Thyroid function and effect of aging in combined hetero/homozygous mice deficient in thyroid hormone receptors alpha and beta genes.

The maintenance of thyroid hormone (TH) homeostasis is dependent on the synthesis and secretion of TH regulated by TSH. This is achieved, in turn, by the negative feedback of TH on TSH secretion and synthesis, which requires the interaction with TH receptors (TRs). Derived by alternative splicing of two gene transcription products, three TRs (TRbeta1, TRbeta2 and TRalpha1) interact with TH while another, TRalpha2, binds to DNA but not to TH. In this study we compare the results of thyroid function tests in mice with deletions of the TRalpha and TRbeta genes alone and present novel data on mice that are double homozygous and combined heterozygous. Homozygous deletions of both the TRalpha and TRbeta in the same mouse (TRalphao/o; TRbeta-/-) resulted in serum TSH values only slightly lower than those in athyreotic, Pax8 knockout mice. Whereas the absence of TRalpha alone does not cause resistance to TH, the absence of TRbeta in the presence of TRalpha results in a 205, 169, 544% increase in serum thyroxine (T(4)), triiodothyronine (T(3)) and TSH concentrations respectively. However, in the absence of TRbeta, loss of one TRalpha allele can worsen the resistance to TH with a 243 and 307% increase in T(4) and T(3) respectively. Similarly, while the heterozygous mouse with a single TRbeta allele shows no alteration in thyroid function, the concomitant deletion of TRalpha brings about mild but significant resistance to TH. Furthermore, the severity of the resistance to TH was noted to decrease with age in parallel with the decrease in serum free T(4) values also seen in wild-type mice. These results demonstrate that (1) unliganded TRalpha or TRbeta are not absolutely necessary for the upregulation of TSH; (2) TRbeta but not TRalpha is sufficient for TH-mediated downregulation of TSH; and (3) TRalpha may partially substitute for TRbeta in mediating a partial TH-dependent TSH suppression.

Increased sensitivity to thyroid hormone in mice with complete deficiency of thyroid hormone receptor alpha.

Only three of the four thyroid hormone receptor (TR) isoforms, alpha1, beta1, and beta2, bind thyroid hormone (TH) and are considered to be true TRs. TRalpha2 binds to TH response elements on DNA, but its role in vivo is still unknown. We produced mice completely deficient in TRalpha (TRalpha(o/o)) that maintain normal serum thyroid-stimulating hormone (TSH) concentration despite low serum thyroxine (T(4)), suggesting increased sensitivity to TH. We therefore examined the effects of TH (L-3,3',5-triiodothyronine, L-T3) given to TH-deprived and to intact TRalpha(o/o) mice. Controls were wild-type (WT) mice of the same strain and mice resistant to TH due to deficiency in TRbeta (TRbeta(-/-)). In liver, T3 produced significantly greater responses in TRalpha(o/o) and smaller responses in TRbeta(-/-) as compared with WT mice. In contrast, cardiac responses to L-T3 were absent or reduced in TRalpha(o/o), whereas they were similar in WT and TRbeta(-/-) mice, supporting the notion that TRalpha1 is the dominant TH-dependent TR isoform in heart. 5-Triiodothyronine (L-T3) given to intact mice produced a greater suppression of serum T(4) in TRalpha(o/o) than it did in WT mice and reduced by a greater amount the TSH response to TSH-releasing hormone. This is an in vivo demonstration that a TR deficiency can enhance sensitivity to TH. This effect is likely due to the abrogation of the constitutive "silencing" effect of TRalpha2 in tissues expressing the TRbeta isoforms.

Improved radioimmunoassay for measurement of mouse thyrotropin in serum: strain differences in thyrotropin concentration and thyrotroph sensitivity to thyroid hormone.

We report an improved heterologous radioimmunoassay (RIA) for the measurement of thyrotropin (TSH) in mouse serum. The assay components are: antirat thyrotropin (rTSH) serum from the National Hormone and Pituitary Program, a commercial [125I]-labeled rTSH and mouse thyrotropin (mTSH) serum standards produced by dilution of a serum pool from hypothyroid mice with high TSH with a serum pool from mice treated with excess levothyroxine (LT4) (mTSH-0). Sensitivity was increased by reducing the amount of antibody and tracer and by taking advantage of the disequilibrium technique. Accuracy was greatly improved by the preparation of mouse serum TSH standards. TSH in serial dilutions of individual mice with high TSH of different etiologies paralleled the mTSH standard curve but not that of rTSH or a crude mouse TSH/luteinizing hormone (LH) reference preparation. The high-mTSH-serum standard contained 20 mU TSH per milliliter, measured in a bioassay utilizing a cell line stably transfected with human TSH receptor cDNA, and a relative TSH concentration of 40 ng/mL. The sensitivity of the RIA is 0.01 to 0.02 ng/mL, depending on the quality of the tracer and the preparation of mTSH-0 serum. The intra-assay and interassay coefficients of variations were, respectively: 16% and 27% at 0.04 ng/mL; 6.3% and 8.2% at 0.4 ng/mL; 5.4% and 9.8% at 1.7 ng/mL; 10% and 24% at 4.0 ng/mL. The mean TSH concentration in serum of 60-80-day-old male mice was four-fold higher than that in females of the same age. The assay was able to distinguish differences in serum TSH concentrations in five different strains of mice. Baseline serum TSH concentrations (mean +/- SD) of 70-day-old male mice were: 0.143 +/- 0.065 ng/mL in the CD-1 strain; 0.229 +/- 0.042 ng/mL in C57BL/6 mice; 0.084 +/- 0.017 ng/mL in SWR/J mice; 0.133 +/- 0.057 ng/mL in NOD SCID mice, and 0.266 +/- 0.122 ng/mL in FVB mice. Mean serum thyroxine (T4) concentrations were also significantly different among the mouse strains but did not correlate with the serum TSH level. Administration of levotriiodothyronine (LT3) suppressed the serum TSH to a greater degree in mice with higher baseline TSH values. Suppression of the thyroidal radioiodide uptake with LT3 correlated with that of serum TSH.

Thyroid hormone action on liver, heart, and energy expenditure in thyroid hormone receptor beta-deficient mice.

Thyroid hormone (TH) responsive genes can be both positively and negatively regulated by TH through receptors (TR) alpha and beta expressed in most body tissues. However, their relative roles in the regulation of specific gene expression remain unknown. The TR beta knockout mouse, which lacks both TR beta1 and TR beta2 isoforms, provides a model to examine the role of these receptors in mediating TH action. TR beta deficient (TR beta-/-) mice that show no compensatory increase in TR alpha, and wild-type (TR beta+/+) mice of the same strain were deprived of TH by feeding them a low iodine diet containing propylthiouracil, and were then treated with supraphysiological doses of L-T3 (0.5, 5.5, and 25 microg/day/mouse). TH deprivation alone increased the serum cholesterol concentration by 25% in TR beta+/+ mice and reduced it paradoxically by 23% in TR beta-/- mice. TH deprivation reduced the serum alkaline phosphatase (AP) concentration by 31% in TR beta+/+ mice but showed no change in the TR beta-/- mice. Treatment with L-T3 (0.5 to 25 microg/mouse/day) caused a 57% decrease in serum cholesterol and a 231% increase in serum AP in the TR beta+/+ mice. The TR beta-/- mice were resistant to the L-T3 induced changes in serum cholesterol and showed increase in AP only with the highest L-T3 dose. Basal heart rate (HR) in TR beta-/- mice was higher than that of TR beta+/+ mice by 11%. HR and energy expenditure (EE) in both TR beta+/+ and TR beta-/- mice showed similar decreases (49 and 46%) and increases (49 and 41%) in response to TH deprivation and L-T3 treatment, respectively. The effect of TH on the accumulation of messenger RNA (mRNA) of TH regulated liver genes was also examined. TH deprivation down regulated spot 14 (S14) mRNA and showed no change in malic enzyme (ME) mRNA in both TR beta+/+ and TR beta-/- mice. In contrast treatment with L-T3 produced an increase in S14 and ME but no change in TR beta-/- mice. From these results, it can be concluded that regulation of HR and EE are independent of TR beta. With the exception of serum cholesterol concentration and liver ME mRNA accumulation, all other markers of TH action examined during TH deprivation exhibited the expected responses in the absence of TR beta. Thus, as previously shown for serum TSH, TR beta is not absolutely necessary for some changes typical of hypothyroidism to occur. In contrast, except for HR and EE, the full manifestation of TH-mediated action required the presence of TR beta.

Thyrotropin regulation by thyroid hormone in thyroid hormone receptor beta-deficient mice.

Thyroid hormone responsive genes can be both positively and negatively regulated by thyroid hormone. TSH is down-regulated by thyroid hormone and rises during thyroid hormone deprivation. Because both thyroid hormone receptor (TR) alpha and beta genes are expressed in the pituitary gland, it is unclear what the relative roles of TR alpha and TR beta are in TSH regulation. Experiments using over expression of artificial genes have yielded conflicting results. The TR beta knock-out mouse that lacks both TR beta1 and TR beta2 isoforms provides a model to examine the role of these receptors in TSH regulation. TR beta deficient (TR beta-/-) and wild-type (TR beta+/+) mice of the same strain were deprived of thyroid hormone by feeding them a low iodine diet containing propylthiouracil and were then treated with different doses of L-T3 and L-T4. Thyroid hormone deprivation rapidly increased the serum TSH level in both TR beta+/+ and TR beta-/- mice, reaching a similar level in the absence of thyroid hormone. In contrast, the decline of serum TSH by treatment with both L-T3 and L-T4 was severely blunted in TR beta-/- mice, and full suppression was not achieved with the maximal L-T3 dose of 25 microg/day x mouse. These data indicate that TR beta is not required for the up-regulation of TSH in thyroid hormone deficiency. However, although TR alpha alone can mediate thyroid hormone induced TSH suppression, TR beta enhances the sensitivity of TSH down-regulation and may be essential for the complete suppression of TSH.