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Departments of Physiology, Microbiology, and Medicine (M.J.S., S.N.F., E.S.G., D.L.S.G., V.A.G.), Dartmouth Medical School, Lebanon, New Hampshire 03756; Nuclear Medicine and Medical Services (B.T., S.W.), Department of Veterans’ Affairs Medical Center, Long Beach, California 90822; and Harbor-UCLA Medical Center (A.F.P.), Torrance, California 90509
Address all correspondence and requests for reprints to: Valerie Anne Galton, Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756. E-mail: val.galton{at}dartmouth.edu.
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, the D1 can act on a broad range of iodothyronines. It can carry out with efficiency the 5'D of rT3 and 3,3'-diiodothyronine (3,3'-T2), and their sulfated derivatives (rT3S and 3,3'-T2S). It can also convert T4 to T3, although with a relatively low efficiency as judged by in vitro kinetic analysis. In addition, the sulfated derivatives of T4 and T3, but not the native hormones, can be efficiently deiodinated at the inner ring by the D1 (2, 3).
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Thus, the role of the D1 is incompletely understood. With its dual catalytic capability and its ability to deiodinate a broad range of iodothyronines, the D1 may have more than one function, and the complexity of its catalytic activity makes a complete delineation of its role in thyroid physiology difficult.
The present study uses a mouse rendered D1 deficient by targeted disruption of the Dio1 gene to explore directly the role of the D1 in thyroid hormone economy and to assess the function of the D1 in some of the major organs in which it is expressed.
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Materials and Methods |
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3.2 kb) with the neomycin/G-418 resistance cassette (Neo) (Fig. 2). To accomplish this, a Dio1 genomic clone, in the pBelo BAC II plasmid, was obtained from Incyte Genomics Inc. (St. Louis, MO). This clone was identified by screening an SvJ-129 mouse embryonic stem (ES)-cell library by PCR using an oligonucleotide primer pair capable of producing a 134-bp product from exon 2. Within the identified genomic clone, two tandem, 5' to 3' SacI fragments (10 and 9 kb in length, respectively) were identified and found to span the bulk of the Dio1 gene. The 9-kb fragment, which contained exons 3 and 4, was ligated to the right of Neo in pBluescript SK(–) (10), and an intronic 4.5-kb BamHI fragment, excised from the 10-kb SacI fragment, served as the left arm of the targeting construct. The structure of the completed construct was such that the orientation of Neo was opposite to that of the two Dio1 arms. The targeting vector, linearized with XhoI, was electroporated into the DS2A line of mouse ES cells, and positive selection for Neo-containing colonies was achieved by addition of G-418 to the culture medium.
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). This reaction was designed to generate an approximately 5.5-kb PCR product only if the template contained DNA in which homologous recombination had occurred. (As a validation of this screening approach, PCR was carried out successfully using wild-type (WT) ES cell DNA, the same sense primer, and an antisense primer based on the sequence in an equivalent position of the native gene.) The PCR products were separated by electrophoresis, and when a 5.5-kb band was detected, the eight samples of DNA in that pool were subjected individually to the same PCR procedure to determine in which clone(s) homologous recombination had occurred.
Three colonies were identified from a total of 392 and verified by Southern analysis of SacI-digested genomic DNA, using as the probe a 1.5-kb SacI/BamHI fragment (Fig. 2). The WT and targeted alleles were expected to produce a 10- and 7.7-kb band, respectively. ES cells from one colony were injected into blastocysts, which in turn were implanted into CD-1 pseudopregnant mice. The resulting chimeric male pups were crossed with C57BL/6 females for determination of germ line transmission of the mutated Dio1 allele in the F1 generation. All genotyping was accomplished by Southern analysis of tail tip DNA prepared using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). The founder males were then bred to the +/– F1 females, and the –/– D1 knockout (D1KO) and +/+ (WT) genotypes were identified in the offspring. Mice of the same genotype were then bred to generate and maintain colonies of WT and D1KO mice.
Animals
All mice were housed under conditions of controlled lighting and temperature in the barrier section of the Dartmouth Medical School animal research facility. In addition to the WT and D1KO mice generated in this study, D2-deficient (D2KO) mice from our established colony were employed in some experiments (10). Details of all WT and D1KO births, including litter size, birth abnormalities, and neonatal deaths were recorded. Mice were weighed at regular intervals. Some mice were made hypothyroid by placing them on drinking water containing 0.1% methimazole and 1% KClO4 (MMI/ClO4) for a minimum of 4 wk before study. Other mice were made hyperthyroid by injecting T3 (15 µg/100 g body weight, ip) daily for 15 d. In some experiments, mice were injected with either [125I]T4 (2 µg/100 g body weight) or [125I]T3 (1.5 µg/100 g body weight) 24–48 before euthanasia. [125I]T4 (specific activity, 969 Ci/mmol) and [125I]T3 (specific activity, 2200 Ci/mmol) were obtained from PerkinElmer Inc. (Norwalk, CT) and were purified by chromatography using Sephadex LH-20 (Sigma Chemical Co., St. Louis, MO) before use. All animal protocols were approved by the Institutional Review Board of Dartmouth Medical School.
Tissue preparation
The mice were euthanized with CO2, the abdomen was immediately opened, and blood was taken directly into a syringe from the inferior vena cava. In the case of neonates at postpartum d 10 (P10) and P15, trunk blood, obtained after decapitation, was pooled from two to three pups. The serum was aspirated after centrifugation and then stored at –20 C for subsequent assay. The following tissues were harvested: liver, kidney, heart, skin, thyroid, pituitary, and brain. The brain was rapidly sectioned into four parts for individual analysis: cerebellum, cerebral cortex, hypothalamus, and the remainder, which for this study is termed the midbrain. For determination of deiodinase activities, aliquots of liver, kidney, skin, and brain parts were homogenized immediately in ice-cold deiodinase buffer [0.25 mM sucrose, 20 mM Tris-HCl (pH 7.6) containing 5 mM dithiothreitol (DTT)] as previously described (12), to yield an approximately 1:5 homogenate (wt/vol). Pituitaries and thyroids were homogenized by hand in 0.5 ml of the same buffer using a ground glass homogenizer. The homogenates were centrifuged in the cold at 1000 x g for 15 min and the supernatants stored at –20 C for subsequent assay of 5'D and 5D activities. For other studies, the tissues were snap frozen on dry ice and stored at –20 C for future use.
Total RNA was isolated from liver using a commercial RNA isolation reagent (TRIzol solution; Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.
Determination of 5'D and 5D activities
5'D and 5D activities were assayed in homogenates of liver, kidney, skin, thyroid, brain, and pituitary according to our published methods (13, 14). Briefly, for 5'D activity, the reaction mixture (total volume, 50 µl) consisted of 2–100 µg tissue protein in deiodinase buffer containing 1.2 mM EDTA and 20 mM DTT. The substrate was 1.0 nM of either [125I]rT3 (specific activity, 546 Ci/mmol) or [125I]T4. Incubations were carried out for 1 h at either 37 or 0 C. The percent deiodination of substrate that occurred at 37 C was corrected for any nonenzymic deiodination by subtracting any deiodination that occurred during the same time period at 0 C. In determining 5'D activity, the percent iodide generated was multiplied by 2 because the substrates were labeled randomly in either the 3' or 5' positions. Thus, the specific activities of the labeled products were only half that of the substrate. In pituitary and brain, tissues that express both D1 and D2, the 5'D assays were carried out in the presence and absence of 1 mM PTU; at this concentration, PTU inhibits the activity of D1 but not that of D2. Pituitary and brain were also assayed for 5'D activity using [125I]T4 as substrate. T4 is the preferred substrate for D2, and at the 1.0 nM concentration employed in the assay, none of the 5'D activity was PTU sensitive, indicating that it was all attributable to the D2.
For determination of 5D activity, the reaction mixture (50 µl) did not contain EDTA, the substrate was 1.0 nM [125I]T3, and the cofactor was 50 mM DTT. Products were separated using paper chromatography (14). In both the 5D and the 5'D assays, protein concentrations were adjusted to ensure that deiodination was less than 20%. Protein concentrations of the liver, kidney, skin, and brain homogenates were determined according to the method of Comings and Tack (15), using BSA as the standard. Deiodination was expressed as picomoles or femtomoles of iodide or product generated per hour per milligram protein or, in the cases of pituitary and thyroid, per whole gland.
Assays for serum T4, T3, rT3, and TSH concentrations
Serum total T4 concentration was determined using the Coat-A-Count RIA total T4 kit (Diagnostic Systems Laboratories, Inc., Webster, TX) according to the manufacturer’s instructions. Tests with serum obtained from thyroidectomized mice indicated that there was no nonspecific effect of mouse serum in this T4 assay. The minimal detectable concentration of T4 in the assay was 0.25 µg/100 ml.
Serum total T3 concentration was determined using the nonequilibrium RIA assay procedure previously described (16), using a T3 antibody obtained from a commercial source (Fitzgerald Industries International, Inc., Concord, MA; catalog no. 20-TR45; cross-reactivity with T4 < 0.38%). Briefly, the RIA buffer consisted of 0.2 M glycine, 0.13 M sodium acetate (pH 8.6) containing 0.02% BSA, and 1% sodium salicylate. Serum (10 µl) was assayed directly, and an equivalent amount of thyroid hormone-depleted serum (17) was included in the standard curve. A combined polyethylene glycol/second-antibody separation step was employed. Assay sensitivity was approximately 2 pg/tube.
Serum rT3 concentration was measured by RIA using a method described previously (18). Serum samples were extracted with 3 vol of 63% ethanol before assay. The lower limit of detection was 5 ng/100 ml. Cross-reactivities were as follows: T4 less than 0.027%, T3 less than 0.001%, 3,3'-T2 less than 1.4%, and 3'-T1 less than 0.01%.
An index of the circulating levels of thyroid hormone carrier proteins was obtained by measuring the residual capacity of the serum to bind [125I]T3, using the Coat-A-Count RIA T3 uptake kit (Diagnostic Systems) according to the manufacturer’s instructions.
Mouse serum TSH levels were determined using a highly sensitive double-antibody method, developed by A. F. Parlow. The details of this assay have recently been published (10).
Assay for serum cholesterol concentration
The level of total cholesterol in serum was determined using the Infinity cholesterol reagent (Sigma Diagnostics, Inc., St. Louis, MO) according to the manufacturer’s instructions.
Analysis of mRNA levels by real-time PCR
Aliquots of RNA (90 µg) from each sample were adsorbed onto QIAamp columns contained in the QIAGEN (Valencia, CA) RNeasy mini kit, and subjected to DNase treatment with the QIAGEN RNase-free DNase set. Two micrograms of RNA from each column eluate were reverse transcribed to cDNA using SuperScript first-strand synthesis system for RT-PCR (Invitrogen). Real-time PCR was carried out using 0.33 µl of the resulting cDNA samples as template with the SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), according to the manufacturer’s protocol. Samples were run in duplicate. As a standard, cyclophilin cDNA (nine 5-fold dilutions from 1 ng to 2.56 fg) was coamplified in duplicate. The following primers used were: 5'-CCCCTGGTGTTGAACTTTG-3' and 5'-CTGTGGCGTGAGCTTCTTC-3' for D1, 5'-GTGTGCGATACCTCCAGAAG-3' and 5'GTTGTGTTGTCCGTCATAGTAG-3' for 
-glycerol phosphate dehydrogenase, 5'-GGCTTCCGTCTCCCTCTG-3' and 5'-CCTTGCTGCCTCGTGAAC-3' for Spot14, and 5'-GGTCCTCCTGCATCTCTTTG-3' and 5'-GGCTAGGCAGTATGGGATAAG-3' for glucose-6-phosphatase. The Bio-Rad (Hercules, CA) I-Cycler 9000 was programmed as follows: 95 C for a 6-min delay and then 40 cycles of 15 sec at 95 C, 15 sec at 60 C, and 30 sec at 72 C. The data generated by the I-Cycler software were expressed in terms of femtograms of DNA amplified per sample, where the amount of cyclophilin DNA was derived from a standard curve based on the nine known amounts of the standard entered into the cycler program. The data were then transferred to an Excel spreadsheet and the experimental values divided by that of the cyclophilin standard.
Determination of total T4 and T3 content of the thyroid gland
Thyroid glands were incubated in 300 µl Puregene cell lysis solution (Gentra, Minneapolis, MN) with 2 µl proteinase K solution (20 mg/ml) (Roche Diagnostics, Indianapolis, IN) at 55 C for 3 h. The hydrolysates were then mixed with 0.5 ml of 100% ethanol and held at room temperature for 10 min with intermittent vortexing. After centrifugation at 12,000 x g, the supernatants were removed to fresh tubes and the pellets extracted twice with 0.25 ml ethanol. The pooled supernatants were then evaporated to dryness in a Rotovac apparatus and the residue dissolved in 250 µl RIA buffer. The T4 content of the samples was determined using the Coat-A-Count RIA total T4 kit with the following modification: aliquots of sample (2.5 µl) were diluted to 25 µl, the required sample volume, using the calibrator A solution (no T4) provided for the standard curve. The T3 content in the samples was determined 1) using the Coat-A-Count RIA total T3 kit and 2) using the nonequilibrium T3 RIA. For the Coat-A-Count assay, 2.5-µl samples were diluted to the required volume (100 µl) with the calibrator A solution. For the nonequilibrium assay, the samples were diluted 1:20 in the RIA buffer and 2.5-µl diluted samples were assayed. Results obtained with both T3 assays correlated closely.
Studies of in vivo turnover of T4, T3, and their metabolic products
These studies were carried out in special cages designed to permit the separate collection of urine and feces. Care was taken to ensure that the mice had easy access to food and water, and they were provided with a small dark-colored igloo-shaped shelter to reduce their level of stress.
Three types of experiment were performed. In the first, WT and D1KO mice were injected ip with either [125I]T4 or [125I]T3, 2 and 1.5 µg/100 g body weight, respectively, and urine and feces were collected over the following 48 h. In the T4 study, a group of D2KO mice was included. The radioactive contents of the urine and feces were determined in a 
-counter (model 1195; Amersham Searle, Arlington Heights, IL). To determine the fraction of the radioactivity in the urine that was in the form of [125I]iodide, aliquots of urine were counted before and after passage through a column of Bio-Rad AG 50W-X8(H–), a resin that adsorbs iodothyronines. This procedure was also used with some samples of serum. Although the [125I]T4 and [125I]T3 were purified before use as described above, once diluted for injection, some [125I]iodide was always present (1–2%). To be sure that the urinary [125I]iodide reflected [125I]iodide generated from the injected hormone or its metabolites, the fraction of contaminating iodide in the injection solution was determined using a Bio-Rad AG 50W-X8(H–) column and subtracted from the total [125I]iodide excreted in the urine. At the end of the 48-h period, mice were euthanized and the thyroids removed and counted. No significant amount of 125I was taken up by the thyroid gland.
In the second type of experiment, urine and feces from untreated WT and D1KO mice were collected for a 24-h period and stored frozen for subsequent determination of T4, T3, rT3, and 3,3'-T2 contents. Samples of urine and feces were extracted with 3 and 16 vol of 63% ethanol, respectively, before assay as described previously (18). Using [125I]T4 or [125I]T3 as indicators, the recoveries of T4 and T3 were, respectively, 51.1 ± 0.7 and 54.9 ± 0.5%. There were no significant differences in the extraction efficiencies between the WT and D1KO mice. Twenty-five to 100 µl of extracts were used in the RIAs. Details of the RIAs employed have been described previously (18, 19). The values obtained were not corrected for the extraction efficiencies.
In the third type of experiment, WT and D1KO mice were injected iv with either [125I]T4 or [125I]T3, 2 and 1.5 µg/100 g body weight, respectively. Blood was obtained from the tail after 1 h and, after euthanasia, from the inferior vena cava after 4 or 24 h. The [125I]iodothyronine in each serum sample was determined by counting an aliquot before and after passage through an AG 50W-X8(H–) column. Analysis by paper chromatography showed that essentially all of the [125I]iodothyronine remaining in the serum corresponded to the compound that was injected.
Statistical analyses
Data are expressed as mean ± SE. Statistical analyses were carried out using the GB-Stat PPC 6.5.4 computer program (Dynamic Microsystems, Inc., Silver Spring, MD). For comparison of values obtained in WT and D1KO mice, Student’s t test was used. Serum TSH values were also analyzed using the nonparametric Mann-Whitney rank sum test. For comparisons among three or more groups, one-way ANOVA was performed, and the differences were assessed using Fisher’s least significant difference test (protected t test). Statistical significance is defined as P < 0.05.
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). Detailed studies were not carried out in female mice or in mice older than 19 wk, but weights obtained in a much smaller number of age-paired females and older mice indicated that D1KO mice were consistently heavier than the WT mice (data not shown).
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), in the absence and presence of PTU. High levels of 5'D activity were found in liver, kidney, and thyroid from WT mice and in liver from D1KO heterozygotes. In these tissues, 5'D activity was reduced by more than 99% in the presence of PTU, indicating that it was primarily, if not completely, a result of the D1. No 5'D activity was detected in these tissues in the D1KO mice. 5'D activity was also found in pituitaries from WT mice, and approximately 20% of this activity was not inhibited by PTU, indicating the presence of both the D1 and the D2. In the D1KO pituitary, 5'D activity was much lower than that in the WT pituitary, and this activity was a result solely of the D2 because it was not inhibited in the presence of PTU.
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). In previous studies using tissues from the D2KO mouse, we had detected low but significant levels of 5'D activity that were completely inhibited by PTU (Galton, V. A., unpublished data). The highest level of D2 activity in both WT and D1KO mice was found in pituitary, but activity was also found in brain and skin. The levels of activity found in cerebral cortex, midbrain, and cerebellum were very low but comparable in WT and D1KO mice. Activity in the hypothalamus, when assayed in a single hypothalamus, was too low to be considered significant. However, significant activity was obtained in a later study when a pool of three hypothalami were used (data not shown). Considerable D2 activity was found in skin, although the level varied considerably among mice and was very dependent on the location of the skin sample. The skin used for the data given in Table 2 was taken from the belly. Activity was much lower in skin obtained from the back. No D2 activity was detected in the thyroid gland or the liver.
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). No 5D activity was detected in pituitary or thyroid, but relatively high levels of activity were found in cerebral cortex, midbrain, and hypothalamus. 5D activity was also found in cerebellum and skin. Because in all brain areas and skin, the 5D activity was comparable in WT and D1KO mice and was not reduced when assayed in the presence of PTU, it was attributed to the D3. A low level of 5D activity was found in liver of WT mice, and the level was significantly reduced in the D1KO mice. This reduction, together with the finding that the 5D activity in the WT mice was reduced approximately 70% in the presence of PTU (data not shown), strongly suggests that the major fraction of the 5D activity in liver of WT mice is a result of the D1.
D1 mRNA expression in liver
RNA samples prepared from liver of 10-wk-old euthyroid and hyperthyroid WT and D1KO mice were subjected to real-time PCR using D1 primers. The D1/cyclophilin mRNA signals in WT and hyperthyroid WT mice were 0.034 ± 0.004 and 0.275 ± 0.093, respectively. No signal was obtained in the hepatic RNA samples from the D1KO mice.
Levels of T4, T3, rT3, and TSH in serum from D1KO and WT mice
Compared with values in male WT mice, levels of both T4 and rT3 in serum of 10-wk-old male D1KO mice were significantly elevated. In contrast, serum T3 and TSH levels in WT and D1KO mice were comparable (Fig. 4). A comparable serum hormone profile was seen also in female mice (data not shown). The elevated serum T4 level in association with normal serum T3 and TSH levels occurred as early as P10 (Fig. 5). Values for T3 uptake were comparable in WT and D1KO mice, 53.8 ± 0.7 and 53.6 ± 0.8, respectively, suggesting that free fractions of T4 and T3 are the same in the two strains.
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-glycerol phosphate dehydrogenase, which was significantly reduced in hypothyroid mice, was not significantly different in D1KO and WT mice (Fig 6). The levels of mRNA for glucose-6-phosphatase and Spot14 were also comparable in WT and D1KO mice (data not shown). Likewise the serum cholesterol level and heart weight/body weight ratios in euthyroid D1KO mice were not significantly different from those in euthyroid WT mice (Fig. 7). That these parameters are sensitive to the thyroid status was confirmed by the finding that cholesterol levels and heart weight/body weight ratios were altered in WT hypothyroid and hyperthyroid mice (Fig. 7).
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, pair of bars on the right). In this study, hyperthyroidism was induced by the administration of a daily injection of T3 (15 µg/100 g body weight/d) for 15 d. At 5 h after the last T3 administration, the serum T3 level in WT and D1KO mice was, respectively, 154 ± 6.1 and 176 ± 7.2 ng /100 ml (P < 0.05) and after 24 h was 76 ± 8.9 and 103 ± 4.3 (P < 0.025). Thus, at this dose level of T3, the D1KO mice exhibited both a higher serum T3 level and a greater degree of tissue hyperthyroidism than the WT mice. It was also noted that this treatment suppressed the serum TSH level in both WT and D1KO mice by approximately 20% [WT, 181 ± 10; WT + T3, 140 ± 7 (P < 0.025); D1KO, 195 ± 8; D1KO + T3, 156 ± 4 (P < 0.001)].
Levels of T4 and T3 in thyroid glands of WT and D1KO mice
Substantial D1 activity is present in the mouse thyroid gland (Table 1), and thus there are two possible mechanisms available for the synthesis of T3 in this organ. It is well established that T3 is formed by the coupling of monoiodotyrosine and diiodotyrosine residues contained in the thyroglobulin molecule. However, it could be generated from T4 by the action of the D1 in this tissue. If the D1 plays a major role in the synthesis of T3 in the thyroid, then the T3 content of the thyroid in the D1KO mouse should be greatly reduced. Determination of the T4 and T3 contents of the thyroid gland in euthyroid and hypothyroid WT and D1KO mice revealed that the total amount of T3 in the thyroid of the D1KO mouse was not reduced. Indeed, the levels of both T3 and T4 were significantly increased. By comparison, only a minimal amount of T4 was detected in hypothyroid glands, and the T3 content was also greatly reduced (Fig. 8). The thyroid glands of WT and D1KO mice were of comparable size; at 13 wk, WT and D1KO thyroids weighed 4.4 ± 0.5 and 4.3 ± 0.4 mg, respectively.
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). The WT mice excreted 40% of the injected 125I in the urine and 42% in the feces. In contrast, only 10% of the injected 125I was excreted in urine by the D1KO, whereas 70% appeared in the feces. In the D2KO, urinary 125I was reduced to 30%, whereas almost 50% appeared in the feces. Analysis of urine and fecal samples indicated that at least 98% of the radioactivity in the urine was in the form of inorganic iodide, whereas that excreted in the feces was primarily organically bound.
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In contrast, all four iodothyronines were present in much greater amounts in the feces of the D1KO mice than in the WT feces (Fig. 11). The amounts of T4 and T3 in the D1KO feces were increased almost 2- and 3-fold, respectively, whereas those of rT3 and 3,3'-T2 were increased approximately 5-fold.
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One striking feature of the D1KO mouse is that it exhibits an elevated serum T4 level but no significant decrease in the serum level of either T3 or TSH. This hormone profile in serum is evident at least by P10. The normal serum T3 in the D1KO is accompanied by a seemingly euthyroid state in liver and heart as indicated by the findings that the serum cholesterol level, the heart weight/body weight ratio, and the levels of three T3-responsive hepatic genes are comparable in D1KO and WT mice.
The finding that the serum T3 level was maintained in the absence of the D1 was surprising because there is considerable indirect evidence that in the rodent a significant fraction of the serum T3 is generated from T4 by the D1 in peripheral tissues, the liver in particular (1, 4, 5). In addition, it has been shown that in thyroidectomized rats maintained on exogenous T4, inhibition of D1 activity with PTU results in a rise in serum T4 and a concomitant fall in serum T3 (6). However, although it is generally assumed that these two changes are directly related, the fall in serum T3 could also be a result of the lack of thyroidal secretion of the hormone.
It is notable that the D1KO phenotype is similar in some respects to that of the C3H mouse. The C3H mouse has less than 10% of the hepatic D1 activity found in the common C57 strain (23, 24) and it also exhibits normal plasma T3 and TSH levels in association with an elevation in the serum T4 level. In the normal mammal, the two other potential sources of serum T3 are secretion by the thyroid gland and its generation from T4 by the D2 in peripheral tissues. The D1KO, the C3H, and the PTU-treated mice all have the D2 as a potential source of serum T3, but only the D1KO and the C3H mice have intact thyroid glands. The possibility that, in the D1KO and C3H mice, the rate of secretion of T3 from the thyroid gland is increased, thus preventing any fall in the serum T3 that otherwise would result from the reduced hepatic D1 activity, is unlikely in view of the normal serum TSH level in both models. Furthermore, the maintenance of the serum T3 level in the euthyroid D1KO mouse is not the result of a decrease in the rate of degradation of T3. The rate of disappearance of T3 from the circulation and the levels of D3 activity in adult mouse tissues are comparable in WT and D1KO mice. Thus, the results obtained in the D1KO mouse challenge the concept that T3 generated from T4 by the D1 in peripheral tissues such as liver is a significant source of plasma T3 in the euthyroid rodent (1, 4). Indeed they support results obtained in the rat that suggest that the thyroid is the major source of the circulating T3 (8).
It is well established that a substantial part of the negative feedback control of TSH production by the anterior pituitary gland involves the local conversion of T4 to T3, and it has been estimated that 24–50% of the receptor-bound T3 in the rat pituitary is generated in that organ by local 5'D of T4 (25, 26). A considerable body of evidence indicates that the D2 plays a major role in the negative feedback of T4 in the pituitary, especially in the hypothyroid state (1, 5, 27), and this has been confirmed by the finding that the D2KO mouse exhibits a phenotype of pituitary resistance to T4 (10). Thus, compared with WT mice, the D2KO mouse exhibits a 60% increase in the serum T4 level in association with a 2- to 3-fold elevation in the serum TSH level. Furthermore, the elevated serum TSH level in hypothyroid D2KO mice could be suppressed with T3 but, unlike in the WT mouse, not with T4 (10).
However, the pituitary also expresses the D1, most notably in the euthyroid state (28, 29, 30). The present studies have confirmed this and in fact show that when rT3 is the substrate, the 5'D activity measured in pituitary homogenates is primarily because of the D1 (Table 1). However, a role for the D1 in the feedback of T4 has not yet been established, and it has not been clarified which pituitary cell type(s) express this enzyme. It is notable that the D1KO exhibits an increase in serum T4 comparable to that found in the D2KO. Yet in contrast to the situation in the D2KO, the serum TSH level is normal, a result presumably of the normal serum T3 level and conversion of T4 to T3 by the D2. Although this indicates the lack of a major pituitary resistance to T4 in the D1KO such as occurs in the D2KO mouse, it is not clear why, in view of the elevated serum T4 level and normal T3 level, the serum TSH level is not decreased. This is not because of a reduction in pituitary D2 activity, because D2 activity was not significantly decreased in the D1KO mouse despite the elevated serum T4 level (Table 2). Thus, unless some modification in the set-point of the feedback system had occurred during the development of the D1KO, one cannot from these data exclude the possibility that the D1 plays a role, albeit minor, in the negative feedback of T3 at the level of the pituitary in the euthyroid mouse.
The mammalian thyroid gland contains a relatively high level of D1 activity. In the present study, the level of D1 activity in the WT mouse thyroid was found to be almost as high as that in the kidney (Table 2). The thyroidal D1 activity does not appear to play a significant role quantitatively in the synthesis of T3 in thyroglobulin. In the euthyroid rodent, more than 98% of the iodine in the thyroid gland is bound covalently in thyroglobulin (31), and in the present study it was shown that the total amount of T3 present in the thyroid after hydrolysis of the thyroglobulin is not reduced in the D1KO mouse. In fact, for reasons that are not yet defined, the total amounts of both T3 and T4 are higher in the D1KO thyroid than in the WT thyroid. Although the function of the D1 in the thyroid has not been established, it has been suggested that it plays a role in determining the relative amounts of T4 and T3 that are secreted into the circulation after their release from thyroglobulin. This view is based primarily on the finding that the molar ratio of T4 to T3 in the rat thyroid is considerably higher than the estimated ratio of T4 to T3 secreted from the thyroid into the circulation (1, 32). The D1 may also help to ensure that T3 is the major thyroid hormone released into the circulation in iodine-deficient animals. Acute iodine deficiency results in a decrease in the T4 content of the thyroid, whereas the T3 content remains unchanged (33). The thyroidal D1 activity, which is increased several-fold in iodine-deficient mice (Galton, V. A., unpublished data), may provide an additional mechanism for iodine conservation by converting to T3 any T4 released from the thyroglobulin, thus ensuring that the thyroid hormone secreted into the circulation is primarily T3. As has been reported for rats (34), we have found that the serum of mice fed a low iodine diet for several weeks contained a measurable level of T3, whereas T4 was very low or undetectable (Galton, V. A., unpublished data).
The most striking effects of D1 deficiency found to date relate to the way in which thyroid hormones are metabolized and excreted. After administration of [125I]T4 or [125I]T3, the rates of disappearance of the hormones from the circulation and the total amounts of radioactivity excreted were comparable in WT and D1KO mice. However, the ratios of urinary 125I to fecal 125I in the two genotypes were very different. In the WT mice, approximately 40% of the injected radioactivity appeared in the urine, almost exclusively as iodide. In the D1KO, only 10% of the injected dose was excreted in the urine. This iodide must have been generated by the D2, a conclusion supported by the finding that in the D2KO, the percentage of radioactivity in the urine was diminished by 10%. Thus, a deficiency of D1 impairs overall 5'D of iodothyronine substrates to a much greater extent than does a deficiency of D2. Because the total amounts of radioactivity excreted were comparable, the D1KO and the D2KO mice both had elevated levels of radioactivity in the form of organically bound iodide in the feces, and the elevation was considerably greater in the D1KO mice than in the D2KO mice. A similar shift in the normal profile of urinary and fecal excretion of radioactivity was noted when thyroidectomized rats, equilibrated with a daily dose of [125I]T4, were treated with PTU to reduce 5'D activity (35).
Nguyen et al. (7) have demonstrated that in the rat, the D1 and D2 contribute equally to the production of T3 from T4 in peripheral tissues. If this is true also in the mouse, and assuming that the contribution of D2 to iodide generation derives solely from T4 to T3 conversion, a similar amount of iodide generation from the D1 can be attributed to the same process. In the experiment shown in Fig. 9, WT and D1KO mice excreted, respectively, 40 and 10% of the injected dose of [125I]T4 as [125I]iodide in the urine. In the D1KO mice, this urinary [125I]iodide (10% of the injected radioactivity) must have been generated by the D2. Thus, in the WT mouse, 10% of the injected [125I]T4 must have been converted to T3 by the D2 and another 10% converted to T3 by the D1. Because these two reactions account for only half of the [125I]iodide excreted in the urine, the remainder of the urinary [125I]iodide (20% of the administered [125I]T4) must represent 5'D by the D1 of substrates other than T4 (Fig. 1). To the extent that T4 to T3 conversion by the D2 is enhanced in the D1KO mouse because of increased substrate availability in tissues, as implied by the elevated serum T4 level, then the use by the D1 of substrates other than T4 may actually be underestimated by this analysis.
This suggestion that the D1 in vivo primarily 5'-deiodinates substrates other than T4 is supported by the patterns of urinary and fecal excretion of radioactivity in WT and D1KO mice after a single injection of [125I]T3. The D1KO mice excreted markedly less [125I]iodide in the urine than did the WT mice. As was the case after T4 injection, an increased amount of radioactivity was present in the feces. T3 is a very poor substrate for 5'D. Thus, the increased amount of [125I]iodide excreted in the urine of the WT mice must have been generated by the 5'D of T3 metabolites such as 3,3'-T2 or 3,3'-T2S by the D1.
In addition to an elevated serum T4 level, the D1KO also exhibits marked increases in the serum rT3 level and in the amounts of both rT3 and 3,3'-T2 excreted per day in the feces. These iodothyronines and/or their sulfate conjugates serve as excellent substrates for 5'D by the D1 (2) (Fig. 1), and thus the fact that their levels are elevated in the absence of the D1 is not surprising. However, in the absence of the inner-ring deiodinating activity of the D1 they must have been generated by the D3. Although D3 activity in mouse liver is very low, both brain and skin in the adult mouse exhibit substantial D3 activity. Thus, either or both of these tissues may be the site(s) of generation of these inactive metabolites of T4.
On the basis of the findings in the D1KO mouse, it is evident that, under laboratory conditions, the D1 is not essential for life, normal growth, or reproduction. Furthermore, it is not essential for the maintenance of a normal serum T3 level in the rodent. Indeed, as discussed above, it appears that the majority of the 5'D activity of the D1 is applied to substrates other than T4. Thus, one might reasonably question the importance of the D1 to the physical wellbeing of the rodent, at least in the euthyroid state. However, the situation may be very different under conditions when the thyroid status is challenged, for example, by iodine deficiency. In the WT mouse with intact D1 activity, a much higher fraction of the hormonal iodine is released as inorganic iodide than in the D1KO. This iodide would be available for recycling to the thyroid gland if iodide supplies were low. Conversely, in the absence of the D1, the increased excretion of organically bound iodine in the feces could prove detrimental when dietary iodine is limited.
The present data also indicate that the D1KO mouse is compromised in its ability to handle excess thyroid hormone. Although clearance is not impaired in the euthyroid state, in the experiment shown in Fig. 7, treatment with a relatively high daily dose of T3 resulted in a significantly higher serum T3 level and significantly greater physiological responses in D1KO mice than in WT mice. This suggests that the D1 can limit the increase in the serum T3 level in the hyperthyroid state. This role for the D1 in hyperthyroidism may provide a rationale for the otherwise paradoxical observation that D1 expression is induced in hyperthyroidism (1, 2).
Finally, an intriguing role for the D1, and perhaps other deiodinases, may be emerging with regard to the production of iodothyronamine compounds (e.g. 3-iodothyronamine). These metabolites of thyroid hormones, which have recently been demonstrated to cause rapid effects on body temperature and heart rate (36), require 5D and 5'D, as well as decarboxylation, for their production. Given the capability of the D1 to catalyze both types of deiodination, this enzyme may be an important source of these compounds.
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Footnotes |
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First Published Online October 13, 2005
Abbreviations: D1, Type 1 deiodinase; D1KO, D1 knockout; DTT, dithiothreitol; ES, embryonic stem; MMI/ClO4, 0.1% methimazole and 1% KClO4; P10, postnatal d 10; PTU, 6n-propyl-2-thiouracil; T2, diiodothyronine; WT, wild type.
Received June 20, 2005.
Accepted for publication September 26, 2005.
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