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.

Adult onset of type 3 deiodinase deficiency in mice alters brain gene expression and increases locomotor activity.

Constitutive loss of the type 3 deiodinase (DIO3) causes abnormally increased levels of thyroid hormone action in the developing and adult brain, leading to an array of behavioral abnormalities. To determine to what extent those phenotypes derive from a lack of DIO3 in the adult brain, versus developmental consequences, we created a mouse model of conditional DIO3 inactivation. Mice carrying "floxed" Dio3 alleles and a tamoxifen-inducible cre transgene were injected with tamoxifen at two months of age. Compared to oil-injected controls, the brain tissue of these mice showed a 75-80% decrease in DIO3 activity and 85-95% Dio3 mRNA was expressed from recombinant alleles. Mice with adult DIO3 deficiency did not show significant differences in growth, serum thyroid hormone parameters or behaviors related to anxiety and depression. However, female mice exhibited elevated locomotor activity and increased marble-burying behavior. They also manifested relatively modest alterations in the expression of T3-dependent genes and genes related to hyperactivity in a sex- and region-specific manner. Upon thyroid hormone treatment, the expression response of T3-regulated genes was generally more pronounced in DIO3-deficient female mice than in female controls, while the opposite effect of altered genotype was noticed in males. The extent of the molecular and behavioral phenotypes of adult-onset DIO3 deficiency suggests that a substantial proportion of the neurological abnormalities caused by constitutive DIO3 deficiency has a developmental origin. However, our results show that DIO3 in the adult brain also influences behavior and sensitivity to thyroid hormone action in a sexually dimorphic fashion.

Deletion of the Thyroid Hormone-Activating Type 2 Deiodinase Rescues Cone Photoreceptor Degeneration but Not Deafness in Mice Lacking Type 3 Deiodinase.

Type 2 deiodinase amplifies and type 3 deiodinase depletes levels of the active form of thyroid hormone, triiodothyronine. Given the opposing activities of these enzymes, we tested the hypothesis that they counteract each other's developmental functions by investigating whether deletion of type 2 deiodinase (encoded by Dio2) modifies sensory phenotypes in type 3 deiodinase-deficient (Dio3-/-) mice. Dio3-/- mice display degeneration of retinal cones, the photoreceptors that mediate daylight and color vision. In Dio2-/- mice, cone function was largely normal but deletion of Dio2 in Dio3-/- mice markedly recovered cone numbers and electroretinogram responses, suggesting counterbalancing roles for both enzymes in cone survival. Both Dio3-/- and Dio2-/- strains exhibit deafness with cochlear abnormalities. In Dio3-/-;Dio2-/- mice, deafness was exacerbated rather than alleviated, suggesting unevenly balanced actions by these enzymes during auditory development. Dio3-/- mice also exhibit an atrophic thyroid gland, low thyroxine, and high triiodothyronine levels, but this phenotype was ameliorated in Dio3-/-;Dio2-/- mice, indicating counterbalancing roles for the enzymes in determining the thyroid hormone status. The results suggest that the composite action of these two enzymes is a critical determinant in visual and auditory development and in setting the systemic thyroid hormone status.

Type 3 Deiodinase Role on Central Thyroid Hormone Action Affects the Leptin-Melanocortin System and Circadian Activity.

The role of thyroid hormones (THs) in the central regulation of energy balance is increasingly appreciated. Mice lacking the type 3 deiodinase (DIO3), which inactivates TH, have decreased circulating TH levels relative to control mice as a result of defects in the hypothalamic-pituitary-thyroid axis. However, we have shown that the TH status of the adult Dio3-/- brain is opposite that of the serum, exhibiting enhanced levels of TH action. Because the brain, particularly the hypothalamus, harbors important circuitries that regulate metabolism, we aimed to examine the energy balance phenotype of Dio3-/- mice and determine whether it is associated with hypothalamic abnormalities. Here we show that Dio3-/- mice of both sexes exhibit decreased adiposity, reduced brown and white adipocyte size, and enhanced fat loss in response to triiodothyronine (T3) treatment. They also exhibit increased TH action in the hypothalamus, with abnormal expression and T3 sensitivity of genes integral to the leptin-melanocortin system, including Agrp, Npy, Pomc, and Mc4r. The normal to elevated serum levels of leptin, and elevated and repressed expression of Agrp and Pomc, respectively, suggest a profile of leptin resistance. Interestingly, Dio3-/- mice also display elevated locomotor activity and increased energy expenditure. This occurs in association with expanded nighttime activity periods, suggesting a disrupted circadian rhythm. We conclude that DIO3-mediated regulation of TH action in the central nervous system influences multiple critical determinants of energy balance. Those influences may partially compensate each other, with the result likely contributing to the decreased adiposity observed in Dio3-/- mice.

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 Type 3 Deiodinase Is a Critical Determinant of Appropriate Thyroid Hormone Action in the Developing Testis.

Timely and appropriate levels of thyroid hormone (TH) signaling are necessary to ensure normal developmental outcomes in many tissues. Studies using pharmacological models of altered TH status have revealed an influence of these hormones on testis development and size, but little is known about the role of endogenous determinants of TH action in the developing male gonads. Using a genetic approach, we demonstrate that the type 3 deiodinase (D3), which inactivates TH and protects developing tissues from undue TH action, is a key factor. D3 is highly expressed in the developing testis, and D3-deficient (D3KO) mice exhibit thyrotoxicosis and cell proliferation arrest in the neonatal testis, resulting in an approximately 75% reduction in testis size. This is accompanied by larger seminiferous tubules, impaired spermatogenesis, and a hormonal profile indicative of primary hypogonadism. A deficiency in the TH receptor-α fully normalizes testis size and adult testis gene expression in D3KO mice, indicating that the effects of D3 deficiency are mediated through this type of receptor. Similarly, genetic deficiencies in the D2 or in the monocarboxylate transporter 8 partially rescue the abnormalities in testis size and gonadal axis gene expression featured in the D3KO mice. Our study highlights the testis as an important tissue in which determinants of TH action coordinately converge to ensure normal development and identifies D3 as a critical factor in testis development and in testicular protection from thyrotoxicosis.

Genomic imprinting variations in the mouse type 3 deiodinase gene between tissues and brain regions.

The Dio3 gene, which encodes for the type 3 deiodinase (D3), controls thyroid hormone (TH) availability. The lack of D3 in mice results in tissue overexposure to TH and a broad neuroendocrine phenotype. Dio3 is an imprinted gene, preferentially expressed from the paternally inherited allele in the mouse fetus. However, heterozygous mice with paternal inheritance of the inactivating Dio3 mutation exhibit an attenuated phenotype when compared with that of Dio3 null mice. To investigate this milder phenotype, the allelic expression of Dio3 was evaluated in different mouse tissues. Preferential allelic expression of Dio3 from the paternal allele was observed in fetal tissues and neonatal brain regions, whereas the biallelic Dio3 expression occurred in the developing eye, testes, and cerebellum and in the postnatal brain neocortex, which expresses a larger Dio3 mRNA transcript. The newborn hypothalamus manifests the highest degree of Dio3 expression from the paternal allele, compared with other brain regions, and preferential allelic expression of Dio3 in the brain relaxed in late neonatal life. A methylation analysis of two regulatory regions of the Dio3 imprinted domain revealed modest but significant differences between tissues, but these did not consistently correlate with the observed patterns of Dio3 allelic expression. Deletion of the Dio3 gene and promoter did not result in significant changes in the tissue-specific patterns of Dio3 allelic expression. These results suggest the existence of unidentified epigenetic determinants of tissue-specific Dio3 imprinting. The resulting variation in the Dio3 allelic expression between tissues likely explains the phenotypic variation that results from paternal Dio3 haploinsufficiency.

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.

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.

Cerebellar abnormalities in mice lacking type 3 deiodinase and partial reversal of phenotype by deletion of thyroid hormone receptor α1.

Thyroid hormone serves many functions throughout brain development, but the mechanisms that control the timing of its actions in specific brain regions are poorly understood. In the cerebellum, thyroid hormone controls formation of the transient external germinal layer, which contains proliferative granule cell precursors, subsequent granule cell migration, and cerebellar foliation. We report that the thyroid hormone-inactivating type 3 deiodinase (encoded by Dio3) is expressed in the mouse cerebellum at embryonic and neonatal stages, suggesting a need to protect cerebellar tissues from premature stimulation by thyroid hormone. Dio3(-/-) mice displayed reduced foliation, accelerated disappearance of the external germinal layer, and premature expansion of the molecular layer at juvenile ages. Furthermore, Dio3(-/-) mice exhibited locomotor behavioral abnormalities and impaired ability in descending a vertical pole. To ascertain that these phenotypes resulted from inappropriate exposure to thyroid hormone, thyroid hormone receptor α1 (TRα1) was removed from Dio3(-/-) mice, which substantially corrected the cerebellar and behavioral phenotypes. Deletion of TRα1 did not correct the previously reported small thyroid gland or deafness in Dio3(-/-) mice, indicating that Dio3 controls the activation of specific receptor isoforms in different tissues. These findings suggest that type 3 deiodinase constrains the timing of thyroid hormone action during cerebellar development.

Distinct roles of deiodinases on the phenotype of Mct8 defect: a comparison of eight different mouse genotypes.

Mice deficient in the thyroid hormone (TH) transporter Mct8 (Mct8KO) have increased 5'-deiodination and impaired TH secretion and excretion. These and other unknown mechanisms result in the low-serum T(4), high T(3), and low rT(3) levels characteristic of Mct8 defects. We investigated to what extent each of the 5'-deiodinases (D1, D2) contributes to the serum TH abnormalities of the Mct8KO by generating mice with all combinations of Mct8 and D1 and/or D2 deficiencies and comparing the resulting eight genotypes. Adding D1 deficiency to that of Mct8 corrected the serum TH abnormalities of Mct8KO mice, normalized brain T(3) content, and reduced the impaired expression of TH-responsive genes. In contrast, Mct8D2KO mice maintained the serum TH abnormalities of Mct8KO mice. However, the serum TSH level increased 27-fold, suggesting a severely impaired hypothalamo-pituitary-thyroid axis. The brain of Mct8D2KO manifested a pattern of more severe impairment of TH action than Mct8KO alone. In triple Mct8D1D2KO mice, the markedly increased serum TH levels produced milder brain defect than that of Mct8D2KO at the expense of more severe liver thyrotoxicosis. Additionally, we observed that mice deficient in D2 had an unexplained marked reduction in the thyroid growth response to TSH. Our studies on these eight genotypes provide a unique insight into the complex interplay of the deiodinases in the Mct8 defect and suggest that D1 contributes to the increased serum T(3) in Mct8 deficiency, whereas D2 mainly functions locally, converting T(4) to T(3) to compensate for distinct cellular TH depletion in Mct8KO mice.

Type 3 deiodinase deficiency causes spatial and temporal alterations in brain T3 signaling that are dissociated from serum thyroid hormone levels.

The type 3 deiodinase (D3) is an enzyme that inactivates thyroid hormones (TH) and is highly expressed during development and in the central nervous system. D3-deficient (D3KO) mice develop markedly elevated serum T(3) level in the perinatal period. In adulthood, circulating T(4) and T(3) levels are reduced due to functional deficits in the thyroid axis and peripheral tissues (i.e. liver) show evidence of decreased TH action. Given the importance of TH for brain development, we aimed to assess TH action in the brain of D3KO mice at different developmental stages and determine to what extent it correlates with serum TH parameters. We used a transgenic mouse model (FINDT3) that expresses the reporter gene ß-galactosidase (ß-gal) in the central nervous system as a readout of local TH availability. Together with experiments determining expression levels of TH-regulated genes, our results show that after a state of thyrotoxicosis in early development, most regions of the D3KO brain show evidence of decreased TH action at weaning age. However, later in adulthood and in old age, the brain again manifests a thyrotoxic state, despite reduced serum TH levels. These region-specific changes in brain TH status during the life span of the animal provide novel insight into the important role of the D3 in the developing and adult brain. Our results suggest that, even if serum concentrations of TH are normal or low, impaired D3 activity may result in excessive TH action in multiple brain regions, with potential consequences of altered neural function that may be of clinical relevance to neurological and neuroendocrine disorders.

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.

Type 3 deiodinase, a thyroid-hormone-inactivating enzyme, controls survival and maturation of cone photoreceptors.

Maturation of the mammalian nervous system requires adequate provision of thyroid hormone and mechanisms that enhance tissue responses to the hormone. Here, we report that the development of cones, the photoreceptors for daylight and color vision, requires protection from thyroid hormone by type 3 deiodinase, a thyroid hormone-inactivating enzyme. Type 3 deiodinase, encoded by Dio3, is expressed in the immature mouse retina. In Dio3(-/-) mice, approximately 80% of cones are lost through neonatal cell death. Cones that express opsin photopigments for response to both short (S) and medium-long (M) wavelength light are lost. Rod photoreceptors, which mediate dim light vision, remain essentially intact. Excessive thyroid hormone in wild-type pups also eliminates cones. Cone loss is mediated by cone-specific thyroid hormone receptor beta2 (TRbeta2) as deletion of TRbeta2 rescues cones in Dio3(-/-) mice. However, rescued cones respond to short but not longer wavelength light because TRbeta2 under moderate hormonal stimulation normally induces M opsin and controls the patterning of M and S opsins over the retina. The results suggest that type 3 deiodinase limits hormonal exposure of the cone to levels that safeguard both cone survival and the patterning of opsins that is required for cone function.

Life without thyroxine to 3,5,3'-triiodothyronine conversion: studies in mice devoid of the 5'-deiodinases.

Considerable indirect evidence suggests that the type 2 deiodinase (D2) generates T(3) from T(4) for local use in specific tissues including pituitary, brown fat, and brain, whereas the type I deiodinase (D1) generates T(3) from T(4) in the thyroid and peripheral tissues primarily for export to plasma. From studies in deiodinase-deficient mice, the importance of the D2 for local T(3) generation has been confirmed. However, the phenotypes of these D1 knockout (KO) and D2KO mice are surprisingly mild and their serum T(3) level, general health, and reproductive capacity are unimpaired. To explore further the importance of 5'deiodination in thyroid hormone economy, we used a mouse devoid of both D1 and D2 activity. In general, the phenotype of the D1/D2KO mouse is the sum of the phenotypes of the D1KO and D2KO mice. It appears healthy and breeds well, and most surprisingly its serum T(3) level is normal. However, impairments in brain gene expression and possibly neurological function are somewhat greater than those seen in the D2KO mouse, and the serum rT(3) level is elevated 6-fold in the D1/D2KO mouse but only 2-fold in the D1KO mouse and not at all in the D2KO mouse. The data suggest that whereas D1 and D2 are not essential for the maintenance of the serum T(3) level, they do serve important roles in thyroid hormone homeostasis, the D2 being critical for local T(3) production and the D1 playing an important role in iodide conservation by serving as a scavenger enzyme in peripheral tissues and the thyroid.

Minireview: Defining the roles of the iodothyronine deiodinases: current concepts and challenges.

As is typical of other hormone systems, the actions of the thyroid hormones (TH) differ from tissue to tissue depending upon a number of variables. In addition to varying expression levels of TH receptors and transporters, differing patterns of TH metabolism provide a critical mechanism whereby TH action can be individualized in cells depending on the needs of the organism. The iodothyronine deiodinases constitute a family of selenoenzymes that selectively remove iodide from thyroxine and its derivatives, thus activating or inactivating these hormones. Three deiodinases have been identified, and much has been learned regarding the differing structures, catalytic activities, and expression patterns of these proteins. Because of their differing properties, the deiodinases appear to serve varying functions that are important in regulating metabolic processes, TH action during development, and feedback control of the thyroid axis. This review will briefly assess these functional roles and others proposed for the deiodinases and examine some of the current challenges in expanding our knowledge of these important components of the thyroid homeostatic system.

A protective role for type 3 deiodinase, a thyroid hormone-inactivating enzyme, in cochlear development and auditory function.

Thyroid hormone is necessary for cochlear development and auditory function, but the factors that control these processes are poorly understood. Previous evidence indicated that in mice, the serum supply of thyroid hormone is augmented within the cochlea itself by type 2 deiodinase, which amplifies the level of T(3), the active form of thyroid hormone, before the onset of hearing. We now report that type 3 deiodinase, a thyroid hormone-inactivating enzyme encoded by Dio3, is expressed in the immature cochlea before type 2 deiodinase. Dio3-/- mice display auditory deficits and accelerated cochlear differentiation, contrasting with the retardation caused by deletion of type 2 deiodinase. The Dio3 mRNA expression pattern in the greater epithelial ridge, stria vascularis, and spiral ganglion partly overlaps with that of thyroid hormone receptor beta (TRbeta), the T(3) receptor that is primarily responsible for auditory development. The proposal that type 3 deiodinase prevents premature stimulation of TRbeta was supported by deleting TRbeta, which converted the Dio3-/- cochlear phenotype from one of accelerated to one of delayed differentiation. The results indicate a protective role for type 3 deiodinase in hearing. The auditory system illustrates the considerable extent to which tissues can autoregulate their developmental response to thyroid hormone through both type 2 and 3 deiodinases.

Impaired bacterial clearance in type 3 deiodinase-deficient mice infected with Streptococcus pneumoniae.

The activation of type 3 deiodinase (D3) has been postulated to play a role in the reduction of thyroid hormone levels during illness. Using a mouse model of acute bacterial infection, we have recently demonstrated marked D3 immunostaining in neutrophils infiltrating infected organs. These observations suggest a possible additional role for this enzyme in the innate immune response. To further assess the role of D3 in the response to acute bacterial infection, we used null D3 [D3 knockout (D3KO)] and wild type (WT) mice and infected them with Streptococcus pneumoniae. Marked reductions in serum thyroid hormone levels were observed both in D3KO and WT mice. Infection resulted also in a decrease in liver D1 activity in WT, but not in infected D3KO mice. Upon infection, pulmonary neutrophilic influx (measured by myeloperoxidase levels) and IL-6 and TNF concentrations increased equally in D3KO and WT mice, and histological examination of infected mice showed similar pulmonary inflammation in both strains. However, D3KO animals demonstrated significantly higher bacterial load in blood, lung, and spleen compared with WT mice. We conclude that 1) D3 is not required to generate the systemic manifestations of the nonthyroidal illness syndrome in this model; 2) the lack of D3 does not affect the extent of pulmonary inflammation; and 3) bacterial outgrowth in blood, spleen, and lung of D3KO mice is significantly higher than in WT mice. Our results suggest a protective role for D3 in the defense against acute bacterial infection, probably by reinforcing the microbial killing capacity of neutrophils.

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.