiodinase defect. J Clin Endocrinol Metab 66:684688.
Leonard JL, Silva JE, Kaplan MM, Mellen SA, Visser TJ, Larsen PR: 1984. Acute posttranscriptional regulation of cerebrocortical and pituitary iodothyronine 5’-
Safran M, Leonard JL: 1991. Comparison of the physiochemical properties of type I and type II iodothyronine 5’-deiodinase. J Biol Chem 266:3233-3238.
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St. Germain DL: 1988a. Dual mechanism of regulation of type I iodothyronine 5’deiodinase in the rat kidney, liver, and thyroid gland: implications for the treatment of hyperthyroidism with radiographic contrast agents. J Clin Invest 81:14761484. St. Germain interactions the cellular regulation deiodinase. 1868.
DL: 1988b. The effects and of substrates, inhibitors, and thiol-disulfide balance on the of type II iodothyronine 5’Endocrinology 122: 1860-
St. Gerrnain DL, Croteau W: 1989. Ligandinduced inactivation of type I iodothyronine 5’-deiodinase: protection by propylthiouracil in vivo and reversibility in vitro. Endocrinology 125~2735-2744. St. Germain DL, Adler RA, Galton VA: 1985. Thyroxine 5’-deiodinase activity in anterior pituitary glands transplanted under the renal capsule in the rat. Endocrinology 117:55-63. St. Gerrnain DL, Dittrich W, Morganelli CM, Cryns V: 1990. Molecular cloning by hybrid arrest of translation in Xenopus Iaevis oocytes: identification of a cDNA encoding the type I iodothyronine S’deiodinase from rat liver. J Biol Chem 265:20,087-20,090. Santini F, Chopra IJ: 1992. A radioimmunoassay of rat type I iodothyronine 5’monodeiodinase. Endocrinology 13 1:252 l2526. Santini F, Chopra IJ, Hurd RE, Solomon DH, Teco GN: 1992. A study of the characteristics of the rat placental iodothyronine 5-monodeiodinase: evidence that it is distinct from the rat hepatic iodothyronine S-monodeiodinase. Endocrinology 130:23252332.
41
Schoenmakers CHH, Pigmans IGAJ, Poland A, Visser TJ: 1993. Impairment of the selenoenzyme type I iodothyronine deiodinase in C,H/He mice. Endocrinology 132:357361. Sharifi J, St. Germain DL: 1992. The cDNA for the type I iodothyronine 5’-deiodinase encodes an enzyme manifesting both high K,,, and low K, activity. J Biol Chem 267: 12,53912,544. Silva JE, Matthews PS: 1984. Production rates and turnover of triiodothyronine in rat-developing cerebral cortex and cerebellum: responses to hypothyroidism. J Clin Invest 74:1035-1049.
Van Doom J, van der Heide D, Roelfsema F: 1983. Sources and quantities of 3,5,3’triiodothyronine in several tissues of the rat. J Clin Invest 72:1778-1792. Van Stralen PG, van der Hoek HJ, Dotter R, et al.: 1993. Reduced T, deiodination by the human hepatoblastoma cell line HepG2 caused by deficient sulfation. Biochim Biophys Acta 1157:114-118. Visser TJ: hormones.
1988. Metabolism of thyroid In Cooke BA, King RJB, van der
Molen HJ, eds. Hormones and Their Action, Part 1. New York, Elsevier, pp 81-103. Wu S: 1991. Thyroid Hormone Metabolism: Regulation and Clinical Implications. Boston, Blackwell. Wu S, Shyh T, Chopra IJ, Solomon DH, Huang H, Chu P: 1982. Comparison of sodium ipodate (Oragrafin) and propylthiouracil in early treatment of hyperthyroidism. J Clin Endocrinol Metab 54:630TEM 634.
REPRINTS
Silva JE, Gordon MB, Crantz FR, Leonard JL, Larsen PL: 1984. Qualitative and quantitative differences in the pathways of extrathyroidal triiodothyronine generation between euthyroid and hypothyroid rats. J Clin Invest 73:898-907.
Reprints of articles in TEM are available (minimum order: 100). Please contact: Crystal Howard, Advertising Sales Elsevier Science Inc. 655 Avenue of the Americas New York, NY 10010
Toyoda N, Nishikawa M, Mori Y, et al.: 1992. Thyrotropin and triiodothyronine regulate iodothyronine S-deiodinase messenger ribonucleic acid levels in FRTL-5 rat thyroid cells. Endocrinology 131:389-394. Vadhanavikit S, Ganther HE: 1993. Selenium requirements of rats for normal hepatic and thyroidal 5’-deiodinase (type I) activities. J Nutr 123:1124-l 128.
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International Symposium on PUBERTY: BASIC AND CLINICAL ASPECTS National Academy of Medicine Buenos Aires, Argentina, April 6-8,1994 Organiziug Committee C. Bergads and J.A. Moguilevsky [Argentina) HonoraryPresident: S. Taleisnick (Argentina)
& z&o iial-
~~
Presidents:
Scientific Committee M. Grumbach (USA), Chairman, W. Wuttke (Germany), Chairman, J.P. Bourguignon (Belgium) E. Cacciari (Ifaly), D. Cardinali (Argentina), S. Ojeda (USA), R. Rosenfield (USA), and M. Zachmann Preliminary List of Invited Speakers F. Fraschini (Italy) E. Adashi (USA) A. Genazzani (Italy) E. Aguilar (Spain) I.P. Bourguignon (Belgium) P. Goodfellow (UK) R. Gorski (USA) C. Brook (UK) M. Grumbach (USA) H. Burger (Australia) S. Kaplan (USA) E. Cacciari (Italy) R. Kelch (USA) [. Cameron (USA) E. Knobil (USA) W. Crowley (USA) C. Kordon (France) C. Flamigni (Italy) G. Massa (Belgium) D. Foster (USA)
R. Negro Vilar (USA) S. Ojeda (USA) D. Pfaff (USA) T. Plant (USA) R. Rosenfield (USA) M. Rubert (Switzerland) P. Seeburg (Germany) M. Wierman (USA) W. Wuttke (Germany) M. Zachmann (Switzerland)
Scientific Secretaries M.E. Escobar de Lazzari and C. Libertun Panama 2121-(1640) Martinez-Buenos Aires Argentina Tel (1) 793-1302/3398 FAX (1) 763-7219 Sponsor: Serono Symposia For further information, please contact Serono Symposia, Via Ravenna 8, 00161 Rome, Italy. Tel +39 (6) 44291229 / FAX +39 (6) 44291324.
42
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Main Topics Neuroendocrinology of puberty Neurophysiology of LH pulsatility Molecular aspects of sexual differentiation Control of gonadotropin secretion by different factors Role of stimulatory amino acid in the mechanism of puberty Body weight, nutritional factors, exercise, metabolic activity, GH, IGF-I as potential mechanism controlling the initiation of puberty Gn-RH deficiency Polycystic ovary syndrome Delayed and precocious puberty
Useful Information There is no charge for admission to the scientific sessions. Registration fee US $300.00. The fee entitles participants to the proceedings volume and congressional services. A limited number of free communications will be accepted for oral or poster presentation. Abstracts must be sent to the Scientific Secretaries before December IO, 1993 (postmarked).
01994, Elsevier Science Inc., 1043-2760/94/$7.00
TEM Vol. 5, No. 1, 1994
[reviewed by Leonard (1991)]. Recent affinity-labeling studies have confirmed this impression (Safran and Leonard 1991). These three processes have been designated the type I, II, and III iodothyronine deiodinases, and a summary of their tissue distribution and catalytic activity in the rat is shown in Figure 2. Type I Deiodinase
The iodothyronine deiodinases constitute a family of enzymes that catalyze the removal of iodine atoms from various thyroid hormones (THs) in the thyroid gland and extrathyroidal tissues. As such, they are responsible for both the activation and inactivation of these compounds, and are thus important regulators of TH action. Recently, new insights have been gained into the biochemical characteristics of these proteins and their physiologic roles in TH metabolism. In particular, the availability of affinity-labeling techniques, molecular probes, and specific antisera for these enzymes, and the recent identification of the type I deiodinase as a selenoprotein, have ushered in a new era in the study of thyroid hormone deiodination. (Trends Endocrinol Metab 1994;5:3642)
??
Overview of Thyroid Hormone Metabolism
The metabolism of thyroid hormones (THs) in extrathyroidal tissues involves a complex series of enzymatic reactions that result in the deiodination, conjugation, andlor deamination and oxidative decarboxylation of these iodothyronine compounds (Figure 1). The extent to which these reactions occur within a given cell or organ has profound effects upon the circulating and tissue levels of THs and thus their metabolic effects (Wu 1991). Of importance, these metabolic reactions are not mutually exclusive, but may occur sequentially. For example, sulfoconjugation appears to enhance the rate of certain deiodination reactions selectively in some tissues (see the following). Deiodination is the predominant mechanism of TH metabolism and involves the removal of an iodine atom from either the tyrosyl ring (at the 3 or biochemically equivalent 5 position) or the phenolic ring (at the 3’ or 5’ position). Although thyroxine (T,) is the principal secretory product of the thyroid gland, it has a relatively low affinity Donald L. St. Get-main is at the Departments of Medicine and Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA. 36
for the nuclear TH receptors when compared with 3,5,3’-triiodothyronine (T,), and functions primarily as a prohormone. Thus, the 5’-deiodination of T, to form T, represents a key activation step in the action of THs. In contrast, deiodination of T, or T, on the tyrosyl ring results in the formation of the inactive iodothyronines 3,3’,5’-triiodothyronine (reverse T,, rT,) or 3,3’-diiodothyronine (T,), respectively. The physiologic importance of deiodination to overall TH economy is highlighted by the finding that -80% of the secreted T, is deiodinated to form T, and rT, in approximately equimolar amounts (Engler and Burger 1984).
??
Classification and Characteristics of Iodothyronine Deiodinases
Until recently, the methods available for studying the 5’- and 5-deiodinase enzymes (5’-D, 5-D, respectively) that catalyze these reactions have been quite limited, in large part because reliable purification methods for these relatively low-abundance microsomal proteins have not yet been developed. Based on activity measurements in tissue homogenates, three major patterns of deiodination have been identified and, because of their diverse characteristics, have been assumed to result from the catalytic properties of three separate enzymes
01994, Elsevier Science Inc., 1043-2760/94/$7.00
The type I deiodinase is found predominantly in the liver and kidney and can catalyze either 5’- or 5-deiodination (Visser 1988). Both processes are extremely sensitive to inhibition by propylthiouracil (PTU). In euthyroid rats, 70% of the circulating T, appears to be derived from the catalysis of T, at the 5’ position by this enzyme (Silva et al. 1984). In certain species such as humans and rats, type I deiodinase is also present in the thyroid gland and appears to contribute significantly to thyroidal T, production (Beech et al. 1993). The sulfation state of the iodothyronine substrates appears to have a major influence on the type I catalytic reaction; nonsulfated iodothyronines, such as T, and rTT,, undergo predominantly 5’deiodination, whereas T, sulfate and T, sulfate are deiodinated at the 5 position (Visser 1988). As investigated recently in HepG2 cells, sulfation appears to be an obligate step in the 5-deiodination process by the type I enzyme (Van Stralen et al. 1993). Thus, the sulfation pathway(s) in tissues expressing type I activity may play an important role in regulating TH metabolism by dictating the relative extent of tyrosyl versus phenolic ring deiodination. Of potential physiologic importance, the catalytic efficiency with which various substrates are deiodinated by the type I deiodinase is markedly different, as judged by the V,,,/K, ratios of the individual compounds (Visser 1988). T, is a relatively poor substrate for 5’- and 5-deiodination by this enzyme; the V,,,/ K,,, ratio for the 5’-deiodination of rTJ is at least 500 times greater than that of T,, and both T, sulfate and T, sulfate are 5-deiodinated with considerably greater efficiency than unconjugated T,. Such data, derived with dithiothreitol (DTT) used as a cofactor in tissue homogenates, where free hormone concentrations are uncertain, permits only cautious extrapolation to the in vivo setting, but suggests that in tissues such as the
TEM Vol. 5,No.1,1994
liver and kidney, where tissue T, levels are only modest,
the predominant
cata-
lytic processes carried out by the type I enzyme are the 5’-deiodination of rTs and the 5-deiodination
of the sulfated
1 DEIODINATION
conjugates of T, and T,. In the thyroid, however, where intracellular concentrations of T, are high, the 5’-deiodination of T, to T, may be the principal reaction process. Recent studies demonstrating little change in serum T, levels in rats with impaired extmthyroidal type I deiodinase activity are consistent with such an analysis and implicate the thyroid gland as the principal source of circulating T, in this species (Chanoine et al. 1993). Similarly, in the C,H/He mouse, which manifests an impairment in type I deiodinase activity, serum T, levels are no different from those in control mice, but circulating rT, levels are increased -3fold, demonstrating the importance of this enzyme in rTJ clearance (Schoenmakers et al. 1993). Type II Deiodinase The central nervous system, pituitary gland, and brown adipose tissue (BAT) express type II deiodinase activity, which catalyzes deiodination exclusively at the 5’ position (Leonard 1991). Both T, and rT, are efficiently metabolized by this pathway, with the product of T, deiodination being the active TH T,. Unlike the type I deiodinase, this process is relatively resistant to the inhibitory effects of PTU and aurothioglucose. The type II enzyme appears to be broadly distributed in the rat brain, with relatively high levels present in the cerebral cortex, cerebellum, and hypothalamus (Kaplan et al. 1981). Although the cell types expressing this enzyme have not yet been defined in vivo, type II activity is present in cultured astroglial cells (Leonard and Larsen 1985, Courtin et al. 1991) and in the NB41A3 neuroblastoma cultured cell line (St. Germain 1986). Studies in fetal and neonatal rats have demonstrated that this deiodinase is of considerable importance in ensuring the adequate intracellular concentration of T, in the brain during critical periods of development (Silva and Matthews 1984, Ruiz de Oiia et al. 1988). During cold adaptation, a rapid increase in type II deiodinase activity in brown fat contributes significantly to T, levels in that tissue, and thus the THinduced increase in thermogenesis (Bi-
TEM Vol. 5,No.1,1994
I
1
I
m I
I
I T3
I T4
rever.se T3
1 CONJUGATION *
I
I T4
(
1
SO4&-& 1 I T4 sulfate
DEAMINATION I DECARBOXYLATION
1
*-* T4
Tetrac
Figure 1. Enzymatic processes involved in the metabolism of thyroid hormones. anco
and Silva
1988).
In the anterior
pituitary lobe, the local generation of T, by the type II deiodinase appears to contribute significantly to nuclear receptor T, occupancy, suggesting a mechanism whereby pituitary function can be modulated by circulating T4 as well as T, levels (Van Door-n et al. 1983). Of note, transplantation of the anterior pituitary to the kidney capsule results in a marked decrease in the level of type II activity and an increase in type I activity, suggesting that hypothalamic releasing factors may play a role in regulating deiodination in this tissue (St. Germain et al. 1985). Type II deiodinase has also been described in lower species, such as in fish liver (MacLatchy and Eales 1992) and in several tissues of the metamorphosing tadpole (Galton and Hiebert 1988). Type III Deiodinase An exclusively tyrosyl-ring-deiodinating enzyme, the type III deiodinase, is found primarily in the cerebral cortex and skin of adult nonpregnant rats (Kaplan et al. 1983, Huang et al. 1985), but is also expressed in large amounts in the placenta and in several fetal rat tissues,
little affinity
for the nuclear
tors and are metabolically type III deiodinase may
TH recep-
inactive, the serve during
development to protect the fetal brain and other tissues against excessively high levels of active THs. The function of this enzyme in the skin is at present uncertain. Skin contains the highest levels of rT, of any tissue in the adult rat (Huang et al. 1985>, however, suggesting that the high levels of type III activity noted in skin homogenates accurately reflect the activity of this enzyme in vivo. Other Deiodinases A recent report suggests that the classification of deiodinases into the three types described above may need to be broadened. In the kidney of the fish species tilapia (Oreochromis niloticus), a s’Figure 2. Classification and tissue distxibution of the iodothyronine deiodinases as identified in the adult rat. BAT, brown adipose tissue.
such as skeletal muscle, intestine, liver, and cerebellum (Huang et al. 1988). An analogous deiodinase has been demonstrated in human placenta (Roti et al. 1981) and brain (Kodding et al. 1988). The exact physiologic role of this deiodinating pathway is uncertain. However, since the products of 5-deiodination (rT3 converted from T,, and T, from T,) have
01994,
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37
been
it parallels the decrease in activity of the
controlled
described that displays properties of both the type I and the type II deiodinases (Mol et al. 1993). Similar to the type I enzyme, the preferred substrate for the fish deiodinase is rT,, and the K, value in the presence of high concentrations of
type III 5-deiodinase in the brain and skin. In contrast, the activity of the type II 5’-deiodinase in brain, pituitary, and brown fat increases markedly in response to decreases in circulating TH levels, and thus serves to maintain relatively normal T, levels in these tissues.
1993). In the latter conditions, the decreased mRNA levels may reflect tissue hypothyroidism induced by the altered nutritional status of the animal. Because cDNAs for the type II and type III deiodinases have not yet been isolated,
deiodinating
process
has recently
DTT is in the micromolar range. The reaction process, however, is quite resistant to inhibition by PTU and aurothioglucose, which are properties more consistent with a type II deiodinase.
??
Coordinate Regulation of Deiodinases in Response to Alterations in Thyroid Hormone Status
Dramatic changes in deiodinase activity accompany alterations in TH status (Kaplan 1986). From a physiologic viewpoint, these changes appear to be coordinated in such a fashion as to maintain T, levels as normal as possible in the circulation and selected tissues such as the brain, and may have developed as a protective mechanism in response to iodine deficiency. As illustrated in Figure 3, the response in the rat to hypothyroidism includes a marked decrease in the activity of the type I enzyme in liver and kidney. To the extent that this enzyme is involved in 5-deiodinating reactions as noted above, this decrease in activity of an “inactivating” process may serve to protect circulating T, and T, levels, and
Figure 3. Alterations in iodothymnine deiodinase activities in response to hypothyroidism. Changes in the relative size of the boxes and weight of the arrows relative to Figure 2 represent the alterations in the various deiociinase processes and reactions that occur in the hypothyroid state.
TYPE II Brain, Pituitary,
38
T4
TYPE III
T3
m.el”. Sk,”
T3 7.2
BAT
rT3 T2
An exception to this pattern is the type I deiodinase in the thyroid gland, which increases in the hypothyroid state under the influence of TSH stimulation (Erickson et al. 1982). Such a response likely serves to increase the proportion of T, secreted by the thyroid in states of impaired thyroid function such as iodine deficiency and may explain in part the increased T,iT, ratio observed in the hypothyroid state. The administration of T, to experimental animals to induce a hyperthyroid state results in opposite changes in activity of all three deiodinating pathways (Kaplan 1986). In addition to being regulated by TSH, the type I activity in the thyroid gland is stimulated by the thyroid-stimulating immunoglobulins that circulate in Graves’ disease, an effect that likely contributes to the increased thyroidal secretion of T, in this condition (Ishii et al. 1981).
??
Cellular Mechanisms Deiodinase Activity
Pretranslational
Regulating
Regulation
The complex pattern of altered deiodinase activity resulting from changes in TH status involves both pre- and posttranslational regulatory mechanisms. The regulation of type I activity in response to altered TH levels results from changes in the mENA level for this enzyme (Berry et al. 1990, St. Get-main et al. 1990). We have quantified this response by using an RNA solution hybridization assay and have demonstrated in hypothyroid rats an approximate 50-fold increase, over a 72-h time period, in type I deiodinase mRNA level in both liver and kidney in response to T, administration (O’Mara et al. 1993). This increase in mRNA level is accompanied, after an 8- to 24-h delay, by an increase in type I activity of similar magnitude. Alterations in mRNA level are also responsible for the stimulation of enzyme activity by TSH in the thyroid (Toyoda et al. 1992) and the decreases in activity observed in fasting and the poorly
01994.
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diabetic
state (O’Mara
et al.
the pretranslational regulation of these enzymes has not been well defined. Posttranslational
Regulation
In addition to this pretranslational
mode
of regulation, the type I deiodinase, as well as the type II enzyme, are subject to rapid posttranslational inactivation, induced by THs and competitive inhibitors such as sodium ipodate (Leonard et al. 1984, St. Germain 1988a and b). This in vivo loss of catalytic activity is manifested by a decrease in maximal enzyme velocity, and we have termed this process ligand-induced inactivation. The cellular mechanisms mediating this loss of activity have not yet been fully delineated and may vary depending on the enzyme and tissue involved. For the type I deiodinase in liver and kidney, inactivation appears to be closely linked to the sulfhydryl state of the enzyme and may require direct interaction between the ligand and the active site as a triggering mechanism (St. Germain 1988a). PTU, by binding to the active site of the enzyme and inhibiting catalytic cycling, protects the type I deiodinase from inactivation (St. Germain and Croteau 1989). These concepts have recently been confirmed and extended by Santini and Chopra (1992). Using a specific RIA for this enzyme, these investigators have demonstrated that treatment of rats with sodium ipodate is associated with a decreased mass of type I deiodinase in rat liver and kidney, whereas PTU treatment results in an increase in the liver content of this protein. Studies conducted on the GH, rat pituitary tumor cell line suggest that the sulfhydryl status of the cell is also important in the inactivation of the type II deiodinase; treatment of intact cells with the sulfhydryl-oxidizing agent diamide causes a rapid loss of type II catalytic activity, whereas treatment with DTT protects against ligand-induced inactivation (St. Germain 1988b). Leonard et al. (1990) have investigated the inactivation of the type II deiodinase by THs in cultured astroglial cells and have found that this loss of activity is
TEM Vol.5,No. I.1994
inhibited by cytochalasins, implicates
a finding that
the actin cytoskeleton in this In subsequent studies using
process. affinity-labeling
techniques,
it has been
demonstrated that T, rapidly promotes actin polymerization and internalization of the type II deiodinase catalytic subunit to an endosomal pool resulting in its inactivation (Farwell et al. 1990 and 1993). Whether this unique and important extranuclear mechanism of TH action demonstrated in cultured glial cells is applicable to the inactivation of deiodinases in other tissues remains uncertain. For example, in GH, cells, the structural requirements of the iodothyronines for inducing inactivation of the type II deiodinase differ from those observed in astroglial cells (St. Germain 1988b, Safran et al. 1993) and cytochalasins do not influence the inactivation process (St. Germain unpublished observation 1990). Cultured astroglial cells, which express type III deiodinase activity as well as type II activity, have also proved to be an excellent model system for studying the effects of other regulatory mechanisms on deiodinase enzymes. In this cell system, CAMP, phorbol esters, and fibroblast growth factor a have marked stimulatory effects on deiodinase activity (Courtin et al. 1991).
??
Thiol Cofactors
Thiol cofactors are essential components of the deiodination reaction process and interact with the deiodinase enzymes and substrates in either a ping-pong or a sequential kinetic pattern (Leonard 1991). In tissue homogenates, dithiols such as DTT are the most potent in supporting deiodinase activity. Of note, the kinetic characteristics of 5’-deiodination in rat liver and kidney homogenates differ significantly, depending on the cofactor system used in the in vitro assay system. When DTT is used in the reaction mixture, the I$,, values for the 5’-deiodination of T4 and rT, are in the micromolar range. Such concentrations far exceed the intracellular levels of these substrates in liver and kidney, and thus called into question the physiologic significance of this process. This was brought into further relief by the demonstration that physiologic cofactors such as reduced glutathione (GSH) or a reconstituted thioredoxin-NADPH system supported 5’-deiodination in homogenates
TEM Vol. 5, No. 1, 1994
of these tissues nanomolar
with K,
values in the and Rosen-
1988). Indirect evidence suggests a close
1988, Bhat et al. 1989). This sug-
association between PDI and deiodinase enzymes. Initial attempts to isolate a
gested that, in addition to the DTTsupported process, one or more “low K,”
cDNA for the type I enzyme made use of a polyclonal antiserum that was demon-
5’-deiodinases might be present in these tissues. This uncertainty as to the number of 5’-deiodinases in the liver and the kidney was recently resolved by the
strated in vitro to precipitate 5’-deiodinase activity from solubilized liver microsomes. Of potential significance, the use of this antiserum to screen a hgtl 1 cDNA expression library resulted in the isolation of a cDNA for PDI (Boado et al. 1988). More recently, Leonard and colleagues have demonstrated that PDI is
berg
range (Goswami
demonstration that the type I deiodinase, when expressed in Xenopus laevis oocytes by the injection of mRNA prepared in vitro with use of the full-length cDNA for this enzyme as template, manifests both high K, and low K, activity, depending on the thiol cofactor system utilized in the in vitro reaction assay (Sharifi and St. Germain 1992). Of importance, in the absence of added thiols, significant levels of low K, 5’-deiodinase activity are demonstrable in this system, suggesting that this is the kinetic pattern of the enzyme when endogenous cofactors are utilized. Thus, the liver and kidney express only a single 5’-deiodinase, the type I enzyme, whose reaction kinetics differ markedly depending on the available thiol cofactor. Irrespective of the cofactor utilized, however, rTT, is a much more efficient substrate than T, for 5’-deiodination by this enzyme (Sharifi and St. Germain 1992). Of the three deiodinase processes described to date, only type 1 activity has been demonstrated in the presence of a “physiologic” cofactor system such as GSH or thioredoxin; types II and III deiodinase activity have been demonstrated only with relatively high concentrations of DTT, and GSH does not efficiently support deiodination by these latter enzymes in vitro. Thus, the identity of the cofactor system(s) that supports deiodination in vivo remains uncertain.
??
Deiodination Isomerase
and Protein
Disulfide
The study of the deiodinases has recently become intertwined with that of another microsomal enzyme, protein disulfide isomerase (PDI). This multifunctional, 55-kD protein was first identified because of its ability to catalyze the isomerization of protein disulfide bonds in vitro [reviewed in Noiva and Lennarz (1992)]. Of note, PDI binds T, with relatively high affinity and is downregulated by posttranslational mechanisms in response to T, treatment in GH, cells (Obata et al.
01994,
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translocated in glial cells by the actin cytoskeleton in response to T, treatment in a manner analogous to the type II deiodinase (Safran et al. 1992). Although the significance of these observations remains uncertain, they suggest that PDI could be involved in either the posttranslational regulation of the deiodinases as described here, or function as part of the thiol cofactor system necessary for deiodinase catalytic activity. Of note regarding the latter possibility is that PDI contains two thioredoxinlike active sites (Noiva and Lennarz 1992).
??
The Role of Selenium Deiodination
in
The characterization of full-length cDNAs for the rat and human type I deiodinases has confirmed earlier evidence that these enzymes are selenoproteins that, like the glutathione peroxidases, contain a selenocysteine at their active site (Berry et al. 1991, Mandel et al. 1992). Additional studies by Berry and Larsen (1992) have provided important insights into the molecular mechanisms controlling selenocysteine incorporation into proteins during the translational process. Using mutation analysis, these investigators have also demonstrated that the presence of the selenocysteine is responsible for several of the unique properties of the type I enzyme, such as the marked sensitivity to inhibition by PTU and the gold compound aurothioglucose. Indeed, the lack of sensitivity of the type II and III deiodinases to these inhibitors suggests that they may not be selenoproteins or may lack a selenocysteine at the active center of the enzyme (Berry et al. 1991, Safran et al. 1991, Santini et al. 1992). In experimental animals, selenium deficiency leads to a marked reduction in type I deiodinase activity and protein content in the liver and kidney, but,
39
surprisingly, not in the thyroid gland, which appears to be markedly resistant to selenium depletion (Behne et al. 1990, Vadhanavikit and Ganther 1993, Chanoine et al. 1993). The cellular basis for this conservation of selenium within the thyroid gland is uncertain, but it appears to be responsible for the observation that moderate selenium deficiency in the rat does not lower serum T, levels in spite of markedly decreased extrathyroidal type I deiodinase activity. Severe selenium deficiency, however, can impair thyroidal function as well as peripheral TH metabolism, and evidence from both human and animal studies suggests that concurrent selenium deficiency may be an important determinant of the severity of the clinical manifestations of iodine deficiency (Arthur et al. 1992, Berry and Larsen 1992).
??
Clinical Implications
The complexity of the deiodinating pathways has important implications for human thyroid physiology. In Graves’ disease, for example, T, to T, conversion is increased in the thyroid gland and other tissues containing the type I deiodinase, a process that likely contributes to the hyperthyroid state. Thus, high doses of the type I deiodinase inhibitor PTU, or radiographic contrast agents, such as sodium ipodate, which are potent inhibitors of all deiodinase processes, are of therapeutic benefit (Croxson et al. 1977, Wu et al. 1982). To date, very few patients with defects in deiodination have been described, a situation that contrasts with the many case reports of the TH resistance syndromes caused by defective T, receptors (Refetoff et al. 1993). Kleinhaus et al. (1988) described a clinically euthyroid patient with elevated T, and rTs levels accompanied by normal T, and TSH concentrations, findings similar to those found in the C,H/I-Ie mouse with deficient type I deiodinase activity (Berry et al. 1993, Schoenmakers et al. 1993). Thus, mild-to-moderate degrees of type I deficiency may be manifest only by hyperthyroxinemia, with little or no impact on clinical thyroid status (Maxon et al. 1982). The clinical phenotypes of deficient type II or type III deiodinase activity are uncertain, because patients with these defects have not been recognized. Furthermore, selective inhibitors of these
40
processes are not yet available, and animal models with defective activity have not been described. Given the distribution of the these pathways, however, one might expect deficiencies of type II or type III activity to have an adverse effect on fetal development in general and brain maturation in particular.
techniques,
and
biochemical
the investigation
methods
of selenium
in
metabolism.
Biol Trace Elem Res 2627:439447. Berry
MJ,
Larsen
selenium
PR:
in thyroid
1992.
The
hormone
role
action.
of En-
doer Rev 13:207-219. Berry MJ, Kates A, Larsen PR: 1990. Thyroid hormone
regulates
senger
type I deiodinase
RNA in rat liver.
mes-
Mol Endocrinol
4:743-748. ??
Future Studies
Berry
In spite of important
new insights
into
the mechanisms of TH deiodination, considerable work remains to be accomplished before we have a complete understanding of these important metabolic processes. Our knowledge of the physiologic role of these enzymes in individual tissues and in overall TH economy remains incomplete. This is due in part to the complexity of these enzymatic processes, each of which can utilize multiple substrates, and the lack of specific inhibitors for the different deiodinases in various tissues. In this regard, the availability in the future of experimental animal models produced by recombinant DNA techniques with impaired deiodinase activity of specific enzymes in specific tissues should provide important information, particularly regarding the role of deiodination in TH action in the brain and during development. In addition, little is currently known of the thiol cofactor system(s) that supports deiodination in vivo. The interplay of deiodination with other pathways of TH metabolism is likely also to be an important area of future study.
MJ,
Kieffer
Evidence
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PR:
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St. Germain DL: 1988a. Dual mechanism of regulation of type I iodothyronine 5’deiodinase in the rat kidney, liver, and thyroid gland: implications for the treatment of hyperthyroidism with radiographic contrast agents. J Clin Invest 81:14761484. St. Germain interactions the cellular regulation deiodinase. 1868.
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Reprints of articles in TEM are available (minimum order: 100). Please contact: Crystal Howard, Advertising Sales Elsevier Science Inc. 655 Avenue of the Americas New York, NY 10010
Toyoda N, Nishikawa M, Mori Y, et al.: 1992. Thyrotropin and triiodothyronine regulate iodothyronine S-deiodinase messenger ribonucleic acid levels in FRTL-5 rat thyroid cells. Endocrinology 131:389-394. Vadhanavikit S, Ganther HE: 1993. Selenium requirements of rats for normal hepatic and thyroidal 5’-deiodinase (type I) activities. J Nutr 123:1124-l 128.
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International Symposium on PUBERTY: BASIC AND CLINICAL ASPECTS National Academy of Medicine Buenos Aires, Argentina, April 6-8,1994 Organiziug Committee C. Bergads and J.A. Moguilevsky [Argentina) HonoraryPresident: S. Taleisnick (Argentina)
& z&o iial-
~~
Presidents:
Scientific Committee M. Grumbach (USA), Chairman, W. Wuttke (Germany), Chairman, J.P. Bourguignon (Belgium) E. Cacciari (Ifaly), D. Cardinali (Argentina), S. Ojeda (USA), R. Rosenfield (USA), and M. Zachmann Preliminary List of Invited Speakers F. Fraschini (Italy) E. Adashi (USA) A. Genazzani (Italy) E. Aguilar (Spain) I.P. Bourguignon (Belgium) P. Goodfellow (UK) R. Gorski (USA) C. Brook (UK) M. Grumbach (USA) H. Burger (Australia) S. Kaplan (USA) E. Cacciari (Italy) R. Kelch (USA) [. Cameron (USA) E. Knobil (USA) W. Crowley (USA) C. Kordon (France) C. Flamigni (Italy) G. Massa (Belgium) D. Foster (USA)
R. Negro Vilar (USA) S. Ojeda (USA) D. Pfaff (USA) T. Plant (USA) R. Rosenfield (USA) M. Rubert (Switzerland) P. Seeburg (Germany) M. Wierman (USA) W. Wuttke (Germany) M. Zachmann (Switzerland)
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42
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Main Topics Neuroendocrinology of puberty Neurophysiology of LH pulsatility Molecular aspects of sexual differentiation Control of gonadotropin secretion by different factors Role of stimulatory amino acid in the mechanism of puberty Body weight, nutritional factors, exercise, metabolic activity, GH, IGF-I as potential mechanism controlling the initiation of puberty Gn-RH deficiency Polycystic ovary syndrome Delayed and precocious puberty
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