Type 1 Deiodinase Regulates ApoA-I Gene Expression and ApoA-I Synthesis Independent of Thyroid Hormone Signaling.

OBJECTIVE: Plasma levels of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (ApoA-I) are reduced in individuals with defective insulin signaling. Initial studies using liver-specific insulin receptor (InsR) knockout mice identified reduced expression of type 1 deiodinase (Dio1) as a potentially novel link between defective hepatic insulin signaling and reduced expression of the ApoA-I gene. Our objective was to examine the regulation of ApoA-I expression by Dio1. APPROACH AND RESULTS: Acute inactivation of InsR by adenoviral delivery of Cre recombinase to InsR floxed mice reduced HDL-C and expression of both ApoA-I and Dio1. Overexpression of Dio1 in InsR knockout mice restored HDL-C and ApoA-I levels and increased the expression of ApoA-I. Dio1 knockout mice had low expression of ApoA-I and reduced serum levels of HDL-C and ApoA-I. Treatment of C57BL/6J mice with antisense to Dio1 reduced ApoA-I mRNA, HDL-C, and serum ApoA-I. Hepatic 3,5,3'-triiodothyronine content was normal or elevated in InsR knockout mice or Dio1 knockout mice. Knockdown of either InsR or Dio1 by siRNA in HepG2 cells decreased the expression of ApoA-I and ApoA-I synthesis and secretion. siRNA knockdown of InsR or Dio1 decreased activity of a region of the ApoA-I promoter lacking thyroid hormone response elements (region B). Electrophoretic mobility shift assay demonstrated that reduced Dio1 expression decreased the binding of nuclear proteins to region B. CONCLUSIONS: Reductions in Dio1 expression reduce the expression of ApoA-I in a 3,5,3'-triiodothyronine-/thyroid hormone response element-independent manner.

Optimal bone strength and mineralization requires the type 2 iodothyronine deiodinase in osteoblasts.

Hypothyroidism and thyrotoxicosis are each associated with an increased risk of fracture. Although thyroxine (T4) is the predominant circulating thyroid hormone, target cell responses are determined by local intracellular availability of the active hormone 3,5,3'-L-triiodothyronine (T3), which is generated from T4 by the type 2 deiodinase enzyme (D2). To investigate the role of locally produced T3 in bone, we characterized mice deficient in D2 (D2KO) in which the serum T3 level is normal. Bones from adult D2KO mice have reduced toughness and are brittle, displaying an increased susceptibility to fracture. This phenotype is characterized by a 50% reduction in bone formation and a generalized increase in skeletal mineralization resulting from a local deficiency of T3 in osteoblasts. These data reveal an essential role for D2 in osteoblasts in the optimization of bone strength and mineralization.

Thyroid Hormone Metabolism: A Historical Perspective.

In this article, starting with the recognition that iodine is essential for normal thyroid function and is a component of thyroid hormone (TH) molecules, we discuss the many seminal observations and discoveries that have led to identification of various pathways of TH metabolism and their potential roles in TH economy and action. We then recount evidence that TH metabolism participates in maintaining the appropriate content of active hormone in a TH-responsive tissue or cell. Thus, metabolism of the TH is not merely a means by which it is degraded and eliminated from the body, but an essential component of an intricate system by which the thyroid exerts its multiple regulatory effects on almost all organs and tissues. The article ends with a summary of the current concepts and some outstanding questions that are awaiting answers.

The Deiodinases: Their Identification and Cloning of Their Genes.

In this minireview, we provide a historical outline of the events that led to the identification and characterization of the deiodinases, the recognition that deiodination plays a major role in thyroid hormone action, and the cloning of the 3 deiodinase genes. The story starts in 1820, when it was first determined that elemental iodine was important for normal thyroid function. Almost 100 years later, it was found that the primary active principle of the gland, T4, contains iodine. Once radioactive iodine became available in the 1940s, it was demonstrated that the metabolism of T4 included deiodination, but at the time it was assumed to be merely a degradative process. However, this view was questioned after the discovery of T3 in 1952. We discuss in some detail the events of the next 20 years, which included some failures followed by the successful demonstration that deiodination is indeed essential to normal thyroid hormone action. Finally, we describe how the 3 deiodinases were identified and characterized and their genes cloned.

The Intrinsic Activity of Thyroxine Is Critical for Survival and Growth and Regulates Gene Expression in Neonatal Liver.

Background: Thyroxine (T4) is generally considered to be a prohormone that requires conversion to triiodothyronine (T3) to exert biological activity. Although evidence suggests that T4 has intrinsic activity, it is questionable if this activity has any physiological relevance. Methods: To answer this question, triple knockout (KO) mice (Triples) that cannot express the types 1 (D1) and 2 (D2) deiodinase and the Pax8 genes were generated. Thus, they lack a thyroid and cannot convert T4 to T3. Triples were injected on alternate days with either vehicle or physiological doses of T4, T3, or T3+T4 from postnatal days 2-14. They were euthanized at P15, and RNA-seq was employed to profile gene expression in the liver. In another experiment, Pax8KO mice were injected with T3, T4, or T4+T3, and growth rate and survival to P84 were determined. Results: The growth retardation of Triples was not improved by either T3 or T4 alone but was significantly improved by T4+T3. In the liver, T4 significantly regulated the expression of genes that were also regulated by T3, but the proportion of genes that were negatively regulated was higher in mice treated with T4 than in mice treated with T3. Treatment with T4+T3 identified genes that were regulated synergistically by T3 and T4, and genes that were regulated only by T4+T3. Analysis of these genes revealed enrichment in mechanisms related to cell proliferation and cholesterol physiology, suggesting a unique contribution of T4 to these biological functions. Pax8KO mice all survived to P84 when injected with T4 or T4+T3. However, survival rate with T3 was only 50% and 10% at 3.5 and 12 weeks of life, respectively. Conclusions: T4 has intrinsic activity in vivo and is critical for survival and growth. At a physiological level, T4 per se can upregulate or downregulate many T3 target genes in the neonatal liver. While most of these genes are also regulated by T3, subsets respond exclusively to T4 or demonstrate enhanced or normalized expression only in the presence of both hormones. These studies demonstrate for the first time a complex dependency on both T4 and T3 for normal mammalian growth and development.

Type 3 deiodinase is critical for the maturation and function of the thyroid axis.

Developmental exposure to appropriate levels of thyroid hormones (THs) in a timely manner is critical to normal development in vertebrates. Among the factors potentially affecting perinatal exposure of tissues to THs is type 3 deiodinase (D3). This enzyme degrades THs and is highly expressed in the pregnant uterus, placenta, and fetal and neonatal tissues. To determine the physiological role of D3, we have generated a mouse D3 knockout model (D3KO) by a targeted inactivating mutation of the Dio3 gene in mouse ES cells. Early in life, D3KO mice exhibit delayed 3,5,3'-triiodothyronine (T3) clearance, a markedly elevated serum T3 level, and overexpression of T3-inducible genes in the brain. From postnatal day 15 to adulthood, D3KO mice demonstrate central hypothyroidism, with low serum levels of 3,5,3',5'-tetraiodothyronine (T4) and T3, and modest or no increase in thyroid-stimulating hormone (TSH) concentration. Peripheral tissues are also hypothyroid. Hypothalamic T3 content is decreased while thyrotropin-releasing hormone (TRH) expression is elevated. Our results demonstrate that the lack of D3 function results in neonatal thyrotoxicosis followed later by central hypothyroidism that persists throughout life. These mice provide a new model of central hypothyroidism and reveal a critical role for D3 in the maturation and function of the thyroid axis.

Type 3 deiodinase deficiency results in functional abnormalities at multiple levels of the thyroid axis.

The type 3 deiodinase (D3) is a selenoenzyme that inactivates thyroid hormones and is highly expressed during development and in the adult central nervous system. We have recently observed that mice lacking D3 activity (D3KO mice) develop perinatal thyrotoxicosis followed in adulthood by a pattern of hormonal levels that is suggestive of central hypothyroidism. In this report we describe the results of additional studies designed to investigate the regulation of the thyroid axis in this unique animal model. Our results demonstrate that the thyroid and pituitary glands of D3KO mice do not respond appropriately to TSH and TRH stimulation, respectively. Furthermore, after induction of severe hypothyroidism by antithyroid treatment, the rise in serum TSH in D3KO mice is only 15% of that observed in wild-type mice. In addition, D3KO animals rendered severely hypothyroid fail to show the expected increase in prepro-TRH mRNA in the paraventricular nucleus of the hypothalamus. Finally, treatment with T(3) results in a serum T(3) level in D3KO mice that is much higher than that in wild-type mice. This is accompanied by significant weight loss and lethality in mutant animals. In conclusion, the absence of D3 activity results in impaired clearance of T(3) and significant defects in the mechanisms regulating the thyroid axis at all levels: hypothalamus, pituitary, and thyroid.

Thyroid hormone homeostasis and action in the type 2 deiodinase-deficient rodent brain during development.

Considerable indirect evidence suggests that the type 2 deiodinase (D2) generates T3 from T4 for local use in specific tissues such as pituitary, brown fat, and brain, and studies with a D2-deficent mouse, the D2 knockout (D2KO) mouse, have shown this to be the case in pituitary and brown fat. The present study employs the D2KO mouse to determine the role of D2 in the developing brain. As expected, the T3 content in the neonatal D2KO brain was markedly reduced to a level comparable with that seen in the hypothyroid neonatal wild-type mouse. However, the mRNA levels of several T3-responsive genes were either unaffected or much less affected in the brain of the D2KO mouse than in that of the hypothyroid mouse, and compared with the hypothyroid mouse, the D2KO mouse exhibited a very mild neurological phenotype. The current view of thyroid hormone homeostasis in the brain dictates that the T3 present in neurons is generated mostly, if not exclusively, from T4 by the D2 in glial cells. This view is inadequate to explain the findings presented herein, and it is suggested that important compensatory mechanisms must be in play in the brain to minimize functional abnormalities in the absence of the D2.

The ups and downs of the thyroxine pro-hormone hypothesis.

Thyroxine (T4) is the major thyroid hormone in the thyroid gland and the circulation. However, it is widely accepted on the basis of abundant evidence that 3,5,3'-triiodothyronine (T3) is responsible for most, if not all, of the physiological effects of TH in extrathyroidal tissues, and T4 functions as the pro-hormone. Whether T4 has any intrinsic activity per se or is merely a pro-hormone that must be converted to T3 in order to exert any TH action has yet to be resolved. Although there are some physiological actions of T4 that are mediated by receptors at the cell membrane (non-genomic effects), the vast majority of the physiological effects of the THs identified to date involve the binding of T3 to specific nuclear receptors to regulate gene expression (genomic effects). This review examines how the role of T4 in genomic TH action has been viewed and debated during the hundred years since it was first isolated in 1914.

Targeted disruption of the type 1 selenodeiodinase gene (Dio1) results in marked changes in thyroid hormone economy in mice.

The type 1 deiodinase (D1) is thought to be an important source of T3 in the euthyroid state. To explore the role of the D1 in thyroid hormone economy, a D1-deficient mouse (D1KO) was made by targeted disruption of the Dio1 gene. The general health and reproductive capacity of the D1KO mouse were seemingly unimpaired. In serum, levels of T4 and rT3 were elevated, whereas those of TSH and T3 were unchanged, as were several indices of peripheral thyroid status. It thus appears that the D1 is not essential for the maintenance of a normal serum T3 level in euthyroid mice. However, D1 deficiency resulted in marked changes in the metabolism and excretion of iodothyronines. Fecal excretion of endogenous iodothyronines was greatly increased. Furthermore, when compared with both wild-type and D2-deficient mice, fecal excretion of [125I]iodothyronines was greatly increased in D1KO mice during the 48 h after injection of [125I]T4 or [125I]T3, whereas urinary excretion of [125I]iodide was markedly diminished. From these data it was estimated that a majority of the iodide generated by the D1 was derived from substrates other than T4. Treatment with T3 resulted in a significantly higher serum T3 level and a greater degree of hyperthyroidism in D1KO mice than in wild-type mice. We conclude that, although the D1 is of questionable importance to the wellbeing of the euthyroid mouse, it may play a major role in limiting the detrimental effects of conditions that alter normal thyroid function, including hyperthyroidism and iodine deficiency.

MCT8 Deficiency in Male Mice Mitigates the Phenotypic Abnormalities Associated With the Absence of a Functional Type 3 Deiodinase.

Mice deficient in the type 3 deiodinase (D3KO mice) manifest impaired clearance of thyroid hormone (TH), leading to elevated levels of TH action during development. This alteration causes reduced neonatal viability, growth retardation, and central hypothyroidism. Here we examined how these phenotypes are affected by a deficiency in the monocarboxylate transporter 8 (MCT8), which is a major contributor to the transport of the active thyroid hormone, T3, into the cell. MCT8 deficiency eliminated the neonatal lethality of type 3 deiodinase (D3)-deficient mice and significantly ameliorated their growth retardation. Double-mutant newborn mice exhibited similar peripheral thyrotoxicosis and increased brain expression of T3-dependent genes as mice with D3 deficiency only. Later in neonatal life and adulthood, double-mutant mice manifested central and peripheral TH status similar to mice with single MCT8 deficiency, with low serum T4, elevated serum TSH and T3, and decreased T3-dependent gene expression in the hypothalamus. In double-mutant adult mice, both thyroid gland size and the hypothyroidism-induced rise in TSH were greater than those in mice with single D3 deficiency but less than those in mice with MCT8 deficiency alone. Our results demonstrate that the marked phenotypic abnormalities observed in the D3-deficient mouse, including perinatal mortality, growth retardation, and central hypothyroidism in adult animals, require expression of MCT8, confirming the interdependent relationship between the TH transport into cells and the deiodination processes.

The roles of the iodothyronine deiodinases in mammalian development.

The thyroid hormones (TH) are essential for normal development in vertebrate species. This review considers the roles that the three deiodinases, types 1, 2 and 3 (D1, D2, and D3), play in regulating intracellular levels of TH during this critical period. The focus is on rodents and humans with emphasis on brain development. There is little evidence to suggest that the D1 plays a significant role in development and this is substantiated by the absence of any obvious developmental impairment in a D1-deficient mouse model. There is, however, compelling indirect evidence pertaining to the importance of the D2 in development, particularly with respect to that of the brain. However, surprisingly, a D2-deficient mouse model exhibits a very mild phenotype. This, together with the fact that D2 activity is increased in hypothyroidism, suggests that this deiodinase may be of greater importance in development when supplies of thyroxine are limited. The D3 is clearly essential for development in the euthyroid mammal. Information, both indirect and that obtained from a D3-deficient mouse model, strongly suggests that its presence in placenta, uterus and some fetal tissues are critical for limiting exposure of fetal tissues to inappropriate levels of TH.

Insights into the role of deiodinases from studies of genetically modified animals.

The deiodinases function at a pre-receptor level in tissues to modulate the concentrations, and thus the actions, of thyroid hormones. Although much has been learned in the last two decades about the biochemical properties and expression patterns of these enzymes, a complete understanding of their physiologic roles requires study of their actions in the intact animal. To date only a limited number of naturally occurring human or animal models exhibiting excessive or deficient deiodinase activity have been defined. In particular, no human genetic models of deiodinase deficiency have been identified. However, several transgenic animal models involving either loss-of function or gain-of-function of deiodinase activity have been devised and are currently being characterized. This review focuses on the progress being made in using these animal models to define the physiologic functions and significance of this important class of enzymes.

The gene locus encoding iodothyronine deiodinase type 3 (Dio3) is imprinted in the fetus and expresses antisense transcripts.

The mouse Dio3 gene codes for the type 3 iodothyronine deiodinase (D3), a conserved selenocysteine-containing enzyme that inactivates thyroid hormones and is highly expressed during early development. The mouse Dio3 gene and its human homolog map to chromosomal regions that are known to contain imprinted genes. We assessed the allelic expression of the Dio3 using a mouse model in which the gene had been inactivated by the introduction of a critical mutation in the selenocysteine codon. We compared Dio3 gene expression in fetuses that were either wild type or heterozygous (+/-Dio3) for the mutation. D3 enzymatic activities in the head, limbs, liver and body of heterozygous fetuses (E14 to E18) that inherited the mutation from the mother were no different from those found in their wild type littermates. However, D3 activities in heterozygous animals that inherited the mutation from the father were only 18 to 28% of the activities of their wild type littermates in these same tissues. No detectable activity was found in fetuses homozygous for the mutation indicating full inactivation of the enzyme. Northern analysis of mRNA from E15 fetuses showed that the Dio3 mRNA transcripts generated from the paternal allele were at least 5 times more abundant than the transcripts originated from the maternal allele. We conclude that the Dio3 gene is subject to genomic imprinting and preferentially expressed from the paternal allele in the mouse fetus. We also identified a gene that is transcribed antisense from the Dio3 locus. The Dio3 gene likely belongs to the same cluster of imprinted genes detected in mouse chromosome 12 and human chromosome 14 and should be considered as a candidate gene that might play a role in the phenotypic abnormalities associated with uniparental disomy of those chromosomes, a condition in which gene expression is altered due to abnormal genomic imprinting.

Determinants of iodothyronine deiodinase activities in rodent uterus.

The deiodinase types 2 and 3 (D2, D3), which convert T4 to active and inactive metabolites, respectively, are expressed in the rodent uterus and highly induced during pregnancy. To examine the factors regulating the expression of these enzymes in this tissue, we studied D2 and D3 activity in pregnant rats, in pseudopregnant rats before and after the induction of artificial decidualization, and in ovariectomized rats treated with 17beta-estradiol (E2) and/or progesterone (P). Our results demonstrate that induction of D3 activity begins immediately after implantation and increases markedly over the next 72 h. A similar time course and magnitude of D3 induction is noted in the artificially decidualized uterus in pseudopregnant rats, whereas only minimal increases in activity are observed in the nondecidualized control uterine horns in the same animal. In contrast, D2 activity is not induced by a decidualization stimulus. In spontaneously cycling female rats, both D2 and D3 were observed to be 3- to 8-fold higher in proestrus, compared with diestrus. Furthermore, levels of D2 and D3 activity were greatly increased in ovariectomized rats given E2 and P in various combinations. D2 activity was stimulated primarily by E2, whereas E2 and P acted synergistically to increase D3 activity. These results demonstrate that E2 and P regulate thyroid hormone metabolism in the uterus, and that the implantation process is a potent stimulus for the induction of D3 activity in this organ. Such precise and profound changes in deiodinase expression are likely to play important physiological roles in fetal development and may influence uterine function.

The 5'-deiodinases are not essential for the fasting-induced decrease in circulating thyroid hormone levels in male mice: possible roles for the type 3 deiodinase and tissue sequestration of hormone.

Fasting in rodents is characterized by decreases in serum T4 and T3 levels but no compensatory increase in serum TSH level. The types 1 and 2 deiodinases (D1 and D2) are postulated to play key roles in mediating these changes. However, serum T4 and T3 levels in fasted 5'-deiodinase-deficient mice decreased by at least the same percentage as that observed in wild-type mice, whereas serum TSH level was unaffected. D3 activity was increased in kidney, muscle, and liver up to 4-fold during fasting, and the mean serum rT3 level was increased 3-fold in fasted D1-deficient mice, compared with fed animals. In wild-type mice, the tissue contents of T4 and T3 in liver, kidney, and muscle were unchanged or increased in fasted animals, and after the administration of [(125)I]T4 or [(125)I]T3, the radioactive content in the majority of tissues from fasted mice was increased 2- or 4-fold, respectively. These findings suggest that the observed fasting-induced reductions in the circulating T3 and T4 levels are mediated in part by increased D3 activity and by the sequestration of thyroid hormone and their metabolites in tissues. Studies performed in D3-deficient mice demonstrating a blunting of the fasting-induced decrease in serum T4 and T3 levels were consistent with this thesis. Thus, the systemic changes in thyroid hormone economy as a result of acute food deprivation are not dependent on the D1 or D2 but are mediated in part by sequestration of T4 and T3 in tissues and their enhanced metabolism by the D3.

Life without the iodothyronine deiodinases.

The three iodothyronine deiodinases (D1, D2, and D3) play major roles in determining the tissue and cellular content of the active thyroid hormone, T3. The D1 and D2 5'-deiodinate T4 to T3 and the D3 5-deiodinates T4 and T3 to inactive forms. 5'-Deiodinase-deficient mice (D1/D2KO) have a mild gross phenotype, whereas D3-deficient mice (D3KO) exhibit significant phenotypic abnormalities of the hypothalamic/pituitary/thyroid axis and other organ systems and are not viable in some background strains. The goal of this study was to perform an initial assessment of the phenotype of mice devoid of all deiodinases (D1/D2/D3KO) and determine whether the marked phenotypic abnormalities of the D3KO mouse are exacerbated or mitigated by the absence of the D1 and D2. Relative to D3KO mutants, survival, growth, and fertility were improved in the D1/D2/D3KO mice, although considerably impaired relative to wild-type and D1/D2KO animals. The triple deiodinase-deficient mice also demonstrated normal brain T3 content at postnatal day 6, normal cerebellar expression of the T3-responsive gene hairless at postnatal day 21, and near normalization of their serum thyroid hormone levels as adults, parameters that are abnormal in either the D3KO or the D1/D2KO mutants. These studies demonstrate that within the supportive environment of a research vivarium, mice lacking all three deiodinases can be bred and survive and that at least some of the phenotypic abnormalities resulting from a deficiency of either the D3 5-deiodinase, or the D1 and D2 5'-deiodinase, are mitigated by the simultaneous lack of all three enzymes.

American Thyroid Association Guide to investigating thyroid hormone economy and action in rodent and cell models.

BACKGROUND: An in-depth understanding of the fundamental principles that regulate thyroid hormone homeostasis is critical for the development of new diagnostic and treatment approaches for patients with thyroid disease. SUMMARY: Important clinical practices in use today for the treatment of patients with hypothyroidism, hyperthyroidism, or thyroid cancer are the result of laboratory discoveries made by scientists investigating the most basic aspects of thyroid structure and molecular biology. In this document, a panel of experts commissioned by the American Thyroid Association makes a series of recommendations related to the study of thyroid hormone economy and action. These recommendations are intended to promote standardization of study design, which should in turn increase the comparability and reproducibility of experimental findings. CONCLUSIONS: It is expected that adherence to these recommendations by investigators in the field will facilitate progress towards a better understanding of the thyroid gland and thyroid hormone dependent processes.

Is the kidney a major storage site for thyroxine as thyroxine glucuronide?

BACKGROUND: Previous studies have shown that thyroxine (T4) is stored as T4 glucuronide (T4G) in the kidney, and that 24 hours after administration of [(125)I]T4 to mice, 17% of the radioactivity was present in the kidneys, whereas only 4% was found in the liver. The present study was carried out to determine the relative amounts of conjugated and unconjugated T4 and 3,5,3'-triiodothyronine (T3) in the kidney and liver, and whether the conjugated hormones are extracted from tissues using our established extraction protocols, and detected in our radioimmunoassays (RIAs) for T4 and T3. METHODS: Mice were injected with 10 µCi [(125)I]T4 or [(125)I]T3 and 24 hours later, the labeled compounds present in serum, kidney, liver, and urine were extracted and analyzed by paper chromatography before and after treatment with ß-glucuronidase. In addition, the amounts of endogenous T4 and T3 in extracts of mouse kidney and liver were measured by RIA before and after treatment with ß-glucuronidase. RESULTS: After [(125)I]T4, more than 95% of the total kidney and liver radioactivity was extracted, and in the kidney, almost all of it was present in a conjugated form, mostly as T4G. The liver also contained T4G, but none was present in serum or urine. T3 glucuronide (T3G) was also found in the kidney and liver after the administration of [(125)I]T3. Analysis by RIA of the endogenous T4 content in extracts of kidney before and after hydrolysis by ß-glucuronidase revealed that a substantial fraction of the T4 in both tissues was present as T4G, and the T4G was not detected in the RIA. Furthermore, the combined T4+T4G content in the kidney expressed per gram of tissue was significantly higher than that in the liver or serum. In contrast, the kidney content of T3+T3G was very low compared with that of T4+T4G. CONCLUSIONS: In summary, we have shown that the kidney stores a significant amount of T4 as T4G. Since T4G deconjugation can occur rapidly in the kidney, it is possible that this tissue participates in maintaining extrathyroidal serum T4 homeostasis.