Departments of Physiology, Microbiology and Medicine (M.J.S., S.N.F., S.E.P., D.L.S., V.A.G.) Dartmouth Medical School, Lebanon, New Hampshire 03756; and Harbor-University of California Los Angeles Medical Center (A.F.P.), Torrance, California 90509
Address all correspondence and requests for reprints to: Dr. Valerie Anne Galton, Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756. E-mail: val.galton{at}dartmouth.edu
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ABSTRACT |
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INTRODUCTION |
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D1 and D2 can be distinguished by their kinetic properties, substrate preferences, patterns of inhibition by compounds such as 6n-propyl-2-thiouracil (PTU) and aurothioglucose, and their response to changes in thyroid status (1, 2). They also exhibit different profiles of expression in tissues; the highest levels of D1 activity are found in liver, kidney, and thyroid gland while the highest levels of D2 activity are found in pituitary, brown adipose tissue (BAT), and the central nervous system (CNS) (1, 2).
There is indirect evidence to indicate that the deiodinases play a role in the tissue- and cell-specific regulation of intracellular levels of the active thyroid hormone, T3. For example, the finding that the ratio of T3 to T4 varies widely among tissues, but is invariably higher than that in plasma, is likely dependent, at least in part, on the relative levels of activity of the three enzymes in each tissue (8). There is also evidence that the deiodinases are involved in the adaptation of the organism to environmental and internal challenges. Changes in the activities of one or more of the deiodinases have been noted in starvation, illness, exposure to cold, and changes in thyroid status (1, 2, 6, 9).
However, the precise physiological roles of the individual deiodinases, in particular D1 and D2, which both catalyze 5'D, have not been clearly defined. It has been estimated that D1 activity generates T3 from T4 primarily for export to the plasma (10, 11), while D2 activity is thought to generate T3 mainly for local use in tissues. In fact, the presence of D2 in tissues such as pituitary, brain, and BAT has led to the view that it plays a key role in regulating thyroid hormone-dependent processes in these organs, in particular the pituitary feedback mechanism, developmental processes in brain, and thermogenesis in BAT (12, 13, 14). But proof of this concept of separate functions for the two enzymes is complicated by the fact that pituitary and brain express D1 as well as D2, and evidence has been obtained that some of the T3 generated by D2 is also exported to the plasma (15).
One approach to determining the function of an individual deiodinase would be to inhibit specifically its activity. Unfortunately, there is no known pharmacological agent that will specifically and completely inhibit the activity of any of the deiodinases individually. Iopanoic acid is an excellent inhibitor but it inhibits the activity of all three of them. PTU can be employed at levels that greatly reduce the activity of D1 while having little effect on that of D2, but the inhibition is not complete. Furthermore, this compound also inhibits thyroid hormone biosynthesis, and thus it is unsuitable for studies in vivo in animals where intact thyroid function is required.
A second approach is to create an animal model that lacks the active form of a deiodinase enzyme by, for example, targeted disruption of a specific deiodinase gene. We have used this technique successfully to create a mouse that is completely deficient in D2 activity. The methods used in its production, the data confirming that the gene disruption has led to a complete loss of D2 activity in tissues, and details concerning the resulting phenotype are presented herein.
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RESULTS |
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TSH Suppression Studies
The observation that both T4 and TSH levels
are elevated in serum of D2KO mice suggests that the pituitary/thyroid
feedback system is resistant to T4. Presumably,
this is because, in the absence of D2, the pituitary is unable to
convert the T4 to T3,
resulting in lower thyroid hormone receptor occupancy in the D2KO
animals. To test this hypothesis, male WT and D2KO mice were placed on
MMI/ClO4 for 4 wk to raise their circulating TSH
levels. D1 activity was inhibited by the administration of PTU. On the
day that they were killed, the mice were bled from the tail and 1
h later they were injected sc with T4 (3 µg/100
g body wt), T3 (1.2 µg/100 g body wt), or
vehicle. They were killed and serum was obtained 5 h later. At the
time of death, serum T4 levels in the WT and D2KO
mice injected with T4 were 7.8 ± 0.99 and
9.9 ± 0.77 µg/100 ml (ns, P > 0.05),
respectively. Serum T3 levels in the WT and D2KO
mice injected with T3 were 299 ± 32.4 and
366 ± 32.1 ng/100 ml (P < 0.05), respectively.
Serum TSH levels in both WT and D2KO mice 5 h after injection of
hormone were still at least 10 times those seen in corresponding
euthyroid mice. However, values for both the initial and final TSH
levels were very variable within each group, and thus data are
presented as the final TSH level expressed as a percent of the initial
TSH level. Both T4 and T3
significantly suppressed circulating TSH levels in the WT mice. In
contrast, in the D2KO mice, a significant decrease in circulating TSH
was achieved only after the T3 injection;
T4 had no significant inhibitory effect
(Fig. 9).
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DISCUSSION |
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It has been suggested that the mammalian Dio2 gene does not code for a functional protein, and therefore the D2 selenodeiodinase is not responsible for the type 2 deiodinase activity found in rodent tissues (22, 23). This suggestion was based on indirect evidence whereby investigators using an antibody raised against the carboxy terminus of the rat D2 selenodeiodinase were unable to detect a protein of the predicted 30-kDa size in (Bu)2cAMP-stimulated astrocytes, nor was the antibody able to immunoprecipitate D2 activity out of sonicates derived from these same cells. However, the results presented here provide definitive evidence that the product of the Dio2 locus is essential for the expression of D2 activity in mammalian tissues. Given that the expressed protein product of this gene has been demonstrated to catalyze deiodination with properties identical to that of the D2 activity found in native tissues (24), there can be little doubt that the D2 selenodeiodinase is responsible for this enzyme activity in vivo. The inability of antibodies to detect this protein in cell sonicates likely relates to its known very low abundance.
The most striking features of the D2KO mouse are that, compared with the WT mouse, the circulating levels of both T4 and TSH are elevated while those of T3 are unchanged. The fact that the levels of TSH and T4 are both raised in this model suggests that the pituitary thyrotrophs are not responding normally to the plasma level of T4. The most likely explanation for this is that, in the absence of D2 activity, the ability of the pituitary to convert T4 to T3 is impaired. This results in a decrease in the intrapituitary T3 level, and hence nuclear TR occupancy is reduced, which leads to a decreased inhibition of TSH synthesis and secretion.
Direct evidence that the pituitary of the D2KO mouse is resistant to T4 is provided by the finding that plasma TSH levels in hypothyroid WT mice were suppressed significantly after injection of either T4 or T3, whereas in hypothyroid D2KO mice significant suppression of plasma TSH occurred after injection of T3 but not after injection of T4. This lack of suppression by T4 in the D2KO mouse occurred even though a higher serum T4 level was attained in the D2KO mice than in the WT animals. These data provide unequivocal evidence that pituitary D2 activity is essential for the participation of circulating T4 in the pituitary/thyroid feedback mechanism.
The degree of impairment of the pituitary/thyroid feedback mechanism in the euthyroid D2KO mouse is relatively modest since much higher plasma TSH levels were obtained when the mice were rendered hypothyroid. This suggests that the system is only partly dependent on the presence of D2 activity. This was expected since it has been shown previously that only 55% of the T3 bound to the pituitary TRs is generated by 5'D within the pituitary (25). An additional complicating factor is that the euthyroid rat pituitary gland expresses D1 as well as D2 (26, 27, 28, 29). In the present study, PTU-sensitive D1 activity was demonstrated in both WT and D2KO mice using rT3 substrate, but not when 1 nM T4 was substrate. This latter finding supports the concept that, in these mouse tissues, D1 contributes only in a limited fashion to intrapituitary T3. However, one must be cautious about extrapolating to the in vivo situation data obtained in in vitro assays using cofactors and conditions that presumably are not the ones employed in vivo.
There are several possible explanations for the elevated plasma T4 levels in the D2KO mouse. Under normal conditions the steady-state plasma T4 level is determined by the rate of thyroidal secretion of T4, the binding activity of the plasma T4 binding proteins, and the rate of T4 clearance from the circulation by peripheral tissues. The clearance of T4 from plasma is dependent on multiple factors including the activities of the three deiodinases, and of the sulfatases and glucuronidases that catalyze the formation of T4 conjugates, some of which are cleared in bile and feces. The elevated plasma T4 level in the D2KO mouse cannot be attributed to an increase in plasma T4 binding activity since binding activity was shown to be slightly lower in the D2KO compared with the WT mouse. Since the plasma TSH level is elevated in the D2KO mouse, thyroidal secretion of T4 is likely to be increased. However, the plasma T4 level was also elevated in the hypothyroid D2KO mice implanted with a constant release T4 pellet. In these mice, secretion of endogenous T4 had been reduced to levels that were undetectable. While this elevated plasma T4 level could be due to a reduction in the volume of distribution of T4 in the D2KO mouse, this seems unlikely since the WTs and D2KO mice were of comparable weight, and their plasma T4 binding activities were similar. A more likely explanation is that the clearance of T4 from plasma of the D2KO mice was decreased. This reduced rate of clearance cannot be attributed to reduced D1 activity because, under all conditions studied, D1 activity in D2KO mice and corresponding WT mice was comparable. Thus, the reduced clearance of T4 from plasma under the conditions of this experiment likely results, at least in part, from the absence of D2 in peripheral tissues. The reduced clearance of T4 occurred in spite of the increase in brain D3 activity, a change that would be expected to increase the clearance of plasma T4.
Despite the absence of D2 activity, serum T3 was maintained close to WT levels in D2KO mice. One explanation for this finding is that the T3 generated from T4 by D2 activity does not normally contribute to the plasma T3 level. If true, the data would support the view that the role of the D2 is to generate T3 from T4 primarily for use within the cell or tissue in which the deiodination has occurred (30, 31). However, there is considerable indirect evidence consistent with the view that D2 does in fact contribute to plasma T3. Thus, in rats treated with PTU, which greatly reduced hepatic and renal D1 activity, extrathyroidal conversion of T4 to T3 was reduced by only 6070% (32, 33). Furthermore, the D2 pathway appears to be the predominant mechanism for extrathyroidal production of T3 in the euthyroid neonate and the only demonstrable pathway in the hypothyroid neonate (34). In addition, a recent study has provided quantitative evidence that a significant fraction of the plasma T3 in rats is derived from T4 by the action of D2 (15). If this is the case also in mice, then it must be that the deficient production of T3 resulting from the absence of D2 activity is compensated for from other sources. Possible sources include T3 secreted by the thyroid gland per se and T3 generated from T4 by D1 activity. In the D2KO mouse the conditions are such that the supply from both sources could be enhanced. With the elevated plasma TSH levels, thyroidal secretion of T3 would be expected to be elevated. In addition, although the levels of D1 activity in liver and thyroid were not altered in the D2KO mouse, T4 levels are increased, thus providing additional substrate for the generation of T3. Another factor that could contribute to the maintenance of plasma T3 levels in the D2KO mouse is a possible reduction in the rate of clearance of T3 from plasma. Studies are in progress to resolve these issues.
D3 activity was increased approximately 3-fold in cerebral cortex of D2KO mice, a paradoxical increase that would seemingly further compromise T3 levels in the CNS. The stimulus for this increase is not clear. It may result from differences in intracellular levels of T4 and T3 in the CNS. It has been shown that the amphibian D3 gene is up-regulated directly by thyroid hormone (4, 35) and D3 activity in rat brain also appears to be stimulated by thyroid hormone (36). However, the increase in plasma T4 in the D2KO mouse would not be expected to result in an increased level of T3 in the brain, since this organ is thought to derive the majority of its T3 from plasma T4 by local D2 activity (25). One possible explanation for the increase in D3 activity is that T4 per se has a stimulatory effect on the expression of the D3 gene, or an inhibitory effect on the degradation of the enzyme or its mRNA. It is also possible that the set point of D3 activity is altered in the D2KO mice during development. D3 activity in rodent brain decreases markedly in the neonatal period (37), and the extent of this decrease may be altered in mice deficient in D2. Further studies, including those in which intracellular T4 and T3 levels in different parts of the brain are determined, are in progress to resolve this question.
Aside from the increased plasma T4 and TSH levels and the pituitary resistance to T4, the D2KO mouse exhibits a seemingly mild phenotype. Reproductive capacity is unimpaired, any abnormalities in growth are both small and transient, and, outwardly, the mice appear as mobile and healthy as the WT mice. This suggests that the thyroid hormone-dependent processes that participate in these functions are able to derive sufficient T3 from the plasma, a source that is not compromised in the D2KO mouse. However, it is possible that other crucial TH-dependent functions such as hearing, vision, learning, and memory, functions that are less obvious on gross inspection, may be more sensitive than locomotive function to a decrease in the T3 level in brain and may, in fact, be impaired in the D2KO mouse. These parameters are currently being studied.
The D2KO mouse was able to survive in the cold. This is notable since the ability of normal rodents to survive sudden and/or prolonged exposure to cold is thought to be due primarily to increased heat production in BAT (38), a response that does not occur in the absence of T3 (14). In fact, hypothyroid rodents cannot survive in the cold (39). D2 is the only deiodinase expressed in BAT, and BAT D2 activity is up-regulated rapidly and substantially when rodents are placed at 4 C, to enhance the local production of T3 from circulating T4 (40). Thus, the finding that D2 expression in BAT is not essential for survival in the cold indicates that either BAT is not completely dependent on local generation of T3 from T4 or that the D2KO mouse can compensate in some way for impaired thermogenic function in BAT.
In summary, targeted inactivation of the Dio2 selenodeiodinase gene results in the complete loss of D2 activity in all tissues examined. The increased serum levels of T4 and TSH observed in D2KO mice, together with the finding that T4 is ineffective in reducing the plasma TSH level in D2KO mice, demonstrate that the D2 is of critical importance in the pituitary/thyroid feedback regulation of TSH secretion. Studies are in progress to determine the extent to which other thyroid hormone-dependent processes, particularly those in the CNS, are impaired in the D2KO mouse.
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MATERIALS AND METHODS |
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The Dio2 targeting vector was constructed in pBluescript
SK(-) plasmid, in which a 1.7-kb neomycin resistance (Neo)
cassette had been ligated into the EcoRI/HindIII
restriction enzyme site in the sense orientation (provided by Dr. Nancy
Speck, Dartmouth Medical School). The cassette contained the
Neo coding region flanked by the promoter and 3'-
untranslated region of the phosphoglycerokinase gene. As diagrammed
in Fig. 10, a 5.7-kb,
BglII/SpeI restriction fragment of the
Dio2 intron was ligated to the 5'-end of the Neo
cassette, and a 5.0-kb StuI fragment located 3' to the D2
coding region was ligated to the 3'-end. The thymidine kinase gene (not
shown) was ligated 3' to the latter, and in the same orientation. The
targeting vector did not contain the
2.6 kb SpeI to
StuI fragment of the Dio2 gene, and hence 72% of
the D2 coding region was deleted, including the TGA codon, which codes
for selenocysteine.
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Positive PCR results were confirmed by Southern analysis. The ES cell
genomic DNA samples were digested with StuI, electrophoresed
on 0.8% agarose, and transferred to Nytran (Schleicher & Schuell, Inc., Keene, NH.). Blots were probed with the 1.2-kb
StuI/BglII fragment that is located in the
Dio2 gene just 5' of the recombination site (Fig. 10).
Because there is a StuI site in the Neo cassette,
the D2 knockout and wild-type alleles yield bands of 7 and 9.3 kb,
respectively.
Two targeted ES clones were obtained and injected into blastocysts, which were in turn 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 allele in the F1 generation. The genotyping was accomplished by Southern analysis (see above) of genomic DNA obtained from tail tips and prepared using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). The founder males were then bred to the +/- F1 females, and the -/- (D2KO) and +/+ (WT) genotypes identified in the offspring. Mice of the same genotype were then bred to generate and maintain colonies of WT and D2KO mice.
Animals
In addition to the mice described above, 10-wk-old male and
female mice of each of the two background strains, 129SV and C57Bl/6,
were purchased from The Jackson Laboratory (Bar Harbor,
ME). All mice were housed under conditions of controlled lighting and
temperature in the barrier section of the Dartmouth Medical School
animal research facility. Details of all births, including litter size,
birth abnormalities, and neonatal deaths were recorded. Mice were
weighed at regular intervals. Some mice were exposed to 4 C for 4 or
16 h before study. Others were made hypothyroid by placing them on
drinking water containing 0.1% MMI and 1% KClO4
(MMI/ClO4) for a minimum of 4 wk before study.
Some of the latter mice were also implanted sc with a pellet containing
either 0.0025 or 0.005 mg T4. These pellets were
designed to yield a constant level of plasma T4
over a 21-d period and were custom-made for us by Innovative Research of America (Sarasota, FL). Preliminary studies
indicated that a 0.0025 mg pellet yielded a subnormal plasma
T4 level, whereas a pellet containing 0.005 mg
T4 yielded a plasma T4
level that was approximately twice the value observed in a WT euthyroid
mouse.
In another study, male WT and D2KO mice were placed on
MMI/ClO4 for 5 wk. Five days before the mice were
killed, PTU was added to their drinking water (1 mg/liter). On the day
of death, blood (150 µl) was obtained from the tail after which
they were injected sc with PTU (1 mg/100 g body wt). The PTU was
administered in 0.1 ml of saline containing 0.01 N NaOH.
One hour later they were injected sc with T3 (1.2
µ g/100 g body wt), T4 (3 µg/100 g body wt),
or vehicle. The hormones were administered in 0.1 ml of saline
containing 0.1% BSA (wt/vol). The animals were killed 5 h
later.
Except when stated otherwise, all mice were between 9 and 12 wk of age at the time they were killed for study. All animal protocols were approved by the Institutional Review Board of Dartmouth Medical School.
Tissue Preparation
The mice were killed by ether anesthesia followed by
exsanguination. Once the mice were anesthetized, the abdomen was opened
and blood was taken directly into a syringe from the inferior vena
cava. The serum was obtained by centrifugation and then stored at -20
C for subsequent assays. The following tissues were obtained: liver,
kidney, BAT, thyroid (plus the section of trachea to which it was
attached), cerebrum or cerebral cortex, and pituitary. Liver, kidney,
BAT, and brain tissue were homogenized in 0.25 mM sucrose;
20 mM Tris-HCl, pH 7.6, containing 5 mM
dithiothreitol (DTT) as previously described (42) to yield
approximately a 1:5 homogenate (wt/vol). Pituitary and thyroid (plus
trachea) were homogenized by hand in 0.5 ml of the same buffer using a
ground-glass homogenizer. The homogenates were centrifuged at
1,000 x g for 15 min and the supernatants stored at
-20 C for subsequent assay of 5'D and 5D activities.
In addition, total RNA was isolated from BAT obtained from D2KO and WT mice after exposure to 4 C for 16 h, using a commercial RNA isolation reagent (TRIzol Reagent, Life Technologies, Inc., Gaithersburg, MD), according to the manufacturers instructions. Poly(A)+RNA was isolated from total RNA as previously described (43).
Preparation and Culture of Astrocytes
Cerebral hemispheres were removed from 2-d-old WT and D2KO mice
and primary cultures of astrocytes were prepared as described
previously (44). Briefly, cells were cultured in DMEM
supplemented with 6 g/liter glucose, 2.4 g/liter
NaHCO3, antibiotics (100 U/ml penicillin,
100 µg/liter streptomycin, and 0.25 µg/liter
Amphotericin; Sigma, St. Louis, MO)
and 10% FCS (DMEM/FCS). The medium was changed every 23 d until
cells reached confluency at approximately 10 d. At this stage, the
DMEM/FCS was removed and cells were washed with a 1:1 mixture of DMEM
and Hams F12 (DMEM/F12), supplemented with 5.2 g/liter glucose, 1.8
g/liter NaHCO3, and the antibiotics as listed
above. The cells were cultured for 3 additional days in DMEM/F12
supplemented with 30 nM sodium selenite, 10 µg/ml
insulin, 10 µg/ml transferrin, followed by 1 additional day in
DMEM/F12 supplemented with 30 nM sodium selenite, 1
µM cortisol, and 10 µg/ml transferrin. Some cultures of
astrocytes were treated with 10 µM forskolin for 4 h
and 6 h, or 0.1 µM tetradecanoyl-phorbol-13-acetate
(TPA) for 8 h before harvesting.
At the time of harvesting, the medium was aspirated, the cells were rinsed twice with 3 ml of ice-cold PBS, and the dishes containing the cells were frozen at -80 C. Cells were processed for assay of D2 activity by scraping the cells into 1 ml of Tris/sucrose buffer. Cells were centrifuged at 500 x g for 3 min and then resuspended in 50 µl of Tris/sucrose buffer and sonicated for 5 sec.
Determination of 5'D and 5D Activities
5'D and 5D activities were assayed according to our published
methods (45, 46). Briefly, for 5'D activity the reaction
mixture (total volume 50 µl) contained between 2 and 100 µg tissue
protein and 1.2 mM EDTA. The substrate was 1.0
nM of either
[125I]rT3 or
[125I]T4, and the
cofactor was 20 mM DTT. 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 that which took place during the same time period at 0 C.
For liver, kidney, BAT, cerebrum, and astrocytes, 5'D activity is
expressed as picomoles or femtomoles iodide generated/h·mg protein.
For pituitary and thyroid, it is expressed per individual thyroid or
pituitary gland. In determining 5'D activity, the percent iodide
generated was multiplied by 2 since the specific activities of the
labeled products were only half that of the substrate. In pituitary and
cerebrum, 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 cerebrum 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
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. Assays were conducted in the presence and absence of 1 mM PTU. In both the 5D and the 5'D assays, protein concentrations were adjusted to ensure that deiodination was less than 20%.
[125I]rT3,
[125I]T4, and
[125I]T3 (specific
activities 1,000 µCi/µg) were obtained from Perkin-Elmer Corp. (Norwalk, CT) and were purified by chromatography using
Sephadex LH-20 (Sigma) before use. Protein concentrations
of all samples were determined according to the method of Comings and
Tack (47) using BSA as the standard.
Assays for Serum T4, T3, and TSH
Serum total T4 and
T3 levels were determined using the Coat-A-Count
RIA total T4 and total T3
(Diagnostics Systems Laboratories, Inc., Webster, TX). The
total T4 assay was carried out according to the
manufacturers 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. The total T3 assay required
modification for use with mouse serum. Test studies with
charcoal-stripped mouse serum (48), unsupplemented and
supplemented with 50 or 100 ng T3/100 ml,
revealed a significant nonspecific effect of mouse serum in the assay;
the maximum binding value
([125I]T3/antibody
binding in the absence of nonradioactive T3) was
suppressed approximately 12%. This resulted in spuriously high
T3 levels in the supplemented serum samples.
Correction of the maximum binding value for this nonspecific effect
resulted in T3 levels in the supplemented samples
that were within 5% of the estimated levels. This correction was
applied to all mouse serum samples. The minimal detectable level for
T3 was 7 ng/100 ml. The plasma thyroid hormone
binding activities in WT and D2KO sera were compared using the
Coat-A-Count T3 uptake kit purchased from the
same company. In this assay, the samples of sera were incubated with
[125I]T3 in tubes in
which a T3-specific antibody had been immobilized
to the wall. The percent uptake of the
[125I]T3 by the antibody
is determined.
Mouse serum TSH levels were determined using a highly sensitive double antibody method, developed by A. F. Parlow. The assay used a highly purified rat TSH (AFP11542B) as the iodinated ligand, a selected guinea pig antimouse TSH (AFP98991), at a final tube dilution of 1:500,000, as the primary antibody, and a partially purified extract of mouse pituitary containing TSH (AFP5171.8MP) as the reference preparation. Cross-reactivity of either highly purified mouse FSH or mouse LH in this mouse TSH RIA was less than 1%. Displacement curves obtained by testing sera of hypothyroid mice in graded dilutions did not depart significantly from parallelism with displacement curves for the reference preparation. Recovery of exogenous mouse TSH activity added to mouse serum was 80100%. The coefficients of variation within and between assays were 5% and 11%, respectively. Serum of euthyroid WT and D2KO mice were generally tested using a volume of 4080 µl per RIA tube, whereas sera of hypothyroid mice could be tested in volumes as small as 5 µl per RIA tube. All mouse sera were assayed for TSH activity in the absence of knowledge by the tester of the treatment status of the donor mice. Values for serum TSH levels in euthyroid mice obtained using this method, and the finding that levels are significantly higher in male than in female mice, have been reported previously (49).
Northern Analysis
Northern analysis was carried out as described previously
(43). Between 56 µg of
Poly(A)+RNA were loaded into each lane. The blot
was probed with a 950-bp mouse D2 cDNA that contained the entire coding
region plus some 5'-untranslated region (17). To document
the relative amounts of RNA in each lane, the blot was also probed with
a cDNA for the unregulated gene, cyclophilin. Hybridizations were
carried out at 42 C and the final washes were performed at 60 C.
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 D2KO and WT mice, the t test was used.
For multigroup comparisons, one-way ANOVA was performed, and the
differences were assessed using Fishers least significance difference
test. Statistical significance is defined as P <
0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: BAT, Brown adipose tissue; CNS, central nervous system; D1, D2, D3, types 1, 2, and 3 deiodinase, respectively; 5D, 5-deiodination; 5'D, 5' deiodination; D2KO, D2 knockout; DTT, dithiothreitol; ES, embryonic stem; MMI, methimazole; PTU, 6n-propyl-2-thiouracil; TPA, tetradecanoyl-phorbol-13-acetate; WT, wild-type.
Received for publication May 18, 2001. Accepted for publication August 16, 2001.
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REFERENCES |
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