Effect of streptozotocin-induced diabetes mellitus on type 1 deiodinase (D1) in inherited D1-deficient mice.

We investigated the effects of streptozotocin (STZ)-induced diabetes on thyroid hormone levels, type 1 deiodinase (D1) activity and messenger RNA (mRNA) levels in inherited D1 deficient C3H mice in a comparative manner with control C57 mice. The apparent maximum velocity (Vmax) D1 values in C3H mice were 3% (liver) and 26% (kidney) of those in C57 mice. In C3H mice, similar serum T3, slightly higher T4, and 2.6-fold higher rT3 levels were observed compared with C57 mice. In STZ-induced diabetes, serum T4 level markedly decreased in both C3H and C57 mice. Serum T3 levels in STZ-C3H mice similarly decreased as in STZ-C57 mice. On the other hand, serum rT3 levels increased to 3.3-fold higher in STZ-C3H than in STZ-C57 mice. The Vmax values were decreased to 12% (STZ-C3H) and to 30% (STZ-C57) in liver, and decreased to 33% (both STZ-C3H and STZ-C57) in kidney. The changes in D1 mRNA levels in diabetes versus control were comparable to those of D1 activities in both strains. In summary, similar mechanism(s) to those which decrease the D1 expression and the serum T3 level in diabetes, function in D1 deficient C3H mice as in C57 mice. It appears that hepatic and renal D1 activity alone can not explain the similar reduction in T3 level in STZ-C3H mice and STZ-C57 mice.

Age- and sex-related changes in type-1 iodothyronine deiodinase messenger ribonucleic acid in rat liver and kidney.

To evaluate the age- and sex-related changes in Type 1 iodothyronine deiodinase gene expression in the liver and kidneys, we measured 5'-deiodinating activity and deiodinase mRNA in developing rats. The activity in the liver increased after birth, and that in neonates was approximately half that in adults. In contrast, the activity in neonatal kidneys remained very low. The relative importance of activity in male kidneys compared to the liver increased from the ages of 1 to 20 days. The male adult rat liver showed a higher level of activity than the female liver. Deiodinase mRNA in the male liver gradually increased from 1 to 20 days, in correlation with the activity. In kidneys, deiodinase mRNA was low before day 20, and there was no significant sex difference in all age groups. In orchiectomized male rats, the activity and mRNA in the liver was similar to the low levels found in females; however, the levels in the kidneys were not significantly different than those of normal males. These data suggest that the age- and sex-related changes in iodothyronine deiodinase gene expression are regulated mainly at the pretranslational level, and that the relative importance of kidneys to liver in iodothyronine deiodinase increases from birth to age 20 days due to the difference in the gene expression.

Effect of nicotine on type 2 deiodinase activity in cultured rat glial cells.

Intracellular generation of triiodothyronine (T3) from thyroxine (T4) by type 2 deiodinase (D2) in the mammalian brain, plays a key role in thyroid hormone action. The presence of D2 in rat astrocytes suggests the importance of glial cells in the regulation of intracellular T3 levels in the rat central nervous system (CNS). To analyze further the factors that regulate D2 activity in the CNS, we investigated the effects of nicotine and of mecamylamine, which inhibits the binding of nicotine with nicotinic acetylcholine receptors, on D2 activity in cultured mixed glial cells of the rat brain. We incubated cultured mixed glial cells obtained from neonatal Wistar rats in the presence of 10 mM dithiothreitol, 2 nM [125I] reverse T3 and 1 mM 6-N-propyl-2-thiouracil for 2 h at 37 degrees C, and the released 125I- was counted in a gamma counter. D2 activity of cultured cells was dependent on the temperature and the amount of protein. The basal D2 activity of rat mixed glial cells was 1.9 +/- 0.2 fmol of I- released/mg protein/h (mean +/- SEM). The addition of 10(-11), 2 x 10(-11), 10(-10), and 10(-9) M nicotine significantly increased D2 activity to approximately 2.2-, 2.4, 3.5- and 2.9-fold the basal level, respectively. D2 activity stimulated by 10(-8) M nicotine (2.5-fold) reached a peak after 9 h incubation. The stimulatory effect of nicotine was completely blocked by 10(-6) M mecamylamine. In conclusion, nicotine increases D2 activity probably via nicotinic acetylcholine receptors, and may influence brain function, at least in part, by affecting thyroid hormone metabolism.

Type 1 iodothyronine deiodinase in heart --effects of triiodothyronine and angiotensin II on its activity and mRNA in cultured rat myocytes.

We previously demonstrated that iodothyronine 5'-deiodination (5'D) activity is present and increased by triiodothyronine (T3) and angiotensin II (Ang II) in cultured rat cardiac myocytes. To further elucidate the stimulatory mechanism of Ang II, we investigated the effect of intracellular Ca2+ and protein kinase C on myocardial 5'D activity. Moreover, to elucidate the molecular mechanism of the stimulatory effect of T3 and Ang II, we detected the mRNA levels by means of a reverse-transcriptase polymerase chain reaction (RT-PCR). 5'D activity was increased by adding Bay-k 8644, Ca2+ channel agonist and the effect of Bay-k 8644 was completely blocked by nifedipine, a Ca2+ channel antagonist. 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C activator, similarly stimulated 5'D activity. The addition of a high concentration (20-40 mM) of K+, which caused the depolarization of the membrane had significant stimulatory effects on 5'D activity. Type 1 deiodinase (D1) mRNA was evident in myocardial cells by RT-PCR in a single 758 bp band similar to that in the liver. Cardiac fibroblasts did not express the D1 mRNA. A significant increase in D1 mRNA was also evident after adding T3 and Ang II. These findings indicate that 5'D activity in myocardial cells is increased by activating the voltage sensitive Ca2+ channel, protein kinase C, and membrane depolarization, and that the D1 mRNA is present in cardiac myocytes and is increased by T3 and Ang II. This study therefore suggests that Ang II could affect the action of thyroid hormone on the heart by increasing the D1 gene expression.

Quantitative measurements for type 1 deiodinase messenger ribonucleic acid in human peripheral blood mononuclear cells: mechanism of the preferential increase of T3 in hyperthyroid Graves' disease.

To evaluate the regulatory mechanism of human Type 1 iodothyronine deiodinase (D1) gene expression, we measured the D1 mRNA levels in peripheral blood mononuclear cells (PBMC) in normal control subjects and in patients with Graves' disease. We used competitive reverse transcriptase-polymerase chain reaction with the deleted complimentary RNA of D1 as the standard for quantification. The D1 mRNA levels in PBMC were increased significantly in patients with Graves' disease compared with that in normal controls. There was a significant (p < 0.01) positive correlation (r=0.698) between D1 mRNA level and serum T3 concentration. When PBMC from the normal volunteers were cultured with various doses of T3, the quantity of D1 mRNA increased significantly in a dose-dependent manner. These findings indicate that PBMC D1 mRNA is actually up-regulated by T3 in vivo, and we postulate that a vicious spiral of increasing T3 and D1 is responsible for the exacerbation of thyrotoxicosis in hyperthyroid Graves' disease.

Induction of type 2 deiodinase activity by cyclic guanosine 3',5'-monophosphate in cultured rat glial cells.

We investigated the effects of cyclic guanosine 3',5'-monophosphate (cGMP) on type 2 iodothyronine deiodinase (D2) in cultured rat glial cells. Rat glial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum. When cells were cultured in the presence of 8-bromo cGMP (8-Br cGMP), an analogue of cGMP, D2 activity was increased in a time- and concentration-dependent manner. Lineweaver-Burk plots revealed that the stimulation of D2 activity by 8-Br cGMP (10(-3) M) was associated with fivefold increase in maximum velocity but without a significant change in Michaelis-Menten constant, suggesting that cGMP increases D2 activity via new enzyme synthesis. Both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are well known to increase the intracellular cGMP level via their guanylate cyclase-linked receptors in rat glial cells. In the present study, ANP (10(-6) M) and CNP (10(-6) M) significantly increased the D2 activity in rat glial cells (1.9-fold [ANP] or 2.3-fold [CNP] compared with control activity, respectively). Northern blot analysis demonstrated that D2 mRNA level increased in the presence of 8-Br cGMP (10(-3) M), and reached a plateau (six-fold) after 4 hours of incubation. The increment of D2 mRNA level by 8-Br cGMP was comparable with the increase of the D2 activity by this agent. Our data suggest that cGMP induces rat D2 activity, at least in part, at the pretranslational level, and that ANP and CNP increase D2 activity most likely via their guanylate cyclase-linked receptors in rat glial cells.

A case of iatrogenic growth retardation induced by a corticosteroid-containing anti-allergic drug.

A nine-year old boy developed reduced growth velocity at the age of seven. The peak plasma growth hormone (GH) response to 3,4-dihydroxyphenylalanine, GH-releasing factor and insulin was 10.2, 8.1 and 7.6 micrograms/l, respectively, suggesting that the GH reserve was slightly reduced. Serum cortisol was undetectable and urinary excretion of 17-hydroxycorticosteroid was low (0.22-0.31 mg/day), but there were no physical or biochemical signs of adrenocortical insufficiency. He had taken an anti-allergic drug containing 0.25 mg of betamethasone and 2 mg of d-chlorpheniramine maleate per tablet for about 2 years to treat allergic rhinitis. Catch-up growth occurred when this drug was stopped. The present case suggests that daily administration of 0.25 mg of betamethasone can induce growth retardation and that ingestion of corticosteroid-containing preparations needs to be excluded in children who develop short stature without other symptoms.

Correlation of orbital muscle changes evaluated by magnetic resonance imaging and thyroid-stimulating antibody in patients with Graves' ophthalmopathy.

To evaluate the relationship between eye changes and autoantibody to the thyrotropin receptor in patients with Graves' disease, we evaluated the eye changes using magnetic resonance imaging and the results were correlated with thyroid-stimulating antibody, thyrotropin binding inhibitor immunoglobulin and thyroid growth activity. Subjects were 15 patients with Graves' disease who had Graves' ophthalmopathy, including exophthalmos and other signs and symptoms, and nine patients without ophthalmopathy; all were maintained in a euthyroid state by antithyroid drugs. The thyrotropin-binding inhibitor immunoglobulin was measured by a kit, and thyroid-stimulating antibody and thyroid growth activity were evaluated by cyclic adenosine 3',5'-monophosphate production and [3H]thymidine incorporation, respectively, by cultured functional rat thyroid lined cells. The sum of the swelling ratios (muscle thickness to the diameter of the optic nerve) of the four extraocular muscles correlated well with the degree of exophthalmos. The thyrotropin-binding inhibitor immunoglobulin was positive in nine out of 15 patients with ophthalmopathy; however, no correlation was observed between the activity and exophthalmos or muscle swelling. No significant correlation was observed between muscle changes and thyroid growth activity either. On the other hand, thyroid-stimulating antibody (642 +/- 91%) in Graves' patients with ophthalmopathy was significantly (p < 0.02) higher than that (315 +/- 84%) in patients without ophthalmopathy. Moreover, the level of stimulating activity in Graves' patients with ophthalmopathy showed a significant (p < 0.02) positive correlation with the sum of the swelling ratios of the individual eight eye muscles. These results suggest that thyroid-stimulating antibody has a close relation to Graves' ophthalmopathy.

Thyrotropin and triiodothyronine regulate iodothyronine 5'-deiodinase messenger ribonucleic acid levels in FRTL-5 rat thyroid cells.

We investigated the regulation of type I iodothyronine 5'-deiodinase (5'-D) gene expression by TSH and T3 in FRTL-5 rat thyroid cells. Northern blot analysis revealed that these cells express a 5'-D messenger RNA (mRNA) species of 2.1 kilobases. Readdition of TSH to FRTL-5 cells, precultured in both thyroid hormones and TSH-depleted medium for 4 days, increased 5'-D mRNA levels, reaching a maximum (2.8-fold compared to control) after 12 h of TSH (10 microU/ml) stimulation. Dibutyryl cAMP (DBC) and forskolin mimicked this stimulatory effect of TSH on 5'-D mRNA levels. T3 also increased the 5'-D mRNA levels, reaching a maximum (2-fold compared to control) after 8 h of T3 (10(-9) M) stimulation. Addition of TSH (10 microU/ml) or DBC (1 mM) together with T3 (10(-9) M) further increased 5'-D mRNA levels, reaching a maximum (5-fold compared to control) after 12 h of stimulation. Examination of the rate of disappearance of 5'-D mRNA levels after inhibition of mRNA transcription by actinomycin-D revealed that neither TSH nor T3 significantly affected the rate of disappearance. Cycloheximide, a protein synthesis inhibitor, almost completely blocked the induction of 5'-D mRNA by TSH and DBC, but did not block the induction by T3. These results suggest that both TSH and T3 increase 5'-D mRNA levels probably by increasing transcription rate, and that TSH regulates it, in part via the second messenger cAMP, for which cycloheximide-sensitive de novo protein synthesis is required, whereas T3 does without requiring it.

Identification of a 27-kilodalton protein with the properties of type I iodothyronine 5'-deiodinase in human thyroid gland.

N-Bromoacetyl-[125I]T4(BrAc[125I]T4) was used as affinity label to identify type I 5'-deiodinase (5'-D) in human thyroid glands. Affinity labeled proteins were analyzed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Human thyroid microsomes labeled with BrAc[125I]T4 showed the most prominent radiolabeled band of protein at a mol wt of approximately 27,000 (p27). BrAc[125I]T4 incorporations into p27 were significantly higher in both Graves' and follicular adenomas than in normal thyroids. On the other hand, four cases out of five carcinomas were lower than the least value of normal thyroids. Furthermore, an excellent correlation was observed between 5'-D activities and quantities of p27 in all cases (r = 0.96; P less than 0.001). Labeling of p27 was strongly inhibited by preferred type I 5'-D substrate rT3, but to a lesser extent by poor substrate T4 or T3, and the type I 5'-D inhibitor, propylthiouracil and iopanoic acid, also inhibited the p27 labeling in normal and various diseases. In addition, the rate of enzyme inactivation by BrAcT4 equaled the rate of p27 labeling. These data suggest that p27 may be a type I 5'-D itself or at These data suggest that p27 may be a type I 5'-D itself or at least the substrate-binding subunit of this enzyme in human thyroid, and that both Graves' and follicular adenoma thyroids contain larger amounts of it, and papillary adenocarcinoma thyroids smaller than normal amounts.