Frequency and clinical implication of the R450H mutation in the thyrotropin receptor gene in the Japanese population detected by Smart Amplification Process 2.

In Japanese pediatric patients with thyrotropin (TSH) resistance, the R450H mutation in TSH receptor gene (TSHR) is occasionally observed. We studied the frequency and clinical implication of the R450H mutation in TSHR in the general population of Japanese adults using smart amplification process 2 (SmartAmp2). We designed SmartAmp2 primer sets to detect this mutation using a drop of whole blood. We analyzed thyroid function, antithyroid antibodies, and this mutation in 429 Japanese participants who had not been found to have thyroid disease. Two cases without antithyroid antibodies were heterozygous for the R450H mutation in TSHR. Thus, the prevalence of this mutation was 0.47% in the general population and 0.63% among those without antithyroid antibodies. Their serum TSH concentrations were higher than the average TSH concentration not only in subjects without antithyroid antibodies but also in those with antithyroid antibodies. The R450H mutation in TSHR is relatively common in the Japanese population and potentially affects thyroid function. The present study demonstrates that the SmartAmp2 method is useful to detect the R450H mutation in TSHR, which is one of the common causes of TSH resistance in the Japanese population.

A novel mechanism for the inhibition of type 2 iodothyronine deiodinase by tumor necrosis factor α: involvement of proteasomal degradation.

Thyroxine (T4) needs to be converted to 3,5,3'-triiodothyronine (T3) by iodothyronine deiodinase to exert its biological activity. Recent studies revealed the presence of type 2 iodothyronine deiodinase (D2) in human thyroid tissue, human skeletal muscle and other tissues, suggesting that D2 is involved in maintaining plasma T3 level in human. Tumor necrosis factor α (TNFα) is an inflammatory cytokine of which production is elevated in patients with nonthyroidal illness. Although several lines of evidence suggest the causal role of TNFα in nonthyroidal illness, detailed nature of the effect of TNFα on D2 remains unclear. In the present study, we identified D2 activity and D2 mRNA in TCO-1 cells, which were derived from human anaplastic thyroid carcinoma, and studied the mechanisms involved in the regulation of D2 expression by TNFα. The characteristics of the deiodinating activity in TCO-1 cells were compatible with those of D2 and Northern analysis demonstrated that D2 mRNA was expressed in TCO-1cells. D2 activity and D2 mRNA expression were rapidly increased by dibutyryl cAMP ((Bu)2cAMP). TNFα showed an inhibitory effect on (Bu)2cAMP-stimulated D2 activity in spite of little effect on (Bu)2cAMP-stimulated D2 mRNA expression. MG132, a proteasome inhibitor abolished TNFα suppression of D2 activity whereas BAY11-7082 or 6-amino-4-(4-phenoxyphenylethylamino) quinazoline, inhibitors of nuclear factor-κB (NF-κB) failed to attenuate the effect of TNFα on D2 activity. These data suggest that a posttranslational mechanism through proteasomal degradation but not NF-κB activation is involved in the suppression of D2 by TNFα.

Association between accumulation of visceral fat and the combination of ß3 adrenergic receptor Trp64Arg, ß2 adrenergic receptor Arg16Gly and uncoupling protein 1 -3826A>G polymorphisms detected by Smart Amplification Process 2.

ß2 and ß3 adrenergic receptors (ß2AR, ß3AR) and uncoupling protein 1 (UCP1) have been considered as candidate genes for obesity. Although each polymorphism of ß3AR Trp64Arg, ß2AR Arg16Gly and UCP1 -3826A>G is known to be associated with obesity, the interaction among these polymorphisms is not fully understood. We analyzed ß3AR Trp64Arg, ß2AR Arg16Gly and UCP1 -3826A>G polymorphisms by the Smart Amplification Process 2 in 222 Japanese subjects without the medication of hypertension, dyslipidemia or diabetes, and investigated the association between the physical and metabolic characteristics and the combination of these polymorphisms. In analysis of the genotypes combination, only the carriers of both ß2AR Arg/Arg and UCP1 G/G genotypes had significantly higher waist to hip ratio (p=0.014). In analysis of the alleles combination, a significant difference was observed in waist to hip ratio among the groups stratified by the carrying number of the alleles of ß3AR Arg, ß2AR Arg and UCP1 G (p=0.026), and the waist to hip ratio was significantly higher in the carriers of four and five risk alleles than in the carriers from zero to three risk alleles (p=0.005). The present study demonstrated the interaction among ß3AR Trp64Arg, ß2AR Arg16Gly and UCP1 -3826A>G for the accumulation of visceral fat.

Oxidative stress associated with rapid weight reduction decreases circulating adiponectin concentrations.

The effect of stress associated with acute weight reduction on adipocytokine production is incompletely understood. In the present study, we have investigated the changes in circulating adipocytokine concentrations and urinary concentrations of stress markers in male collegiate wrestlers during acute weight reduction for a competition. Twenty healthy Japanese male wrestlers (18-22 years of age) who participated in the national collegiate wrestling tournament were studied. Body weight, body fat amount, serum testosterone, serum leptin, serum adiponectin, urinary 8-hydroxy-2'- deoxyguanosine (8-OHdG) and urinary biopyrrins were analyzed during acute weight reduction for the competition. Body weight, body fat amount and the serum concentrations of testosterone, leptin and adiponectin significantly decreased on the day of weigh-in compared with the levels 12 days before weigh-in. In contrast, urinary concentrations of 8-OHdG and biopyrrins significantly increased on the day of weigh-in compared with the concentrations 12 days before weigh-in. A positive correlation was observed between the serum concentrations of adiponectin and testosterone, and a negative correlation was observed between the concentrations of serum adiponectin and urinary biopyrrins. The present results suggest that rapid weight reduction increases the urinary concentrations of stress markers, which is associated with a decrease in serum concentrations of adiponectin.

Identification and functional analysis of novel inactivating thyrotropin receptor mutations in patients with thyrotropin resistance.

OBJECTIVE: We identified and analyzed novel thyrotropin (TSH) receptor mutations in three Japanese families with resistance to TSH. DESIGN: The TSH receptor gene was sequenced and the mutations were determined. The mutant TSH receptors were transfected into COS-7 cells, and their functions were analyzed. PATIENTS: The patients were compound-heterozygotes for the R450H mutation and novel mutations in the TSH receptor gene. The first patient was a compound-heterozygote for R450H and V473I. The second sibling possessed R450H and R519C. The third sibling had R450H and R519G. RESULTS: The R450H mutant exhibited moderately impaired receptor functions and a moderately decreased cell surface expression in agreement with previous results. The V473I mutant exhibited an almost normal TSH binding, a slightly decreased cyclic adenosine monophosphate (cAMP) response, a moderately decreased inositolphosphate (IP) response, and an almost normal cell surface expression. TSH binding and TSH stimulation of cAMP and IPs were markedly decreased in the R519C and R519G mutants. Cell surface expression was decreased in the R519C mutant and negligible in the R519G mutant. All of these mutants showed normal intracellular synthesis of TSH receptors. CONCLUSIONS: These novel inactivating mutations contribute to understanding of the structure-function relationship of the TSH receptor. To date, all of the patients with TSH resistance resulting from TSH receptor mutations identified in Japan possessed the R450H mutation at least in one allele. These observations suggest that the R450H mutation is a commonly observed TSH receptor mutation in patients with TSH resistance in Japan.

Expression of type 2 iodothyronine deiodinase in human osteoblast is stimulated by thyrotropin.

Thyroid hormones play important roles in bone growth, development, and turnover. To exert its biological activity, T(4) needs to be converted to T(3) by iodothyronine deiodinase. In human thyroid gland as well as rat brown adipose tissue, type 2 iodothyronine deiodinase (D2) expression is regulated by a TSH receptor-cAMP-mediated mechanism. TSH receptor knockout mice demonstrated the direct effects of TSH on bone via TSH receptors found on osteoblast and osteoclast precursors. In the present study we investigated the possible expression and function of iodothyronine deiodinase and TSH receptors in human osteoblast-like osteosarcoma (SaOS-2) cells and normal human osteoblast (NHOst) cells. Iodothyronine deiodinase activity was detected in SaOS-2 cells and NHOst cells, and all of the characteristics of deiodinating activity were compatible with those of D2. Northern analysis demonstrated D2 mRNA expression in SaOS-2 cells and NHOst cells. D2 mRNA levels as well as D2 activities were rapidly increased by dibutyryl cAMP or forskolin in SaOS-2 cells and NHOst cells. TSH receptor mRNA was demonstrated in SaOS-2 cells and NHOst cells, and D2 mRNA and D2 activity were stimulated by TSH in both cells. In addition, all T(3) receptor isoforms were detected by RT-PCR in SaOS-2 cells and NHOst cells. The present results indicate the expression of functional TSH receptors and D2 in human osteoblasts and suggest previously unrecognized roles of TSH receptors and local T(3) production by D2 in the pathophysiology of human osteoblasts.

Expression of type 2 iodothyronine deiodinase in corticotropin-secreting mouse pituitary tumor cells is stimulated by glucocorticoid and corticotropin-releasing hormone.

We identified the presence of iodothyronine deiodinase in AtT-20 mouse pituitary tumor cells that secrete corticotropin. Iodothyronine deiodinating activity in AtT-20 cells fulfills all the characteristics of type 2 iodothyronine deiodinase (D2), including the inhibition by thyroid hormones, the insensitivity to inhibition by 6-propyl-2-thiouracil, and the low Michaelis-Menten constant value for T4. Northern analysis using mouse D2 cRNA probe demonstrated the hybridization signal of approximately 7.0 kb in size in AtT-20 cells. D2 activity and D2 mRNA were stimulated by glucocorticoid in a dose-dependent manner but were not stimulated by testosterone or beta-estradiol. D2 expression was stimulated by (Bu)2cAMP, and CRH in a dose-dependent manner in the presence of dexamethasone. These results suggest the previously unrecognized role of local thyroid hormone activation by D2 in the regulation of pituitary corticotrophs.