Synthesis and Metabolism of Thyroid Hormones Is Preferentially Maintained in Selenium-Deficient Transgenic Mice
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《内分泌学杂志》
Institute for Experimental Endocrinology (L.S., C.R., M.M., E.G., U.S., J.K.), Neurobiology of Selenium (U.S.), Charite-Universitaetsmedizin Berlin, D-10117 Berlin, Germany
Department of Endocrinology (M.O.K.), Centre Hospitalier et Universitaire de Nancy, 54042 Nancy, France
Institut de Physique Biologique (R.S.), Unite d’Analyses Endocriniennes, Hopital Civil, 67091 Strasbourg Cedex, France
Abstract
The thyroid gland is rich in selenium (Se) and expresses a variety of selenoproteins that are involved in antioxidative defense and metabolism of thyroid hormones (TH). Se deficiency impairs regular synthesis of selenoproteins and adequate TH metabolism. We recently generated mice that lack the plasma Se carrier, selenoprotein P (SePP). SePP-knockout mice display decreased serum Se levels and manifest growth defects and neurological abnormalities partly reminiscent of thyroid gland dysfunction or profound hypothyroidism. Thus, we probed the TH axis in developing and adult SePP-knockout mice. Surprisingly, expression of Se-dependent 5'-deiodinase type 1 was only slightly altered in liver, kidney, or thyroid at postnatal d 60, and 5'-deiodinase type 2 activity in brain was normal in SePP-knockout mice. Thyroid gland morphology, thyroid glutathione peroxidase activity, thyroid Se concentration, and serum levels of TSH, T4, or T3 were within normal range. Pituitary TSH transcripts and hepatic 5'-deiodinase type 1 mRNA levels were unchanged, indicating regular T3 bioactivity in thyrotropes and hepatocytes. Cerebellar granule cell migration as a sensitive indicator of local T3 action during development was undisturbed. Collectively, these findings demonstrate that low levels of serum Se or SePP in the absence of other challenges do not necessarily interfere with regular functioning of the TH axis. 5'-deiodinase isozymes are preferentially supplied, and Se-dependent enzymes in the thyroid are even less-dependent on serum levels of Se or SePP than in brain. This indicates a top priority of the thyroid gland and its selenoenzymes with respect to the hierarchical Se supply within the organism.
Introduction
THE HUMAN THYROID gland is known to be among the organs with highest content of the trace element selenium (Se) (1, 2). Se is an essential micronutrient in the diet of mammals (3, 4). Se deficiency is associated with the predisposition to certain conditions including cancer, atherosclerosis, arthritis, male infertility, immunological defects, and diseases of accelerated aging (5, 6). Accordingly, Se supplementation holds great promises for chemoprevention (7) and in improving the immune system in which it turned out to be beneficial in reducing antibody load, e.g. in autoimmune thyroiditis (8, 9). Se is considered to exert its biological activity mainly via selenoproteins, which contain Se in their active centers as part of the 21st proteinogenic amino acid selenocysteine (Sec) (10).
Sec is incorporated cotranslationally into at least 25 or 24 different gene products in humans and rodents, respectively (11). Most of the Sec-containing proteins that have been functionally characterized are enzymes that catalyze redox-reactions including members of the family of glutathione peroxidases (GPx), thioredoxin reductases or iodothyronine deiodinases (Dio) (12). Interestingly, there is a certain hierarchy among different selenoproteins with respect to Se supply and resynthesis after Se depletion, i.e. important selenoproteins like Dio1 are preferentially supplied when the trace element is limiting, whereas others like GPx1 rank low and are dependent on high Se supply (13, 14).
A second hierarchy in Se biology is observed with respect to Se in different tissues. Endocrine organs and the brain are preferentially supplied, whereas serum, liver, kidney, or muscles readily lose their Se stores on dietary Se deprivation. Especially the thyroid gland retains the trace element very efficiently (15). It synthesizes a variety of Sec-containing enzymes including GPx and thioredoxin reductases (16, 17) and the Dio isozymes that control thyroid hormone (TH) metabolism (18, 19). Se deficiency is thus believed to compromise the TH system by two important routes, i.e. the antioxidative defense capacity of thyrocytes is compromised and TH metabolism is disturbed both within the gland and systemically (18, 19).
Recently transgenic mice that lack the main Se-containing plasma protein, i.e. selenoprotein P (SePP), have been established (20, 21). SePP serves as a plasma Se carrier and nicely mirrors the overall Se status of an individual (22, 23). Accordingly, SePP-knockout (SePP-KO) mice display reduced Se levels in the tissues. Se concentrations are reduced to 22% of control in serum, 64% in kidney, 25% in brain, and 47% in testis, whereas liver displays an increased Se concentration of 1.5-fold above control (21). The phenotypes and the reduced tissue Se concentrations can be rescued by suitable Se supplementation regimen (20, 24).
Because these mice present with growth deficits and neurological phenotypes including ataxia, we speculated that a Se-dependent derangement of the TH axis could underlie the observed pathologies. Surprisingly, expression of Se-dependent enzymes in thyroid, serum levels of TSH and TH, and T3-dependent transcriptional and developmental processes turned out to be undisturbed. These findings position the thyroid gland and TH metabolism upmost in the hierarchy of Se-dependent tissues and Se-dependent reactions.
Materials and Methods
Materials
All chemicals were of analytical grade and obtained from Sigma (München, Germany) or Merck (Darmstadt, Germany). [32P]dCTP was from Hartmann Analytic (Braunschweig, Germany) and [125I]rT3 was from NEN Life Science Products DuPont/PerkinElmer (Koeln, Germany). Oligonucleotides were from MWG Biotech (Ebersberg, Germany), restriction endonucleases were from New England Biolabs (Frankfurt, Germany), and Taq polymerases were from QIAGEN (Hilden, Germany). A TA-cloning system was purchased from Promega (Mannheim, Germany), and DNA sequencing was accomplished by a DyeTerminator cycle sequencing kit (Applied Biosystems, Weiterstadt, Germany).
Sample collection and preparation of tissue homogenates
Mice were raised on regular lab chow (Altromin, Lage, Germany) containing on average 0.24 ppm Se. A 12-h light, 12-h dark cycle was controlled automatically. SePP mice used had been backcrossed for four to seven generations onto C57BL/6CR strain. SePP-KO mice were generated by mating heterozygous SePP (±) mice, and genotyping was performed with DNA from ear clippings as described earlier (21). At the age of 35 or 60 d, animals were deeply anesthetized with carbon dioxide, the chest was opened, and blood was drawn from the heart or they were perfusion fixed for histological analyses. After storage at 4 C for 1 h, blood samples were centrifuged (4 C; 10,000 x g; 10 min) and serum was collected. Tissues were removed, frozen in liquid nitrogen, and stored at –80 C until analysis. Animal experimentations were approved by the animal welfare committee of the local governmental authorities in Berlin, Germany. For enzyme activity assays and Se determinations, the tissues were pulverized under liquid nitrogen using a dismembrator (Braun, Melsungen, Germany). Aliquots were homogenized on ice in approximately 5 volumes of 20 mM HEPES (pH 7.0), containing 250 mM sucrose and 1 mM EDTA using a glass/Teflon homogenizer. The homogenates were centrifuged at 10,000 x g for 20 min, the supernatants were collected, and pellets were resuspended in homogenization buffer supplemented with 1 mM dithiothreitol. Protein concentrations were determined by a modified Bradford assay using IgG as standard (Bio-Rad, München, Germany).
Fluorometric Se determination
The Se concentrations of the thyroid glands were determined from the supernatants of the tissue homogenates. The method used was essentially as described before (21) with minor modifications. Due to the limited starting material available, all components of the assay were halved, i.e. volumes of only 50 μl homogenate or standard were digested by only 250 μl HNO3/HClO4 (4:1,vol/vol), reduced by 250 μl HCl, complexed by 1 ml of EDTA (2.5 mM), supplemented with 250 μl 2,3-diaminonaphtalene in 0.1 M HCl, extracted with 0.5 ml cyclohexane, and the fluorescent piazselenols formed were quantified as described (21). A commercially available pooled human serum standard (Sero AS, Billingstad, Norway) along with a Se atomic absorption standard solution (1000 μg/ml; Sigma) was used to validate the method.
Dio assay
The resuspended pellet fractions were assayed for Dio activities in a reaction mixture containing 20 μg (liver and kidney), 100 μg (brain), or 5 μg (thyroid) protein (25). Dithiothreitol (10 mM) served as cofactor and [125I]rT3 was added as tracer (50,000 cpm/tube, specific activity: 25 TBq/mmol, PerkinElmer). The substrate concentration was 1.0 μM rT3 (liver), 500 nM rT3 (kidney), 200 nM rT3 (thyroid), or 1 nM rT3 (brain). Reaction volume was 100 μl, the reaction proceeded at 37 C for 1 or 2 h, and the determinations were performed in triplicates. Conditions had been optimized such that final substrate deiodination was less than 15%. Dio1 and Dio2 activities were distinguished by the inclusion of 1 mM 6-n-propyl-2-thiouracil in the incubation mixture. The fraction of iodide release blocked by 6-n-propyl-2-thiouracil was assigned to Dio1, residual background-corrected activity to Dio2. [125I]rT3 was purified from free [125I] by chromatography using Sephadex LH-20 (Sigma-Aldrich, Muenchen, Germany) before use.
TSH, T4, and T3 determination
Serum was obtained as described above and stored at –80 C until analysis.
TSH determination.
Guinea pig antimouse TSH antibody (AFP98991) and a crude TSH/LH reference preparation (AFP51718MP) were obtained from Dr. A. F. Parlow (National Hormone and Peptide Program, Harbor-UCLA Medical Center, Los Angeles, CA). Commercially available 125I-labeled rat TSH (RPA 554) was obtained from Amersham Biosciences (Freiburg, Germany). Normal guinea pig IgG as carrier and goat antiguinea pig IgG as precipitating antibody were provided by Linco Research (St. Charles, MO; references 3020 and 2020, respectively). A calibration standard at a nominal concentration of 10,000 ng/ml was prepared in PBS/BSA [2% (wt/vol) BSA in PBS] and progressively diluted in PBS/BSA to give rise to nine standard levels ranging from 19 to 5000 ng/ml. The samples (50 μl serum sample or standard diluted with 50 μl PBS) were preincubated overnight at room temperature with 100 μl guinea pig antimouse TSH antibody (1/150,000). Then 100 μl 125I-labeled rat TSH was added and incubated for 4 h at room temperature. Afterward normal guinea pig IgG carrier (100 μl) and the precipitating goat antiguinea pig IgG antibody (100 μl) were added and incubated for 2 h at 4 C. The RIA tubes were centrifuged at 3000 x g for 30 min at 4 C. After aspiration of the supernatant, the precipitated radioactivity in each tube was measured using a PerkinElmer Wizard -counter. A standard curve was obtained by plotting the fraction of precipitated 125I-TSH over total 125I-TSH (B/B0) against the standard nominal concentrations. Intra- and interassay coefficients of variation were 4% at 137.5 ± 5.4 ng/ml and 7.4% at 124.5 ± 9.2 ng/ml, respectively.
Total T4 (TT4) determination.
A competitive RIA was performed (RIA kit CA-1535M; DiaSorin, Rome, Italy). Ten microliters of each sample (serum or standard) were incubated for 45 min with 1 ml of tracer-buffer reagent in a mouse monoclonal anti-T4-coated tube at room temperature. Tracer-buffer reagent was obtained by adding 10 ml of [125I]T4 to 10 ml of Tris-buffered saline, 4 mM 8-anilino-1-naphthalene sulfonic acid (ANS), 6 mM sodium salicylate, 0.2% sodium azide, and 90 ml distilled water. Then all tubes were aspirated except for total count tubes, and the radioactivity in each tube was measured for 1 min using a PerkinElmer Wizard -counter. Standard concentrations of T4 ranged from 1 to 20 μg/dl. Intraassay coefficients of variation were 6.4% at 2.64 μg/dl and 4.8% at 6.85 μg/dl. Interassay coefficients of variation were 11.2% at 2.85 μg/dl and 5.2% at 6.41 μg/dl.
Total T3 (TT3) determination.
A similar competitive RIA was performed (DiaSorin RIA kit CA-1541) that also relied on the use of ANS to displace T3 from serum binding proteins (26). For this technique, 50 μl of each sample (serum or standard) were incubated for 1 h with 1 ml of tracer-buffer reagent in an anti-T3-coated tube at 37 C. The tracer-buffer reagent was prepared as above with [125I]T3 in PBS containing ANS and sodium azide. Standard concentrations of T3 ranged from 0.5 to 8 ng/ml. Intraassay coefficients of variation were 7.9% at 0.89 ng/ml and 3.6% at 1.69 ng/ml. Interassay coefficients of variation were 8.3% at 0.84 ng/ml and 4.8% at 1.68 ng/ml.
Histological analysis
Wild-type and age-matched homozygous SePP-KO mice were deeply anesthetized and perfusion fixed transcardially with 0.1 M Na-phosphate buffer (pH 7.4), followed by 4% paraformaldehyde in the same buffer. Thyroid glands were excised together with the trachea, postfixed overnight in the same fixative, dehydrated, and embedded in paraffin. Sections of 2 μm were cut on a rotating microtome (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin by standard procedures. Tissue sections were examined under a light microscope (Axioskop, Zeiss, Oberkochen, Germany), and pictures were taken using a digital camera (ActionCam, Agfa, Koeln, Germany). Cerebelli were sectioned at 15 μm in the sagittal plane. Neuronal cell bodies were revealed by cresylviolett staining. Immunostainings against calbindin (polyclonal; Swant, Bellinzona, Switzerland), parvalbumin (polyclonal; Swant), calretinin (monoclonal, Swant), and NeuN (monoclonal, Chemicon, Temecula, CA) were revealed using Vector reagents (Vector Laboratories, Burlingame, CA) and NovaRed peroxidase substrate according to the manufacturer’s recommendations.
Northern blot analysis
Total RNA was isolated from the tissues by the use of peqGOLD TriFast (PEQLAB, Erlangen, Germany) according to the instructions of the manufacturer. Aliquots were size fractionated in denaturing formaldehyde/agarose gels, capillary transferred to nylon membranes (Nytran NY 12 N; Schleicher & Schuell, Dassel, Germany) and analyzed under high-stringency conditions as described earlier (27). For pituitary RNA isolation, single mouse pituitaries were directly homogenized in 200 μl peqGOLD TriFast and isopropanol precipitations were performed after adding 1 μg yeast tRNA as carrier. cDNA fragments encompassing the open reading frames of mouse SePP, TSH, Dio1, -actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified by PCR, subcloned, sequenced, and randomly labeled with 32P-dCTP (Hartmann Analytic, Braunschweig, Germany) to high specific activities (>109 cpm/μg). After hybridization and extensive washings at 62 C, Northern results were quantified by analysis with a phosphor imager (Cyclone storage phosphor system, Packard BioScience, Dreieich, Germany). The signals obtained for GAPDH mRNA were used to calculate relative expression levels. For analysis of thyroid RNA preparations, -actin was used as standard because it turned out to mirror relative loading amounts more faithfully than GAPDH.
Statistical analysis
Values are reported as mean ± SE. P values were calculated by one-way ANOVA followed by Bonferroni’s post hoc test. Statistical significance was defined as P < 0.05 (), P < 0.01 (), or P < 0.001 (). Data of serum TSH and TH levels were analyzed by the Kruskal-Wallis test.
Results
Preserved expression of Dio1 and -2 in SePP-KO mice
The activation of T4 to biologically active T3 is catalyzed by Se-dependent Dio1 expressed mainly in liver, kidney, pituitary, and thyroid gland or Dio2 mainly expressed in the central nervous system, brown adipose tissue, and placenta (18, 19). When raised on regular lab chow, SePP-KO mice display drastically reduced serum Se levels and plasma GPx (GPx3) activities (24). Despite those strong Se-dependent effects, transcript and enzyme levels of Dio1 were only marginally altered in liver or kidney of SePP-KO mice at postnatal d (P) 60 (Fig. 1). The small differences observed appear to follow tissue Se concentrations that are increased in livers of SePP-KO mice and decreased in their kidneys.
These findings are in contrast to our initial results from a small fraction of male SePP-KO mice that we had to kill after weaning at P35 because of poor health status. These animals displayed strongest Se-dependent phenotypes and expressed sharply reduced hepatic and renal Dio1 transcript levels and enzymatic activities (to 20–30% control levels). No such effects were observed in age-matched, apparently healthy female SePP-KO littermates or mice that were analyzed at later time points (not shown).
Unaltered serum levels of TH and TSH
Pituitary synthesis and secretion of TSH as part of the regulatory TH feedback system is known to be most sensitive to changes in T3 concentration (27). Comparing wild-type, heterozygous, and SePP-KO mice, serum levels of TSH, TT3, or TT4 were comparable and did not show SePP-dependent differences (Fig. 2). The values obtained were within a narrow range and well in agreement with reports from other laboratories working with C57BL/6 mice (28). Nevertheless, significant sex-specific differences were observed between TSH levels of male and female mice independent of the SePP genotype involved (Fig. 2).
Regular TH target gene expression
The TSH-specific -subunit is stringently controlled by T3 at the transcriptional level and via its poly-A tract length that controls half-life of the transcripts (27, 29). TSH mRNA from pituitaries of SePP-KO mice was normal in quantity and length (Fig. 3A). Upon quantification, TSH mRNA levels from heterozygous mice seemed to be decreased significantly, compared with wild-type mice, and marginally, compared with SePP-KO littermates, but this effect was rather attributable to slightly increased GAPDH mRNA levels in the pituitaries of these mice. Hepatic Dio1 has been characterized as a most sensitive marker of T3 plasma levels (30). Comparing heterozygous and homozygous SePP-KO mice with wild-type littermates, no differences in Dio1 mRNA levels were observed in livers at P60 (Fig. 3B).
Normal thyroid gland morphology, thyroid Se concentration, and thyroid GPx activity
The thyroid gland expresses Se-dependent enzymes involved in TH metabolism and antioxidative defense. Severe Se deficiency could compromise these functions, leading to tissue destruction, malfunction, or goitrous hyperplasia. Visual inspection of SePP-KO mice did not reveal any abnormalities of the neck region or gland appearance at P35 or P60. Upon necroscopy of male and female thyroid glands, no differences in size, color, or texture were noted. Single thyroid glands were processed for histology, enzyme activity or Northern blot analysis at P60. The follicle cells and cavities looked essentially normal in size, number, and location (Fig. 4A). Thyroid GPx1 mRNA levels as most sensitive markers of intracellular Se levels and Se availability were at wild-type levels (Fig. 4B). Likewise, no differences of total Se concentrations were observed in the homogenates of thyroid tissues from wild-type, heterozygous, or SePP-KO mice (Fig. 4B). GPx activity in thyroids representing mainly GPx1 and GPx3 enzyme levels was also found unaffected by the SePP genotype (Fig. 4C). This is in contrast to other tissues from SePP-KO mice analyzed to date including serum, kidney, testes, or even brain (20, 21). In contrast, Dio1 activity was significantly increased in thyroid glands from heterozygous SePP-KO mice. This finding is in agreement with earlier reports from Se-deprived animals (31). Because heterozygous SePP-KO mice are in general without phenotype despite gradually reduced serum and tissue levels of SePP and Se, the increased Dio1 activity might represent a thyroid-specific physiological response to moderate Se deficiency by a SePP-independent mechanism that helps to maintain normal serum TH levels.
Regular TH-dependent cerebellar development and Dio2 activity in brain
Perinatal hypothyroidism induces a number of anatomical alterations in the rodent brain including abnormal cell migration, dendritic arborization, or defects of synaptogenesis (32, 33). It is well established that especially cerebellar granule cell migration during postnatal brain development critically depends on TH by TR1-dependent mechanisms (34) and that it is delayed in hypothyroidism (35). When we did a respective histological analysis of cerebellar specimen from SePP-KO mice and wild-type littermates, no indications for a migration defect that would point toward local hypothyroidism were found (Fig. 5A). Cerebellar architecture appeared normal, and a well-developed Purkinje cell layer with highly elaborated dendritic trees was observed after calbindin staining. Expression patterns of other marker proteins for the major cerebellar cell types like parvalbumin (Purkinje, basket, and stellar cells), NeuN, or calretinin (granule cells) were unchanged despite the compromised SePP expression and reduced brain Se levels (not shown). Likewise, enzymatic activity of Dio2 in brain of SePP-KO mice was at wild-type levels (Fig. 5B) despite those strongly reduced Se concentrations or GPx activities in brain at P35 and those concomitantly developing neurological defects (21, 36).
Thyroid gland Se metabolism is exceptionally independent from SePP
SePP-KO mice display reduced Se levels in serum, kidney, testes, or brain but not in liver because hepatocytes normally synthesize and secrete SePP for systemic supply and thus accumulate Se in the absence of SePP (21). Whereas Se levels in serum and kidney are dependent on hepatically derived SePP, brain Se is maintained via local SePP synthesis and Se recycling mechanisms within the tissue, which render the central nervous system seemingly independent from serum Se and SePP levels (23). Despite the strong neurological and growth phenotypes of SePP-KO mice that resemble in part those defects that are known for TH deficiency, no alterations of T4, T3, or TSH serum levels were observed; thyroid gland morphology and T3-dependent developmental and regulatory processes were normal. Thus, the thyroid gland was able to accumulate, retain, and recycle Se efficiently, even in the absence of SePP, and to adjust its Dio and GPx expression adequately, even in Se deficiency. This exceptional feature seems to rank the thyroid gland even higher than brain in the hierarchy of tissues with respect to Se supply (Fig. 6).
Discussion
The thyroid gland is essential for mammalian life and depends on two important trace elements for the synthesis and metabolism of TH, i.e. Se and iodine (17, 18). Whereas iodine is needed directly as substrate for biosynthesis of T4 and T3, the importance of Se for the TH axis is less well appreciated. The characterization of GPx as well as the TH metabolizing Dio isozymes as selenoproteins provided the molecular basis that linked Se biology with thyroid gland function and more general aspects of health and disease (6). The results presented here demonstrate that the Dio isozymes are preferentially supplied with Se and that the thyroid gland unexpectedly retained its GPx activity exceptionally well in SePP-deficient mice. Thus, the thyroid gland remains equipped with an effective defense system against the tone of reactive oxygen species even in the absence of SePP. Adequate metabolism of TH by the Dio isozymes is likewise maintained with priority. This preferential supply of Dio1 vs., for example, GPx1 has been observed before in vivo and in cell culture models and is an example for the still enigmatic hierarchy among different selenoproteins (14, 31).
Yet our initial analysis of Dio1 expression in SePP-KO mice yielded conflicting results. We had to kill some of the male animals at P35 because they suffered from acute weight loss and bad overall health status including recurrent seizures. Surprisingly, Dio1 expression was drastically reduced in their livers and kidneys, compared with wild-type or heterozygous littermates. This finding was obviously not in line with the hierarchical expression of selenoproteins. But when we later analyzed male or female SePP-KO mice that survived this critical phase after weaning, expression of Dio1 was again found well preserved as in control littermates. Thus, our initial finding of reduced Dio1 expression might be related to the still enigmatic low-T3 syndrome observed in late stages of critical illness or cachectic conditions and rather represents only an indirect consequence of SePP deficiency.
Nevertheless, both male and female SePP-KO mice display strong neurological phenotypes that we expected to be TH dependent, i.e. atactic movements and sporadic seizures. These phenotypes were never observed before in pure dietary depletion models, even after nutritional Se deprivation over many generations (37). Supplementation studies revealed that these phenotypes of SePP-KO mice correlated with reduced brain Se levels and GPx activities and that they could be prevented by Se-enriched diets (20, 24). We demonstrate here, that even without Se supplementation, Dio2 expression in brain of SePP-KO mice is normal and not decreased due to Se deficiency or elevated as in hypothyroid mice (38). In addition, postnatal cerebellar granule cell migration as a sensitive indicator of local T3 activity during development (39) is undisturbed. Thus, the underlying cause for the observed central defects is currently still not understood, but other mechanisms than gross TH derangements in the brain must be considered.
A second hierarchy in Se biology refers to the graded capability of different tissues to retain Se when the trace element is limiting or to accumulate Se on replenishment (15). Here the brain and the endocrine tissues including the thyroid and pituitary glands turned out to be highest ranking (15, 40). These features seem to guarantee that a moderate or transient Se deficiency elicited by insufficient nutritional supply or impaired uptake does not cause adverse health effects. Accordingly, many studies have revealed that nutritional Se deprivations in adult rodents or even during development cause only moderate effects on the TH axis (41, 42, 43, 44, 45). The same is true for second-generation Se-deficient rats (46), experimental Se-deficient sheep (47), or humans with different eating habits that cause drastically different Se plasma levels (48). These mild effects are in sharp contrast to potentially devastating consequences of a combined Se and iodine deficiency as in myxedematous cretinism (49). Here the phenotype might even aggravate further by, for example, thiocyanate overload due to cassava root consumption as found in endemic goitre areas in central Africa (12). Currently we cannot predict the importance of SePP in such pathological settings. Our findings indicate that SePP-KO mice can have a regular functioning thyroid gland and TH feedback system per se if not additionally provoked and their phenotypes are not caused by hypothyroidism. But this does not exclude that exceptional challenges that stimulate TH synthesis and activate thyroid cells beyond a certain degree cause overwhelming defects. Probably high age, low iodine diet, or mild goitrogen treatment might suffice to cause excessive symptoms in SePP-KO mice due to impaired adaptive capacity of the thyroid glands. Such mechanisms might have accelerated the severe illness that we observed with those moribund male SePP-KO mice shortly after weaning. Albeit, the underlying defect, molecular mechanisms, and gender-specific aspect of the severe health conditions still need to be analyzed further.
We observed before that the brain was dependent on SePP for normal GPx expression and undisturbed privileged Se metabolism (21). The tissue-specific deletion of hepatic selenoprotein synthesis including SePP by genetic inactivation of the Sec-specific tRNA resulted in reduced serum and kidney Se levels and GPx activities but did not impact negatively on brain Se or brain GPx expression (23, 50). Thus, serum and kidney were dependent on hepatically derived SePP, but brain needed mainly its locally synthesized SePP pool for efficient Se accumulation and retention against an adverse plasma gradient (51, 52). In contrast, the normal functioning of the thyroid gland in SePP-KO mice indicates that it was able to accumulate and retain Se in the form of selenoenzymes, even without SePP. It depended on neither high Se or SePP plasma levels nor locally produced and retained SePP (23). These findings indicate an evenly graded hierarchy among the preferentially supplied tissues with the thyroid gland ranking more independent and thus higher than the central nervous system.
The mutual interdependence of a functional TH feedback system with undisturbed selenoprotein biosynthesis was just described in humans with inherited mutations in the gene encoding selenocysteine insertion sequence-binding protein 2, a critical factor for selenoprotein translation. Here the synthesis of all selenoproteins analyzed was hampered, and the main clinical condition observed was a disturbed TH feedback system most likely because of reduced expression of the Dio isozymes (53). Together with the findings presented here, one can assume that evolution has optimized the hierarchical principles in Se biology primarily to sustain thyroid gland functioning and adequate TH metabolism, even during transient, dietary, or illness-related shortages of Se.
Acknowledgments
We are grateful to Katja Schreiber, Silke Kappler, Anita Kinne, Vartiter Seher, Birgit Hollenbach, and Metin Yenilmaz for excellent technical assistance. Special thanks go to our colleagues in the animal facility for their support. We also thank Peter Hofmann, Inka Hamann, Cornelia Schmutzler, Birgit Mentrup, and Branislaw Radovic for stimulating discussions; Dr. A. F. Parlow for the generous supply of TH-specific reagents; and the Deutsche Krebshilfe and the Deutsche Forschungsgemeinschaft for financial support.
Footnotes
This work was supported in part by the Deutsche Krebshilfe (Grant 10-1792 SchoI) and the Deutsche Forschungsgemeinschaft (Grant SCHO 849/1-1 and Grant KO 922/8-3).
The authors have nothing to declare.
First Published Online December 1, 2005
Abbreviations: ANS, 8-Anilino-1-naphthalene sulfonic acid; Dio, 5'-deiodinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPx, glutathione peroxidase; P, postnatal day; Se, selenium; Sec, selenocysteine; SePP, selenoprotein P; SePP-KO, SePP-knockout; TH, thyroid hormone(s); TT3, total T3; TT4, total T4.
Accepted for publication November 18, 2005.
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Department of Endocrinology (M.O.K.), Centre Hospitalier et Universitaire de Nancy, 54042 Nancy, France
Institut de Physique Biologique (R.S.), Unite d’Analyses Endocriniennes, Hopital Civil, 67091 Strasbourg Cedex, France
Abstract
The thyroid gland is rich in selenium (Se) and expresses a variety of selenoproteins that are involved in antioxidative defense and metabolism of thyroid hormones (TH). Se deficiency impairs regular synthesis of selenoproteins and adequate TH metabolism. We recently generated mice that lack the plasma Se carrier, selenoprotein P (SePP). SePP-knockout mice display decreased serum Se levels and manifest growth defects and neurological abnormalities partly reminiscent of thyroid gland dysfunction or profound hypothyroidism. Thus, we probed the TH axis in developing and adult SePP-knockout mice. Surprisingly, expression of Se-dependent 5'-deiodinase type 1 was only slightly altered in liver, kidney, or thyroid at postnatal d 60, and 5'-deiodinase type 2 activity in brain was normal in SePP-knockout mice. Thyroid gland morphology, thyroid glutathione peroxidase activity, thyroid Se concentration, and serum levels of TSH, T4, or T3 were within normal range. Pituitary TSH transcripts and hepatic 5'-deiodinase type 1 mRNA levels were unchanged, indicating regular T3 bioactivity in thyrotropes and hepatocytes. Cerebellar granule cell migration as a sensitive indicator of local T3 action during development was undisturbed. Collectively, these findings demonstrate that low levels of serum Se or SePP in the absence of other challenges do not necessarily interfere with regular functioning of the TH axis. 5'-deiodinase isozymes are preferentially supplied, and Se-dependent enzymes in the thyroid are even less-dependent on serum levels of Se or SePP than in brain. This indicates a top priority of the thyroid gland and its selenoenzymes with respect to the hierarchical Se supply within the organism.
Introduction
THE HUMAN THYROID gland is known to be among the organs with highest content of the trace element selenium (Se) (1, 2). Se is an essential micronutrient in the diet of mammals (3, 4). Se deficiency is associated with the predisposition to certain conditions including cancer, atherosclerosis, arthritis, male infertility, immunological defects, and diseases of accelerated aging (5, 6). Accordingly, Se supplementation holds great promises for chemoprevention (7) and in improving the immune system in which it turned out to be beneficial in reducing antibody load, e.g. in autoimmune thyroiditis (8, 9). Se is considered to exert its biological activity mainly via selenoproteins, which contain Se in their active centers as part of the 21st proteinogenic amino acid selenocysteine (Sec) (10).
Sec is incorporated cotranslationally into at least 25 or 24 different gene products in humans and rodents, respectively (11). Most of the Sec-containing proteins that have been functionally characterized are enzymes that catalyze redox-reactions including members of the family of glutathione peroxidases (GPx), thioredoxin reductases or iodothyronine deiodinases (Dio) (12). Interestingly, there is a certain hierarchy among different selenoproteins with respect to Se supply and resynthesis after Se depletion, i.e. important selenoproteins like Dio1 are preferentially supplied when the trace element is limiting, whereas others like GPx1 rank low and are dependent on high Se supply (13, 14).
A second hierarchy in Se biology is observed with respect to Se in different tissues. Endocrine organs and the brain are preferentially supplied, whereas serum, liver, kidney, or muscles readily lose their Se stores on dietary Se deprivation. Especially the thyroid gland retains the trace element very efficiently (15). It synthesizes a variety of Sec-containing enzymes including GPx and thioredoxin reductases (16, 17) and the Dio isozymes that control thyroid hormone (TH) metabolism (18, 19). Se deficiency is thus believed to compromise the TH system by two important routes, i.e. the antioxidative defense capacity of thyrocytes is compromised and TH metabolism is disturbed both within the gland and systemically (18, 19).
Recently transgenic mice that lack the main Se-containing plasma protein, i.e. selenoprotein P (SePP), have been established (20, 21). SePP serves as a plasma Se carrier and nicely mirrors the overall Se status of an individual (22, 23). Accordingly, SePP-knockout (SePP-KO) mice display reduced Se levels in the tissues. Se concentrations are reduced to 22% of control in serum, 64% in kidney, 25% in brain, and 47% in testis, whereas liver displays an increased Se concentration of 1.5-fold above control (21). The phenotypes and the reduced tissue Se concentrations can be rescued by suitable Se supplementation regimen (20, 24).
Because these mice present with growth deficits and neurological phenotypes including ataxia, we speculated that a Se-dependent derangement of the TH axis could underlie the observed pathologies. Surprisingly, expression of Se-dependent enzymes in thyroid, serum levels of TSH and TH, and T3-dependent transcriptional and developmental processes turned out to be undisturbed. These findings position the thyroid gland and TH metabolism upmost in the hierarchy of Se-dependent tissues and Se-dependent reactions.
Materials and Methods
Materials
All chemicals were of analytical grade and obtained from Sigma (München, Germany) or Merck (Darmstadt, Germany). [32P]dCTP was from Hartmann Analytic (Braunschweig, Germany) and [125I]rT3 was from NEN Life Science Products DuPont/PerkinElmer (Koeln, Germany). Oligonucleotides were from MWG Biotech (Ebersberg, Germany), restriction endonucleases were from New England Biolabs (Frankfurt, Germany), and Taq polymerases were from QIAGEN (Hilden, Germany). A TA-cloning system was purchased from Promega (Mannheim, Germany), and DNA sequencing was accomplished by a DyeTerminator cycle sequencing kit (Applied Biosystems, Weiterstadt, Germany).
Sample collection and preparation of tissue homogenates
Mice were raised on regular lab chow (Altromin, Lage, Germany) containing on average 0.24 ppm Se. A 12-h light, 12-h dark cycle was controlled automatically. SePP mice used had been backcrossed for four to seven generations onto C57BL/6CR strain. SePP-KO mice were generated by mating heterozygous SePP (±) mice, and genotyping was performed with DNA from ear clippings as described earlier (21). At the age of 35 or 60 d, animals were deeply anesthetized with carbon dioxide, the chest was opened, and blood was drawn from the heart or they were perfusion fixed for histological analyses. After storage at 4 C for 1 h, blood samples were centrifuged (4 C; 10,000 x g; 10 min) and serum was collected. Tissues were removed, frozen in liquid nitrogen, and stored at –80 C until analysis. Animal experimentations were approved by the animal welfare committee of the local governmental authorities in Berlin, Germany. For enzyme activity assays and Se determinations, the tissues were pulverized under liquid nitrogen using a dismembrator (Braun, Melsungen, Germany). Aliquots were homogenized on ice in approximately 5 volumes of 20 mM HEPES (pH 7.0), containing 250 mM sucrose and 1 mM EDTA using a glass/Teflon homogenizer. The homogenates were centrifuged at 10,000 x g for 20 min, the supernatants were collected, and pellets were resuspended in homogenization buffer supplemented with 1 mM dithiothreitol. Protein concentrations were determined by a modified Bradford assay using IgG as standard (Bio-Rad, München, Germany).
Fluorometric Se determination
The Se concentrations of the thyroid glands were determined from the supernatants of the tissue homogenates. The method used was essentially as described before (21) with minor modifications. Due to the limited starting material available, all components of the assay were halved, i.e. volumes of only 50 μl homogenate or standard were digested by only 250 μl HNO3/HClO4 (4:1,vol/vol), reduced by 250 μl HCl, complexed by 1 ml of EDTA (2.5 mM), supplemented with 250 μl 2,3-diaminonaphtalene in 0.1 M HCl, extracted with 0.5 ml cyclohexane, and the fluorescent piazselenols formed were quantified as described (21). A commercially available pooled human serum standard (Sero AS, Billingstad, Norway) along with a Se atomic absorption standard solution (1000 μg/ml; Sigma) was used to validate the method.
Dio assay
The resuspended pellet fractions were assayed for Dio activities in a reaction mixture containing 20 μg (liver and kidney), 100 μg (brain), or 5 μg (thyroid) protein (25). Dithiothreitol (10 mM) served as cofactor and [125I]rT3 was added as tracer (50,000 cpm/tube, specific activity: 25 TBq/mmol, PerkinElmer). The substrate concentration was 1.0 μM rT3 (liver), 500 nM rT3 (kidney), 200 nM rT3 (thyroid), or 1 nM rT3 (brain). Reaction volume was 100 μl, the reaction proceeded at 37 C for 1 or 2 h, and the determinations were performed in triplicates. Conditions had been optimized such that final substrate deiodination was less than 15%. Dio1 and Dio2 activities were distinguished by the inclusion of 1 mM 6-n-propyl-2-thiouracil in the incubation mixture. The fraction of iodide release blocked by 6-n-propyl-2-thiouracil was assigned to Dio1, residual background-corrected activity to Dio2. [125I]rT3 was purified from free [125I] by chromatography using Sephadex LH-20 (Sigma-Aldrich, Muenchen, Germany) before use.
TSH, T4, and T3 determination
Serum was obtained as described above and stored at –80 C until analysis.
TSH determination.
Guinea pig antimouse TSH antibody (AFP98991) and a crude TSH/LH reference preparation (AFP51718MP) were obtained from Dr. A. F. Parlow (National Hormone and Peptide Program, Harbor-UCLA Medical Center, Los Angeles, CA). Commercially available 125I-labeled rat TSH (RPA 554) was obtained from Amersham Biosciences (Freiburg, Germany). Normal guinea pig IgG as carrier and goat antiguinea pig IgG as precipitating antibody were provided by Linco Research (St. Charles, MO; references 3020 and 2020, respectively). A calibration standard at a nominal concentration of 10,000 ng/ml was prepared in PBS/BSA [2% (wt/vol) BSA in PBS] and progressively diluted in PBS/BSA to give rise to nine standard levels ranging from 19 to 5000 ng/ml. The samples (50 μl serum sample or standard diluted with 50 μl PBS) were preincubated overnight at room temperature with 100 μl guinea pig antimouse TSH antibody (1/150,000). Then 100 μl 125I-labeled rat TSH was added and incubated for 4 h at room temperature. Afterward normal guinea pig IgG carrier (100 μl) and the precipitating goat antiguinea pig IgG antibody (100 μl) were added and incubated for 2 h at 4 C. The RIA tubes were centrifuged at 3000 x g for 30 min at 4 C. After aspiration of the supernatant, the precipitated radioactivity in each tube was measured using a PerkinElmer Wizard -counter. A standard curve was obtained by plotting the fraction of precipitated 125I-TSH over total 125I-TSH (B/B0) against the standard nominal concentrations. Intra- and interassay coefficients of variation were 4% at 137.5 ± 5.4 ng/ml and 7.4% at 124.5 ± 9.2 ng/ml, respectively.
Total T4 (TT4) determination.
A competitive RIA was performed (RIA kit CA-1535M; DiaSorin, Rome, Italy). Ten microliters of each sample (serum or standard) were incubated for 45 min with 1 ml of tracer-buffer reagent in a mouse monoclonal anti-T4-coated tube at room temperature. Tracer-buffer reagent was obtained by adding 10 ml of [125I]T4 to 10 ml of Tris-buffered saline, 4 mM 8-anilino-1-naphthalene sulfonic acid (ANS), 6 mM sodium salicylate, 0.2% sodium azide, and 90 ml distilled water. Then all tubes were aspirated except for total count tubes, and the radioactivity in each tube was measured for 1 min using a PerkinElmer Wizard -counter. Standard concentrations of T4 ranged from 1 to 20 μg/dl. Intraassay coefficients of variation were 6.4% at 2.64 μg/dl and 4.8% at 6.85 μg/dl. Interassay coefficients of variation were 11.2% at 2.85 μg/dl and 5.2% at 6.41 μg/dl.
Total T3 (TT3) determination.
A similar competitive RIA was performed (DiaSorin RIA kit CA-1541) that also relied on the use of ANS to displace T3 from serum binding proteins (26). For this technique, 50 μl of each sample (serum or standard) were incubated for 1 h with 1 ml of tracer-buffer reagent in an anti-T3-coated tube at 37 C. The tracer-buffer reagent was prepared as above with [125I]T3 in PBS containing ANS and sodium azide. Standard concentrations of T3 ranged from 0.5 to 8 ng/ml. Intraassay coefficients of variation were 7.9% at 0.89 ng/ml and 3.6% at 1.69 ng/ml. Interassay coefficients of variation were 8.3% at 0.84 ng/ml and 4.8% at 1.68 ng/ml.
Histological analysis
Wild-type and age-matched homozygous SePP-KO mice were deeply anesthetized and perfusion fixed transcardially with 0.1 M Na-phosphate buffer (pH 7.4), followed by 4% paraformaldehyde in the same buffer. Thyroid glands were excised together with the trachea, postfixed overnight in the same fixative, dehydrated, and embedded in paraffin. Sections of 2 μm were cut on a rotating microtome (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin by standard procedures. Tissue sections were examined under a light microscope (Axioskop, Zeiss, Oberkochen, Germany), and pictures were taken using a digital camera (ActionCam, Agfa, Koeln, Germany). Cerebelli were sectioned at 15 μm in the sagittal plane. Neuronal cell bodies were revealed by cresylviolett staining. Immunostainings against calbindin (polyclonal; Swant, Bellinzona, Switzerland), parvalbumin (polyclonal; Swant), calretinin (monoclonal, Swant), and NeuN (monoclonal, Chemicon, Temecula, CA) were revealed using Vector reagents (Vector Laboratories, Burlingame, CA) and NovaRed peroxidase substrate according to the manufacturer’s recommendations.
Northern blot analysis
Total RNA was isolated from the tissues by the use of peqGOLD TriFast (PEQLAB, Erlangen, Germany) according to the instructions of the manufacturer. Aliquots were size fractionated in denaturing formaldehyde/agarose gels, capillary transferred to nylon membranes (Nytran NY 12 N; Schleicher & Schuell, Dassel, Germany) and analyzed under high-stringency conditions as described earlier (27). For pituitary RNA isolation, single mouse pituitaries were directly homogenized in 200 μl peqGOLD TriFast and isopropanol precipitations were performed after adding 1 μg yeast tRNA as carrier. cDNA fragments encompassing the open reading frames of mouse SePP, TSH, Dio1, -actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified by PCR, subcloned, sequenced, and randomly labeled with 32P-dCTP (Hartmann Analytic, Braunschweig, Germany) to high specific activities (>109 cpm/μg). After hybridization and extensive washings at 62 C, Northern results were quantified by analysis with a phosphor imager (Cyclone storage phosphor system, Packard BioScience, Dreieich, Germany). The signals obtained for GAPDH mRNA were used to calculate relative expression levels. For analysis of thyroid RNA preparations, -actin was used as standard because it turned out to mirror relative loading amounts more faithfully than GAPDH.
Statistical analysis
Values are reported as mean ± SE. P values were calculated by one-way ANOVA followed by Bonferroni’s post hoc test. Statistical significance was defined as P < 0.05 (), P < 0.01 (), or P < 0.001 (). Data of serum TSH and TH levels were analyzed by the Kruskal-Wallis test.
Results
Preserved expression of Dio1 and -2 in SePP-KO mice
The activation of T4 to biologically active T3 is catalyzed by Se-dependent Dio1 expressed mainly in liver, kidney, pituitary, and thyroid gland or Dio2 mainly expressed in the central nervous system, brown adipose tissue, and placenta (18, 19). When raised on regular lab chow, SePP-KO mice display drastically reduced serum Se levels and plasma GPx (GPx3) activities (24). Despite those strong Se-dependent effects, transcript and enzyme levels of Dio1 were only marginally altered in liver or kidney of SePP-KO mice at postnatal d (P) 60 (Fig. 1). The small differences observed appear to follow tissue Se concentrations that are increased in livers of SePP-KO mice and decreased in their kidneys.
These findings are in contrast to our initial results from a small fraction of male SePP-KO mice that we had to kill after weaning at P35 because of poor health status. These animals displayed strongest Se-dependent phenotypes and expressed sharply reduced hepatic and renal Dio1 transcript levels and enzymatic activities (to 20–30% control levels). No such effects were observed in age-matched, apparently healthy female SePP-KO littermates or mice that were analyzed at later time points (not shown).
Unaltered serum levels of TH and TSH
Pituitary synthesis and secretion of TSH as part of the regulatory TH feedback system is known to be most sensitive to changes in T3 concentration (27). Comparing wild-type, heterozygous, and SePP-KO mice, serum levels of TSH, TT3, or TT4 were comparable and did not show SePP-dependent differences (Fig. 2). The values obtained were within a narrow range and well in agreement with reports from other laboratories working with C57BL/6 mice (28). Nevertheless, significant sex-specific differences were observed between TSH levels of male and female mice independent of the SePP genotype involved (Fig. 2).
Regular TH target gene expression
The TSH-specific -subunit is stringently controlled by T3 at the transcriptional level and via its poly-A tract length that controls half-life of the transcripts (27, 29). TSH mRNA from pituitaries of SePP-KO mice was normal in quantity and length (Fig. 3A). Upon quantification, TSH mRNA levels from heterozygous mice seemed to be decreased significantly, compared with wild-type mice, and marginally, compared with SePP-KO littermates, but this effect was rather attributable to slightly increased GAPDH mRNA levels in the pituitaries of these mice. Hepatic Dio1 has been characterized as a most sensitive marker of T3 plasma levels (30). Comparing heterozygous and homozygous SePP-KO mice with wild-type littermates, no differences in Dio1 mRNA levels were observed in livers at P60 (Fig. 3B).
Normal thyroid gland morphology, thyroid Se concentration, and thyroid GPx activity
The thyroid gland expresses Se-dependent enzymes involved in TH metabolism and antioxidative defense. Severe Se deficiency could compromise these functions, leading to tissue destruction, malfunction, or goitrous hyperplasia. Visual inspection of SePP-KO mice did not reveal any abnormalities of the neck region or gland appearance at P35 or P60. Upon necroscopy of male and female thyroid glands, no differences in size, color, or texture were noted. Single thyroid glands were processed for histology, enzyme activity or Northern blot analysis at P60. The follicle cells and cavities looked essentially normal in size, number, and location (Fig. 4A). Thyroid GPx1 mRNA levels as most sensitive markers of intracellular Se levels and Se availability were at wild-type levels (Fig. 4B). Likewise, no differences of total Se concentrations were observed in the homogenates of thyroid tissues from wild-type, heterozygous, or SePP-KO mice (Fig. 4B). GPx activity in thyroids representing mainly GPx1 and GPx3 enzyme levels was also found unaffected by the SePP genotype (Fig. 4C). This is in contrast to other tissues from SePP-KO mice analyzed to date including serum, kidney, testes, or even brain (20, 21). In contrast, Dio1 activity was significantly increased in thyroid glands from heterozygous SePP-KO mice. This finding is in agreement with earlier reports from Se-deprived animals (31). Because heterozygous SePP-KO mice are in general without phenotype despite gradually reduced serum and tissue levels of SePP and Se, the increased Dio1 activity might represent a thyroid-specific physiological response to moderate Se deficiency by a SePP-independent mechanism that helps to maintain normal serum TH levels.
Regular TH-dependent cerebellar development and Dio2 activity in brain
Perinatal hypothyroidism induces a number of anatomical alterations in the rodent brain including abnormal cell migration, dendritic arborization, or defects of synaptogenesis (32, 33). It is well established that especially cerebellar granule cell migration during postnatal brain development critically depends on TH by TR1-dependent mechanisms (34) and that it is delayed in hypothyroidism (35). When we did a respective histological analysis of cerebellar specimen from SePP-KO mice and wild-type littermates, no indications for a migration defect that would point toward local hypothyroidism were found (Fig. 5A). Cerebellar architecture appeared normal, and a well-developed Purkinje cell layer with highly elaborated dendritic trees was observed after calbindin staining. Expression patterns of other marker proteins for the major cerebellar cell types like parvalbumin (Purkinje, basket, and stellar cells), NeuN, or calretinin (granule cells) were unchanged despite the compromised SePP expression and reduced brain Se levels (not shown). Likewise, enzymatic activity of Dio2 in brain of SePP-KO mice was at wild-type levels (Fig. 5B) despite those strongly reduced Se concentrations or GPx activities in brain at P35 and those concomitantly developing neurological defects (21, 36).
Thyroid gland Se metabolism is exceptionally independent from SePP
SePP-KO mice display reduced Se levels in serum, kidney, testes, or brain but not in liver because hepatocytes normally synthesize and secrete SePP for systemic supply and thus accumulate Se in the absence of SePP (21). Whereas Se levels in serum and kidney are dependent on hepatically derived SePP, brain Se is maintained via local SePP synthesis and Se recycling mechanisms within the tissue, which render the central nervous system seemingly independent from serum Se and SePP levels (23). Despite the strong neurological and growth phenotypes of SePP-KO mice that resemble in part those defects that are known for TH deficiency, no alterations of T4, T3, or TSH serum levels were observed; thyroid gland morphology and T3-dependent developmental and regulatory processes were normal. Thus, the thyroid gland was able to accumulate, retain, and recycle Se efficiently, even in the absence of SePP, and to adjust its Dio and GPx expression adequately, even in Se deficiency. This exceptional feature seems to rank the thyroid gland even higher than brain in the hierarchy of tissues with respect to Se supply (Fig. 6).
Discussion
The thyroid gland is essential for mammalian life and depends on two important trace elements for the synthesis and metabolism of TH, i.e. Se and iodine (17, 18). Whereas iodine is needed directly as substrate for biosynthesis of T4 and T3, the importance of Se for the TH axis is less well appreciated. The characterization of GPx as well as the TH metabolizing Dio isozymes as selenoproteins provided the molecular basis that linked Se biology with thyroid gland function and more general aspects of health and disease (6). The results presented here demonstrate that the Dio isozymes are preferentially supplied with Se and that the thyroid gland unexpectedly retained its GPx activity exceptionally well in SePP-deficient mice. Thus, the thyroid gland remains equipped with an effective defense system against the tone of reactive oxygen species even in the absence of SePP. Adequate metabolism of TH by the Dio isozymes is likewise maintained with priority. This preferential supply of Dio1 vs., for example, GPx1 has been observed before in vivo and in cell culture models and is an example for the still enigmatic hierarchy among different selenoproteins (14, 31).
Yet our initial analysis of Dio1 expression in SePP-KO mice yielded conflicting results. We had to kill some of the male animals at P35 because they suffered from acute weight loss and bad overall health status including recurrent seizures. Surprisingly, Dio1 expression was drastically reduced in their livers and kidneys, compared with wild-type or heterozygous littermates. This finding was obviously not in line with the hierarchical expression of selenoproteins. But when we later analyzed male or female SePP-KO mice that survived this critical phase after weaning, expression of Dio1 was again found well preserved as in control littermates. Thus, our initial finding of reduced Dio1 expression might be related to the still enigmatic low-T3 syndrome observed in late stages of critical illness or cachectic conditions and rather represents only an indirect consequence of SePP deficiency.
Nevertheless, both male and female SePP-KO mice display strong neurological phenotypes that we expected to be TH dependent, i.e. atactic movements and sporadic seizures. These phenotypes were never observed before in pure dietary depletion models, even after nutritional Se deprivation over many generations (37). Supplementation studies revealed that these phenotypes of SePP-KO mice correlated with reduced brain Se levels and GPx activities and that they could be prevented by Se-enriched diets (20, 24). We demonstrate here, that even without Se supplementation, Dio2 expression in brain of SePP-KO mice is normal and not decreased due to Se deficiency or elevated as in hypothyroid mice (38). In addition, postnatal cerebellar granule cell migration as a sensitive indicator of local T3 activity during development (39) is undisturbed. Thus, the underlying cause for the observed central defects is currently still not understood, but other mechanisms than gross TH derangements in the brain must be considered.
A second hierarchy in Se biology refers to the graded capability of different tissues to retain Se when the trace element is limiting or to accumulate Se on replenishment (15). Here the brain and the endocrine tissues including the thyroid and pituitary glands turned out to be highest ranking (15, 40). These features seem to guarantee that a moderate or transient Se deficiency elicited by insufficient nutritional supply or impaired uptake does not cause adverse health effects. Accordingly, many studies have revealed that nutritional Se deprivations in adult rodents or even during development cause only moderate effects on the TH axis (41, 42, 43, 44, 45). The same is true for second-generation Se-deficient rats (46), experimental Se-deficient sheep (47), or humans with different eating habits that cause drastically different Se plasma levels (48). These mild effects are in sharp contrast to potentially devastating consequences of a combined Se and iodine deficiency as in myxedematous cretinism (49). Here the phenotype might even aggravate further by, for example, thiocyanate overload due to cassava root consumption as found in endemic goitre areas in central Africa (12). Currently we cannot predict the importance of SePP in such pathological settings. Our findings indicate that SePP-KO mice can have a regular functioning thyroid gland and TH feedback system per se if not additionally provoked and their phenotypes are not caused by hypothyroidism. But this does not exclude that exceptional challenges that stimulate TH synthesis and activate thyroid cells beyond a certain degree cause overwhelming defects. Probably high age, low iodine diet, or mild goitrogen treatment might suffice to cause excessive symptoms in SePP-KO mice due to impaired adaptive capacity of the thyroid glands. Such mechanisms might have accelerated the severe illness that we observed with those moribund male SePP-KO mice shortly after weaning. Albeit, the underlying defect, molecular mechanisms, and gender-specific aspect of the severe health conditions still need to be analyzed further.
We observed before that the brain was dependent on SePP for normal GPx expression and undisturbed privileged Se metabolism (21). The tissue-specific deletion of hepatic selenoprotein synthesis including SePP by genetic inactivation of the Sec-specific tRNA resulted in reduced serum and kidney Se levels and GPx activities but did not impact negatively on brain Se or brain GPx expression (23, 50). Thus, serum and kidney were dependent on hepatically derived SePP, but brain needed mainly its locally synthesized SePP pool for efficient Se accumulation and retention against an adverse plasma gradient (51, 52). In contrast, the normal functioning of the thyroid gland in SePP-KO mice indicates that it was able to accumulate and retain Se in the form of selenoenzymes, even without SePP. It depended on neither high Se or SePP plasma levels nor locally produced and retained SePP (23). These findings indicate an evenly graded hierarchy among the preferentially supplied tissues with the thyroid gland ranking more independent and thus higher than the central nervous system.
The mutual interdependence of a functional TH feedback system with undisturbed selenoprotein biosynthesis was just described in humans with inherited mutations in the gene encoding selenocysteine insertion sequence-binding protein 2, a critical factor for selenoprotein translation. Here the synthesis of all selenoproteins analyzed was hampered, and the main clinical condition observed was a disturbed TH feedback system most likely because of reduced expression of the Dio isozymes (53). Together with the findings presented here, one can assume that evolution has optimized the hierarchical principles in Se biology primarily to sustain thyroid gland functioning and adequate TH metabolism, even during transient, dietary, or illness-related shortages of Se.
Acknowledgments
We are grateful to Katja Schreiber, Silke Kappler, Anita Kinne, Vartiter Seher, Birgit Hollenbach, and Metin Yenilmaz for excellent technical assistance. Special thanks go to our colleagues in the animal facility for their support. We also thank Peter Hofmann, Inka Hamann, Cornelia Schmutzler, Birgit Mentrup, and Branislaw Radovic for stimulating discussions; Dr. A. F. Parlow for the generous supply of TH-specific reagents; and the Deutsche Krebshilfe and the Deutsche Forschungsgemeinschaft for financial support.
Footnotes
This work was supported in part by the Deutsche Krebshilfe (Grant 10-1792 SchoI) and the Deutsche Forschungsgemeinschaft (Grant SCHO 849/1-1 and Grant KO 922/8-3).
The authors have nothing to declare.
First Published Online December 1, 2005
Abbreviations: ANS, 8-Anilino-1-naphthalene sulfonic acid; Dio, 5'-deiodinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPx, glutathione peroxidase; P, postnatal day; Se, selenium; Sec, selenocysteine; SePP, selenoprotein P; SePP-KO, SePP-knockout; TH, thyroid hormone(s); TT3, total T3; TT4, total T4.
Accepted for publication November 18, 2005.
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