Abstract
Background: The Allan-Herndon-Dudley syndrome is a severe psychomotor retardation accompanied by specific changes in circulating thyroid hormone levels (high T<sub>3</sub>, low T<sub>4</sub>). These are caused by mutations in the thyroid hormone transmembrane transport protein monocarboxylate transporter 8 (MCT8). Objective: To test the hypothesis that circulating low T<sub>4</sub> and high T<sub>3</sub> levels are caused by enhanced conversion of T<sub>4</sub> via increased activity of hepatic type I deiodinase (Dio1). Methods: We crossed mice deficient in Mct8 with mice lacking Dio1 activity in hepatocytes. Translation of the selenoenzyme Dio1 was abrogated by hepatocyte-specific inactivation of selenoprotein biosynthesis. Results: Inactivation of Dio1 activity in the livers of global Mct8-deficient mice does not restore normal circulating thyroid hormone levels. Conclusions: Our data suggest that although hepatic Dio1 activity is increased in Mct8-deficient mice, it does not cause the observed abnormal circulating thyroid hormone levels. Since global inactivation of Dio1 in Mct8-deficient mice does normalize circulating thyroid hormone levels, the underlying mechanism and relevant tissues involved remain to be elucidated.
Introduction
The monocarboxylate transporter 8 (MCT8) is the most specific thyroid hormone (TH) transmembrane transporter that is currently known. Mutations in MCT8 lead to a severe form of psychomotor retardation, the Allan-Herndon-Dudley syndrome [1]. Patients present with neurological symptoms including severe hypotonia, lack of speech and poor mental development. Specific endocrine abnormalities in circulating TH levels (low T4, high T3) in the face of normal-to-elevated thyroid-stimulating hormone levels paved the way to the discovery of underlying mutations in MCT8 in these patients [2,3]. Mouse models for Mct8 deficiency have been generated and replicate the endocrine phenotype seen in humans [4,5,6]. Low circulating T4 and high circulating T3 levels lead to the manifestation of local hypo- or hyperthyroidism in different organs and tissues depending on the presence of other TH transmembrane transporters. Tissues like liver [4,5], muscle [7] and kidney [8] are reportedly in a hyperthyroid state in Mct8 deficiency evaluated by deiodinase activities, while the brain shows signs of hypothyroidism measured by reduced uptake of T3 into the brain and increased deiodinase 2 activity [4,5] or mixed hypo- and hyperthyroid changes assessed by behavioral analysis [9].
To date, it is unclear what causes the low circulating T4 and high T3 concentrations. Several explanations have been suggested. Mct8-deficient mice demonstrate enhanced uptake and clearance of TH via the kidney possibly leading to a reduction of T4 and T3 in serum [8]. TH also accumulate in Mct8-deficient thyroid glands and are secreted at a slower rate upon thyroid-stimulating hormone stimulation [10,11]. However, these findings do not seem to account for the low T4 and elevated T3 serum levels since they originate from enhanced metabolism of TH and not from increased loss in the kidney or reduced secretion from the thyroid gland.
In 2011, Liao et al. [12] determined the consequences of combined Mct8 and Dio1 and/or Dio2 deficiency on the hypothalamus-pituitary-thyroid axis. They nicely demonstrated that the global deletion of Dio1 in Mct8-deficient animals leads to a nearly complete normalization of circulating T4 and T3, as well as thyroid-stimulating hormone levels. It was therefore concluded that increased conversion of T4 into T3 by Dio1 is responsible for the elevated circulating T3 and reduced T4 levels in Mct8 deficiency. To directly test this hypothesis, we made use of our previously described hepatocyte-specific selenoprotein-deficient mice (Alb-Cre;Trspfl/fl) that are devoid of deiodinase activity in hepatocytes [13]. Our model revealed that deletion of Dio1 activity in livers of Mct8-deficient mice has no major impact on circulating TH levels and is therefore not the underlying cause for the observed low T4 and high T3 serum levels in Mct8 deficiency.
Material and Methods
Animals
All animal experiments were approved by the local authorities in Berlin, Germany, and have been performed according to local regulations at the Charité-Universitätsmedizin Berlin (Germany). Alb-Cre;Trspfl/fl as well as Mct8-deficient mice have been described before [9,13]. The data presented in this paper were generated using only male mice with the genotypes wild type (wt), Mct8-/y, Alb-Cre;Trspfl/fl and Alb-Cre;Trspfl/fl;Mct8-/y. Mating was set up in a way to obtain animals of all genotypes as littermates.
Type I Deiodinase Assay
Activities of the type I deiodinase (Dio1) were determined in triplicate in liver homogenates (40 µg protein/ml) based on an iodide-release protocol [14] with slight modifications. Liver homogenates were incubated at 37°C for 60 min with 20 mM 1,4-dithiothreitol as cosubstrate, 0.3 µM nonradiolabeled rT3 and 125I-radiolabeled rT3 (PerkinElmer, Hamburg, Germany; 0.82 µCi/pmol) in the absence or presence of 1 mM propylthiouracil (PTU). The reaction was stopped by adding cold 10% BSA, and 0.01 mM PTU and proteins were precipitated by adding 3 volumes of cold 10% trichloroacetic acid. Samples were centrifuged and the supernatant was eluted over a Dowex-50 WX-2 column. The 125I in the eluate was counted using a gamma counter (1277 GammaMaster; LKB Wallac, Turku, Finland). The absence or presence of H2O or PTU in the reaction mixture differentiated between Dio1 activity and total deiodinase activity as the fraction of 125I release blocked by PTU was assigned to Dio1.
TH Assay
Total T4 and T3 levels were measured by competitive radioimmunoassays from DRG Instruments (Marburg, Germany). Samples and calibrators for a standard curve were incubated with 125I-T4 or 125I-T3 as a tracer in antibody-coated tubes for 1 h. Bound radioactivity was determined in a gamma counter (1277 GammaMaster; LKB Wallac, Turku, Finland).
Results
Inactivation of Hepatocyte-Specific Deiodinase Activity in Mct8-Deficient Mice
At present, a mouse model for the conditional inactivation of the Dio1 gene is not available. We therefore took advantage of the fact that deiodinases are selenoenzymes, i.e. enzymes carrying the rare amino acid selenocysteine (Sec). Incorporation of Sec depends on tRNA(Sec), which is encoded by the gene Trsp, of which a mouse model for the conditional inactivation is available. We have previously reported that hepatocyte-specific inactivation of selenoprotein translation abrogated hepatic deiodinase activity in Alb-Cre;Trspfl/fl mice [13]. Expression of all selenoproteins is quantitatively abolished in livers of Alb-Cre;Trspfl/fl mice [15]. Ablation of selenoprotein biosynthesis in hepatocytes does not lead to liver failure or other diseases [16,17]. Hence, we crossed global Mct8-deficient mice with our liver-specific deiodinase-deficient mice in order to test the hypothesis that hepatic deiodinase causes increased T4 to T3 conversion and subsequently low T4, high T3 serum levels in Mct8 deficiency [9,13]. All mice were apparently healthy, as body weight (bw) was not different between the wt, Mct8-/y and Alb-Cre;Trspfl/fl mice and our crossed mouse line. Only Alb-Cre;Trspfl/fl;Mct8-/y mice had a slightly reduced bw at the age of 2-3 months (fig. 1a). Heart weight did not differ between groups when normalized for bw, indicating no hypertrophic effect of TH on the heart in this age group (fig. 1b). As expected, Mct8-deficient mice (Mct8-/y) have higher Dio1 activity in the liver than their littermate controls (fig. 1c). Inactivation of deiodinases in control or Mct8-deficient mice reduced Dio1 activity to levels close to the detection limit (fig. 1c). Residual Dio1 activity most likely stems from the very low amount of Dio1 that is expressed outside of hepatocytes in the liver, possibly in Kupffer cells.
Inactivation of deiodinase activity in Mct8-deficient mice. a wt, Mct8-deficient (Mct8-/y) and hepatocyte-specific deiodinase-deficient (Alb-Cre;Trspfl/fl) mice have normal bw. Only double-knockout hepatocyte-specific deiodinase- and global Mct8-deficient (Alb-Cre;Trspfl/fl;Mct8-/y) mice showed a slightly lower bw compared to control littermates. b Heart weight normalized for bw did not differ between the analyzed groups. c Measuring liver Dio1 activity in all groups led to the expected increase in Mct8-deficient mice. Inactivation of deiodinase activity in wt or in Mct8-deficient livers resulted in the activity being nearly abolished. Data are presented as means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 (Mann-Whitney U test).
Citation: European Thyroid Journal 4, 1_Suppl; 10.1159/000381021
Effect of Hepatic Dio1 Deficiency in Mct8-Deficient Mice on Circulating TH
Since combined global inactivation of Dio1 and Mct8 led to the normalization of circulating TH levels, we measured circulating levels of total T4 and total T3 to see the impact of hepatic Dio1 inactivation on TH metabolism in global Mct8-deficient mice. Also in this combined mouse model, we can replicate the known endocrine phenotype of Mct8 deficiency with low T4 and high T3 levels in Mct8-/y as compared to littermate controls (fig. 2). Inactivation of hepatic deiodinase activity led to only marginally increased total T4 serum levels in Mct8-deficient mice. A slight increase in circulating T4 levels upon hepatic Dio1 inactivation has been described before and may be related to reduced inactivation of T4[18]. Loss of Dio1 activity in Mct8-deficient livers does also not lead to a normalization of circulating T3 levels (fig. 2). They remain as high in Alb-Cre;Trspfl/fl;Mct8-/y mice as in Mct8-/y mice.
Loss of deiodinase activity in Mct8-deficient livers does not normalize abnormal circulating TH levels. Total circulating T4 and T3 level were measured in wt, Mct8-/y, Alb-Cre;Trspfl/fl and Alb-Cre;Trspfl/fl;Mct8-/y mice. Loss of Mct8 led to the expected low T4 and increased T3 levels in serum. Inactivation of hepatic deiodinase only marginally increased the T4 and T3 levels. Alb-Cre; Trspfl/fl;Mct8-/y mice also displayed a minor increase in T4, while circulating T3 levels were not normalized compared to Mct8-deficient mice. Data are presented as means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 (Mann-Whitney U test).
Citation: European Thyroid Journal 4, 1_Suppl; 10.1159/000381021
Discussion
High circulating T3 concentrations in MCT8-deficient patients are considered to be responsible for increased energy expenditure and muscle wasting. At the same time, feeding the patients adequately is challenging, given their impaired motor capabilities, and weight loss often occurs. Serum TH constellations with high T3 and low T4 concentrations in MCT8-deficient patients are considered to be responsible for a variety of these peripheral phenotypes through hyperthyroid states in MCT8-independent tissues like skeletal muscle and liver. Lowering serum T3 may thus represent a therapeutic goal, but this is difficult to achieve in the presence of abnormally low T4 levels in the patients. Local conversion of T4 to T3 is the major source of cerebral T3. Therefore, treatments potentially lowering T4 are at risk of further reducing cerebral TH uptake and T3 availability. It is thus a pertinent question how these altered serum TH levels are caused.
Although a variety of data have been collected in mouse models of Mct8 deficiency, the mechanism for the manifestation of these altered serum TH levels is still unclear. Loss of TH through the kidney was proposed [8]. How increased total T3 could be maintained while T4 is selectively lost is difficult to envision at present. Reduced secretion of TH from the thyroid gland itself has also been proposed [10,11]. How the release of T4 could be lowered while at the same time T3 release would be increased from the thyroid gland is again not clear. Moreover, a patient with a mutation in MCT8 treated with levothyroxine after a complete thyroidectomy maintained the high T3, low T4 levels in serum [6]. Increased conversion of T4 to T3 by deiodinases is thus a possible explanation. Combined deletion of Mct8 and Dio1 in mice resulted in a normalization of serum TH parameters and subsequent improvement of brain T3 content [12]. In contrast, genetic inactivation of Dio2 in Mct8-deficient mice did not improve TH serum concentrations and, on the contrary, increased changes in brain gene expression.
These data suggested that peripheral conversion of T4 to T3 via Dio1 may establish the high T3, low T4 hormonal constellation in Mct8 deficiency. Since increased access of T3 to the liver does not depend on Mct8 and further stimulates Dio1 expression, hepatic Dio1-mediated conversion of T4 represented a plausible mechanism of establishing the abnormal TH levels outlined before. Nonetheless, our data presented here appear to refute this attractive hypothesis. Targeted inactivation of hepatic Dio1 activity neither normalized T3 nor T4 levels in Mct8- deficient mice. The mild increase in serum T4 levels in Alb-Cre;Trspfl/fl;Mct8-/y mice, which is also seen in Alb-Cre;Trspfl/fl mice, instead hints to reduced T4 degradation as in Dio1-/- mice because it does not alter T3 levels. Whether increased Dio1 activity in other organs like kidney or other mechanisms underlie the abnormal TH serum concentrations will be a matter of future studies. The genesis of the abnormal TH constellation in serum upon Mct8 deficiency still remains an open question.
Acknowledgements
The authors would like to thank Gabriele Böhm, Anja Fischbach and Antje Kretschmer for excellent technical assistance. Conditional Trsp knockout mice were kindly provided by Brad Carlson and Dr. Dolph Hatfield, Molecular Biology of Selenium, Basic Research Laboratory, NCI, NIH, Bethesda, Md., USA. Funding for this project has been provided by Deutsche Forschungsgemeinschaft (WI3768/1-1 and WI3768/2-1 Thyroid Trans Act).
Disclosure Statement
The authors declare no conflict of interest.
Footnotes
verified
References
- 1↑
Allan W, Herndon CN, Dudley FC: Some examples of the inheritance of mental deficiency: apparently sex-linked idiocy and microcephaly. Am J Ment Defic 1944;48:325-334.
- 2↑
Friesema EC, Grueters A, Biebermann H, Krude H, von Moers A, Reeser M, Barrett TG, Mancilla EE, Svensson J, Kester MH, Kuiper GG, Balkassmi S, Uitterlinden AG, Koehrle J, Rodien P, Halestrap AP, Visser TJ: Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation. Lancet 2004;364:1435-1437.
- 3↑
Dumitrescu AM, Liao XH, Best TB, Brockmann K, Refetoff S: A novel syndrome combining thyroid and neurological abnormalities is associated with mutations in a monocarboxylate transporter gene. Am J Hum Genet 2004;74:168-175.
- 4↑
Trajkovic M, Visser TJ, Mittag J, Horn S, Lukas J, Darras VM, Raivich G, Bauer K, Heuer H: Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8. J Clin Invest 2007;117:627-635.
- 5↑
Dumitrescu AM, Liao XH, Weiss RE, Millen K, Refetoff S: Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (mct) 8-deficient mice. Endocrinology 2006;147:4036-4043.
- 6↑
Wirth EK, Sheu SY, Chiu-Ugalde J, Sapin R, Klein MO, Mossbrugger I, Quintanilla-Martinez L, Hrabě de Angelis M, Krude H, Riebel T, Rothe K, Köhrle J, Schmid KW, Schweizer U, Grüters A: Monocarboxylate transporter 8 deficiency: altered thyroid morphology and persistent high triiodothyronine/thyroxine ratio after thyroidectomy. Eur J Endocrinol 2011;165:555-561.
- 7↑
Di Cosmo C, Liao XH, Ye H, Ferrara AM, Weiss RE, Refetoff S, Dumitrescu AM: Mct8-deficient mice have increased energy expenditure and reduced fat mass that is abrogated by normalization of serum T3 levels. Endocrinology 2013;154:4885-4895.
- 8↑
Trajkovic-Arsic M, Visser TJ, Darras VM, Friesema EC, Schlott B, Mittag J, Bauer K, Heuer H: Consequences of monocarboxylate transporter 8 deficiency for renal transport and metabolism of thyroid hormones in mice. Endocrinology 2010;151:802-809.
- 9↑
Wirth EK, Roth S, Blechschmidt C, Hölter SM, Becker L, Racz I, Zimmer A, Klopstock T, Gailus-Durner V, Fuchs H, Wurst W, Naumann T, Bräuer A, Hrabě de Angelis M, Köhrle J, Grüters A, Schweizer U: Neuronal 3′,3,5-triiodothyronine (T3) uptake and behavioral phenotype of mice deficient in Mct8, the neuronal T3 transporter mutated in Allan-Herndon-Dudley syndrome. J Neurosci 2009;29:9439-9449.
- 10↑
Trajkovic-Arsic M, Müller J, Darras VM, Groba C, Lee S, Weih D, Bauer K, Visser TJ, Heuer H: Impact of monocarboxylate transporter-8 deficiency on the hypothalamus- pituitary-thyroid axis in mice. Endocrinology 2010;151:5053-5062.
- 11↑
Di Cosmo C, Liao XH, Dumitrescu AM, Philp NJ, Weiss RE, Refetoff S: Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion. J Clin Invest 2010;120:3377-3388.
- 12↑
Liao XH, Di Cosmo C, Dumitrescu AM, Hernandez A, Van Sande J, St Germain DL, Weiss RE, Galton VA, Refetoff S: Distinct roles of deiodinases on the phenotype of Mct8 defect: a comparison of eight different mouse genotypes. Endocrinology 2011;152:1180-1191.
- 13↑
Streckfuss F, Hamann I, Schomburg L, Michaelis M, Sapin R, Klein MO, Köhrle J, Schweizer U: Hepatic deiodinase activity is dispensable for the maintenance of normal circulating thyroid hormone levels in mice. Biochem Biophys Res Commun 2005;337:739-745.
- 14↑
Leonard JL, Rosenberg IN: Iodothyronine 5′-deiodinase from rat kidney: substrate specificity and the 5′-deiodination of reverse triiodothyronine. Endocrinology 1980;107:1376-1383.
- 15↑
Seeher S, Atassi T, Mahdi Y, Carlson BA, Braun D, Wirth EK, Klein MO, Reix N, Miniard AC, Schomburg L, Hatfield DL, Driscoll DM, Schweizer U: Secisbp2 is essential for embryonic development and enhances selenoprotein expression. Antioxid Redox Signal 2014;21:835-849.
- 16↑
Schweizer U, Streckfuss F, Pelt P, Carlson BA, Hatfield DL, Köhrle J, Schomburg L: Hepatically derived selenoprotein P is a key factor for kidney but not for brain selenium supply. Biochem J 2005;386:221-226.
- 17↑
Sengupta A, Carlson BA, Weaver JA, Novoselov SV, Fomenko DE, Gladyshev VN, Hatfield DL: A functional link between housekeeping selenoproteins and phase II enzymes. Biochem J 2008;413:151-161.
- 18↑
Schneider MJ, Fiering SN, Thai B, Wu SY, St Germain E, Parlow AF, St Germain DL, Galton VA: Targeted disruption of the type 1 selenodeiodinase gene (Dio1) results in marked changes in thyroid hormone economy in mice. Endocrinology 2006;147:580-589.
