Biomedical subjects

J E Silva

Publications and source records attributed to J E Silva.

At least 37 records · Page 2Linked to original sources

PubMed1996

Interactions among receptors, thyroid hormone response elements, and ligands in the regulation of the rat uncoupling protein gene expression by thyroid hormone.

Uncoupling protein (UCP) is essential to the thermogenic function of brown adipose tissue (BAT). Thyroid hormone stimulates the rat UCP gene through two thyroid hormone response elements (TRE) located upstream of -2,300 and separated by 27 bp. They are an everted repeat (upstream TRE or upTRE) and a direct repeat (down-stream TRE or dnTRE). The goal of the present studies was to investigate whether these TREs interact and how such an interaction could contribute to explain the UCP responsiveness to T3 in vivo. We therefore aimed to define: the heterodimeric partner of the T3 receptor (T3R); the role of T3 in the receptor-receptor and receptors-DNA interactions; how such in vitro interactions relate to the enhancer function of TREs; and how the two TREs interact. Studies included electrophoretic mobility shift assays, utilizing T3R and retinoid X receptors (RXR); DNA footprinting; and transient transfections of HIB-1B cells, a BAT-derived cell line. As in many previously described TREs, the partner of the T3R is RXR. The unliganded T3Rs bind to the TREs as homodimers, which act as repressors of transcription. T3 reduces the binding of T3R homodimers, hence relieving the repression, and stimulates the binding of heterodimers and transcription in proportion to the heterodimer binding to the elements. Although qualitatively similar in these regards, there were important quantitative differences between both TREs. The upTRE binds more T3R homodimers and less T3R-RXR heterodimers than the dnTRE, and T3 more readily facilitates heterodimer binding to the dn- than to the upTRE. These in vitro characteristics are reflected in a lower efficiency of T3 to relieve T3R homodimer-mediated repression and to stimulate transcription through up-than through dnTRE. There were also significant interactions between the two TREs both in the binding of the receptors, T3R and RXR, and in the responsiveness to T3. By itself, each TRE responded modestly to T3, upTRE with lower sensitivity and dnTRE with higher sensitivity than traditional TREs, whereas together, in the context of the gene sequence, they mediated a response greater than the sum of those mediated by each TRE separately, with an intermediate sensitivity to T3. Thus, two TREs that are inadequate to explain the responsiveness of the UCP gene to T3, together form a complex unit appropriate for the regulation of the gene by thyroid hormone. These interactions represent yet another way TREs can shape up the responsiveness of genes to thyroid hormone.

Adipocytes↗

PubMed1996

A complex retinoic acid response element in the uncoupling protein gene defines a novel role for retinoids in thermogenesis.

Retinoids have been implicated in the control of cell proliferation and differentiation, and in several developmental processes. We report here the molecular bases for a metabolic role of RA, by showing that the expression of the uncoupling protein (UCP), the key element in brown adipose tissue (BAT) thermogenesis, is stimulated by retinoic acid (RA). Both all-trans-RA and 9-cis-RA powerfully increase UCP messenger RNA levels in isolated rat brown adipocytes. Transient transfection experiments in HIB-1B cells, a BAT-derived cell line, identified the sequence -2399/-2490 (called R90) as the RA-responsive sequence in the rat UCP gene. R90 mediated a 20- to 70-fold stimulation of the chloramphenicol acetyl transferase reporter gene by maximal concentrations of all-trans-RA or 9-cis-RA. Non-BAT cells were significantly less responsive. RA effect was also less when chloramphenicol acetyl transferase gene was driven by a heterologous promoter instead of the UCP minimal promoter. By footprinting and site-directed mutagenesis, we identified three discrete sequences as being essential for the RA response within R90, thus defining the complex RA response element (RARE) of this gene. Critical bases in these sequences are arranged in pairs of putative half-sites. RAR gamma-RXR heterodimers can bind to the R90 as revealed by electrophoretic mobility shift assays using in vitro translated receptors, and HIB-1B nuclear extracts with anti-RAR gamma or anti-RXR antibodies. The participation of RAR gamma-RXR heterodimers in RA stimulation is further supported by transient transfection experiments overexpressing selected receptors and dose-response analyses of RA isomers and analogues. These results show that retinoids strongly stimulate the rat UCP gene expression through a complex RARE, composed of three pairs of half-sites, and define a novel role for retinoids in the regulation of facultative thermogenesis and energy expenditure.

Adipocytes↗

PubMed1996

Amplification of the parathyroid hormone-related peptide gene in a colonic carcinoma.

PTH-related peptide (PTHrP) is the major factor responsible for humoral hypercalcemia of malignancy. This paraneoplastic syndrome has been described in association with a number of malignancies, but rarely with carcinoma of the colon. Moreover, little is known about the molecular mechanisms that underlie PTHrP overexpression in tumors. Here we report a patient who presented with hypercalcemia 6 months after resection of a neuroendocrine colonic carcinoma (tumor I). At the time of admission, intact PTH was decreased, circulating PTHrP levels were elevated, and there was tumor recurrence (tumor II). Immunohistochemical staining of paraffin-embedded sections from tumor I did not stain for PTHrP, whereas cells from tumor II stained intensely positive. Southern blot analysis and differential PCR of genomic DNAs from tumor specimens and the patient's leukocytes demonstrated amplification of the PTHrP gene in tumor II. Moreover, staining for p53 protein was evident in tumor II, but not in tumor I, consistent with the presence of a mutant form of p53 and associated loss of tumor suppressor function in the recurrent tumor. PTHrP gene amplification was also detected in one of five other tumors associated with humoral hypercalcemia of malignancy. These findings suggest that a potential mechanism contributing to PTHrP overexpression in malignancies is gene amplification, which could arise from increased genomic instability associated with the progressive stages of neoplasia.

Aged↗

PubMed1995

Thyroid hormone control of thermogenesis and energy balance.

The mechanisms whereby thyroid hormone increases heat production have been analyzed with emphasis in more recent developments. Thyroid hormone increases obligatory thermogenesis as a result of the stimulation of numerous metabolic pathways involved in development, remodeling, and delivery of energy to the tissues. In addition, thyroid hormone may specifically stimulate some thermogenic mechanisms selected during evolution of homeotherms (e.g., Na/K-ATPase, Ca2+ cycling in muscle). Thyroid hormone also plays an essential role in facultative thermogenesis interacting with the sympathetic nervous system (SNS) at various levels. Peripherally, thyroid hormone potentiates the effects of the SNS at the level of the adrenergic receptor and adenylyl cyclase complex as well as distal from this point. Synergistic interactions between T3 and cAMP on the regulation of gene expression have been described. Brown adipose tissue (BAT) T4-5'-deiodinase plays a central role in controlling heat production. When this enzyme is stimulated by norepinephrine in the euthyroid and hypothyroid condition, it provides high concentrations of T3 to BAT; inhibition by T4 in hyperthyroidism may limit brown fat thermogenic responses. Also, thyrotoxicosis uniquely reduces the expression of beta 3-adrenergic receptors in brown adipose tissue, and the increased obligatory thermogenesis of this condition, via afferent neural pathways, may reduce the hypothalamic stimulation of brown fat, providing additional mechanisms to limit brown adipose tissue thermogenesis in hyperthyroidism.

Animals↗

PubMed1995

Delineation of thyroid hormone-responsive sequences within a critical enhancer in the rat uncoupling protein gene.

Uncoupling protein (UCP) is essential to brown adipose tissue (BAT) thermogenesis and, hence, to cold adaptation and energy balance. The sympathetic nervous system, via norepinephrine and cAMP, and thyroid hormone seem to be the major regulators of UCP expression. T3 potentiates the effect of norepinephrine and is essential for the adaptive response of this protein to cold. The goal of the present studies was to investigate whether T3 directly stimulates the transcription of the rat UCP gene, as suggested by in vivo results, and if so, to identify and characterize the sequences involved. We examined the gene sequence between 114 and -3623 by transient transfection analysis in JEG-3 and HIB-1B cells, a BAT-derived cell line. This 3.7-kilobase UCP insert makes the reporter gene responsive to cAMP (4-fold), T3 (4-fold), or both combined (12-fold). We identified an 82-basepair (bp) restriction fragment between -2317 and -2399, which we called thyroid hormone response sequence (THRS), that conferred T3 responsiveness to the UCP minimal promoter (4- to 12-fold) as well as to the thymidine kinase promoter (3- to 6-fold). T3 receptor bound to THRS in vitro, retarding its migration in electrophoretic mobility shift assays. Footprinting of THRS revealed two potential thyroid hormone response elements (TRE) separated by 27 bp: upTRE, -2391/-2376, 5'ACCCCTACTGAGGCAA; and dnTRE, -2348/-2334, 5'AGGGCAGCAAGGTCA. The mutation of these putative TREs caused loss of both T3 receptor binding and transactivation by T3. The analysis of the mutants also demonstrated that both TREs contribute in similar proportion to the T3 responsiveness of the UCP gene and that dnTRE is necessary for the potentiation of the cAMP effect by T3. Both TREs are located within a previously identified 212-bp enhancer element, flanked by sequences considered essential for BAT expression and norepinephrine responsiveness. Although they do not mediate thyroid hormone responsiveness, the sequences flanking THRS increase basal reporter expression and enhance the responses to T3. In conclusion, our results indicate that T3 can stimulate the transcription of the UCP gene and amplify the effect of cAMP acting directly on the gene. The presence of two functional TREs in a location critical to the control of the gene supports the importance of thyroid hormone for its expression and suggests the potential for interactions at the gene level that may explain the complexity of UCP regulation in vivo.

Animals↗

PubMed1995

Effects of thyroid hormone on norepinephrine signaling in brown adipose tissue. I. Beta 1- and beta 2-adrenergic receptors and cyclic adenosine 3',5'-monophosphate generation.

Brown adipose tissue (BAT) thermogenesis is activated by the sympathetic nervous system. BAT responses to norepinephrine are blunted in hypothyroidism and are rapidly restored by thyroid hormone. We examined in rats the effects of thyroid hormone on BAT beta 1- and beta 2-adrenergic receptors (AR) expression and capacity to generate cAMP in response to adrenergic stimulation. Both are reduced in hypothyroidism. The reduction in cAMP generation is equal to or greater than that in beta 1,2-AR; it is the same whether cAMP production is stimulated with norepinephrine, selective beta 3-AR agonists, or forskolin; and it is not affected by the inhibition of phosphodiesterase. Both beta 1,2-AR and the capacity to generate cAMP were slowly corrected by thyroid hormone. T3 normalized beta 1,2-AR between 1 and 2 days, whereas the improvement in cAMP generation lagged 1 or 2 days behind. Within 2 days of acclimation of athyreotic rats at 30 C, the number of beta 1,2-AR reached the euthyroid level, whereas exposure to 4 C decreased these receptors. We reached the following conclusions: 1) BAT beta 1,2-AR and capacity to generate cAMP are reduced in hypothyroidism; 2) the latter, however, is not explained by the reduction in beta 1,2-AR, but, rather, reflects a fault at the postreceptor level; 3) the reduction in beta 1.2-AR number is largely caused by the cold stress derived from the low metabolic rate of the hypothyroid state; and 4) the slow restoration of both receptor number and capacity to generate cAMP after T3 are not consistent with these defects being a significant factor in the previously reported blunted uncoupling protein responses to adrenergic stimulation in hypothyroidism.

Adipose Tissue, Brown↗

PubMed1995

Thyroid hormone and norepinephrine signaling in brown adipose tissue. II: Differential effects of thyroid hormone on beta 3-adrenergic receptors in brown and white adipose tissue.

UNLABELLED: beta 3-adrenergic receptors (AR) are predominantly expressed in brown (BAT) and white adipose tissue (WAT), being more abundant in the former. There is growing interest in these receptors because of their potential to be pharmacologically targeted to control energy expenditure and lipid accretion. We have examined, in BAT and WAT of rats, the effect of thyroid hormone on the expression of beta 3-AR and their contribution to cAMP generation. Receptor density was assessed with the nonselective beta 3-AR ligand [125I]-cyanopindolol ([125I]-CYP) and the highly selective beta 3-AR agonist CL3216,243. beta 3-AR messenger RNA (mRNA) was analyzed by Northern blotting of total BAT and WAT RNA. Generation of cAMP by brown adipocytes in response to norepinephrine (NE) and CL316,243 was measured by RIA. In BAT, beta 3-AR can account for as much as 50% of the maximal cAMP response to NE, whereas in WAT these receptors probably account for all the effect of NE. In hypothyroidism. BAT beta 3-AR number is increased, as are beta 3-AR mRNA (4- to 6-fold) and the relative contribution of these receptors to the maximal cAMP production. In contrast, both beta 3-AR number and mRNA levels are reduced in WAT of hypothyroid rats. The injection of T3 to hypothyroid rats reverts the changes in beta 3-AR within 24 h, and T3 excess causes a greater than 90% reduction in beta 3-AR mRNA in BAT but a 5-fold increase in WAT. In both tissues, hypothyroidism is associated with a marked reduction in the capacity to generate cAMP, but this is not completely restored even after 2 days of a receptor-saturating dose of T3. CONCLUSIONS: 1) thyroid hormone differentially affects the expression of beta 3-AR in BAT and WAT; 2) these effects of T3 are both rapid and marked and seem to take place at a pretranslational level; 3) in both tissues there is a postreceptor defect in the generation of cAMP that is corrected by T3 much later after the changes in beta 3-AR are reversed; and 4) thyroid hormone is among the known factors that most vigorously affect the expression of beta 3-AR.

Adipose Tissue↗

PubMed1992

Transcriptional and posttranscriptional mechanisms in uncoupling protein mRNA response to cold.

Three mechanisms account for the rapid elevation and maintenance of uncoupling protein (UCP) mRNA levels in cold-exposed rats, namely, an increase in the rate of transcription initiation, an increase in the fraction of nascent UCP transcripts undergoing elongation, and stabilization of the mature UCP mRNA. The second mechanism precedes and outlasts the increase in the rate of UCP gene transcription, which is brisk but short lived. After 48 h of cold exposure, mature UCP mRNA levels are maintained elevated solely on the basis of stabilization, since the levels of both transcription initiation and fifth intron-containing transcripts (precursors) have returned to basal. Results in hypothyroid rats given 3,5,3'-triiodothyronine (T3) and in dispersed brown adipocytes show that T3 is involved both in the increase in UCP mRNA precursor level and stabilization of mature UCP mRNA. These mechanisms are rapidly reversed when the rats are returned to thermoneutrality. These coordinated transcriptional and post-transcriptional mechanisms modulating UCP gene expression ensure a rapid increase in the concentration of UCP and prevent further accumulation of the protein as physiologically adequate levels are attained.

Animals↗

PubMed1992

Adenosine 3',5'-monophosphate and thyroid hormone control of uncoupling protein messenger ribonucleic acid in freshly dispersed brown adipocytes.

We intend to develop in vitro model systems to study the hormonal regulation of uncoupling protein (UCP) and its role in brown adipose tissue (BAT) thermogenesis. We report here that UCP mRNA responses to adrenergic and thyroid hormone manipulations in freshly dispersed, mature brown adipocytes mimic in vivo observations. Studies were performed in brown adipocytes obtained from interscapular brown fat of euthyroid or hypothyroid rats. The tissue was dispersed with collagenase, and cells were isolated by floatation over 4% BSA. UCP mRNA in these cells is 2-3 times more abundant than that in the whole tissue, indicating a selection of cells expressing the gene. In cells from euthyroid rats, UCP mRNA is maximally elevated within 2 h of exposure to 1 microM forskolin and 50 ng T3/ml (77 nM total, 0.43 nM free). T3 significantly enhances the effect of forskolin. In the absence of stimulation, UCP mRNA rapidly disappears from euthyroid brown adipocytes, and this can be prevented with the addition of T3 by a mechanism not requiring on-going transcription. In cells from hypothyroid rats, forskolin or isoproterenol plus phenylephrine fail to stimulate UCP mRNA, but within 3 h of exposure to T3, cells recover full responsiveness. As in vivo, a high extracellular concentration of T3 is required for maximal responsiveness of UCP mRNA to cAMP, while T4 can restore responsiveness in physiological concentrations (40 pM). This effect of T4 is prevented by iopanoic acid, a compound that blocks the type II T4 5'-deiodinase. In conclusion, 1) freshly dispersed brown adipocytes retain all of the properties concerning UCP regulation by thyroid hormone and sympathetic nervous system described for brown fat in vivo; 2) the observations made in vivo, thus, represent direct action of norepinephrine and thyroid hormone on these cells; 3) as in vivo, T4 is a better source of intracellular T3 than extracellular T3 for brown adipocytes; hence, the in vivo findings are the result of the cell biology of 5'-deiodinase type II rather than dynamic factors inherent to the in vivo condition; 4) stabilization of mature UCP mRNA by T3 is an important mechanism to maintain the levels of this mRNA elevated under sustained stimulation; and 5) dispersed brown adipocytes and UCP gene products constitute a powerful model to study interactions between the sympathetic nervous system and thyroid hormone at a cellular or molecular level.

Adipose Tissue, Brown↗

PubMed1992

Thyroid hormone receptors and 3,5,3'-triiodothyronine biological effects in FRTL5 thyroid follicular cells.

Specific thyroid hormone (T3) receptors are present in thyroid follicular cells, including the rat FRTL5 clonal line, but little is known about the effects of T3 on the growth and differentiated function of the thyroid. Unlike primary cultures of animal or human thyroid cells, FRTL5 do not secrete appreciable amounts of thyroid hormones. We now have studied the effects of T3 by itself and in combination with TSH and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation into DNA, iodide uptake, and cAMP production in FRTL5. We also have investigated the expression of different c-erbA mRNAs in these cells. Specific binding of T3 to FRTL5 cell nuclei in intact cells occurred with a binding capacity of 0.1-0.15 ng T3/mg DNA and an apparent Kd of 0.4 nM. Using an RNase protection assay on total cellular FRTL5 RNA and specific cRNA probes, we demonstrated the presence of c-erbA alpha and -beta mRNAs, both encoding T3 receptors. Biological effects were assessed in serum-free medium or buffer containing 0.1% BSA after maintaining quiescent culture of cells for at least 5 days in hormone-free medium containing 5% calf serum. T3 alone stimulated a dose-dependent increase in [3H]thymidine incorporation that reached a plateau at 188% of the control value at 10 nM T3. At 10(-11) M TSH, T3 potentiated TSH-stimulated [3H]thymidine incorporation (2.2-fold), but at TSH concentrations greater than 5 x 10(-11) M, T3 had no effect or reduced the response to TSH. T3 potentiated the [3H]thymidine response to 2 and 10 ng/ml IGF-I by 1.5- to 1.7-fold. T3 alone had no effect on iodide uptake, but attenuated iodide uptake stimulated by TSH. T3 was more potent in inhibiting TSH-stimulated iodide uptake than in enhancing TSH-stimulated DNA synthesis. T3 did not affect either basal or TSH-stimulated cAMP accumulation. Thus, in FRTL5 thyroid follicular cells 1) T3 receptors are expressed, as measured by direct binding assays and by the expression of c-erbA mRNAs; and 2) T3 acts as a growth factor and weak antidifferentiation factor. We suggest that T3 may modulate the actions of TSH and growth factors in thyroid epithelium.

Animals↗

PubMed1991

Immunoglobulins of patients recovering from Yersinia enterocolitica infections exhibit Graves' disease-like activity in human thyroid membranes.

Substantial evidence suggests a link between infections with Yersinia enterocolitica (YE) and Graves' disease. We have now examined the sera of 72 patients recovering from YE infection for immunoglobulins that interacted with the TSH receptor in human thyroid membranes. Compared with controls, in concentrations between 1 and 4 mg/mL, patient IgG produced a significant, concentration-dependent inhibition of TSH binding (p less than 0.001) and stimulation of adenylate cyclase activity (p less than 0.005-0.05). Whereas IgG from normal individuals caused no stimulation of adenylate cyclase, IgG from controls caused some concentration-dependent displacement of TSH, as previously reported. However, IgG from convalescents of YE infections was significantly more potent than normal IgG in reducing the binding of TSH to the membrane. Thus, at each examined concentration, YE patients' IgG displaced more TSH than IgG from normal controls. For each milligram per milliliter increment of IgG in the assay, patients' IgG caused a 10.2% inhibition of TSH binding (r -0.90, p less than 0.001), significantly greater than that seen with normal IgG (p less than 0.02). The present studies provide the first demonstration that IgG of patients recovering from YE infections react with the human TSH receptor. The antibodies presumably are produced against the TSH-binding protein present in YE. However, in view of lack of evidence for thyroid dysfunction in the sera of patients recovering from yersiniosis and the presence of TSH-binding proteins in other bacteria, we postulate that infection with YE is neither necessary nor sufficient to cause thyroid autoimmune disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1991

Effect of iodine deficiency and cold exposure on thyroxine 5'-deiodinase activity in various rat tissues.

We measured thyroxine 5'-deiodinase I (T(4)5'D-I) activity in thyroid, liver, and kidney and thyroxine 5'-deiodinase II (T(4)5'D-II) activity in brown adipose tissue (BAT) in rats on a low-iodine diet (LID) to test the possibility that increased deiodinase activity in these tissues might contribute to the maintenance of ther serum 3,5,3'-triiodothyronine (T3) level. Control rats received LID plus KI. Experiments were also performed with LID and LID plus KI rats exposed to cold. T(4)5'D-I activity was greatly increased in the thyroids of LID rats but not in liver or kidney. We consider it likely that increased thyroxine (T4)-to-T3 conversion in the greatly enlarged thyroids of LID rats contributed to the maintenance of serum T3. T(4)5'D-II activity in BAT was markedly increased in LID rats and was further greatly increased on cold exposure. However, we were unable to demonstrate an increase in uncoupling protein mRNA levels in BAT in response to cold in LID rats. We attribute this to the very low serum T4 level, which limits substrate availability. This factor also makes it unlikely that BAT contributes to maintenance of serum T3 in LID rats.

Acclimatization↗

PubMed1991

Central role of brown adipose tissue thyroxine 5'-deiodinase on thyroid hormone-dependent thermogenic response to cold.

As judged by the response of uncoupling protein and key enzymes, brown adipose tissue (BAT) is highly dependent upon the local generation of T3 catalyzed by the tissue type II T4 5'-deiodinase (5'-D-II). In hypothyroid rats treated with T3 or T4, the capacity to withstand cold seems better correlated with the normalization of BAT responses than with the liver thyroid status. 5'D-II is activated by cold via sympathetic nervous system (SNS) stimulation, and the activation generates enough T3 to nearly saturate BAT nuclear T3 receptor (NTR) in euthyroid rats. In hypothyroidism, 5'D-II is highly stimulated by the SNS and hypothyroxinemia. In the present studies we have taken advantage of this situation to test 1) the capacity of 5'D-II to maintain nuclear T3 in rats with various degrees of hypothyroxinemia, and 2) the hypothesis that thyroid hormone-dependent BAT-facultative thermogenesis, rather than the effect of thyroid hormone on obligatory thermogenesis (basal metabolic rate), is the basic mechanism by which thyroid hormone confers protection against acute cold exposure. We treated methimazole-blocked rats (undetectable plasma T4 and T3) for a week with either subreplacement doses of T4 (0.5, 1, 2, and 4 micrograms/kg.day) or replacement doses of T4 or T3 (8 or 3 micrograms/kg.day, respectively). Sources and content of BAT nuclear T3 were studied at 25 C and after 48 h at 4 C by labeling the plasmaborne T3 (T3[T3]) with [131I]T3 and the locally generated T3 (T3[T4]) with [125I]T4. Neither the kinetics of nuclear-plasma exchange of T3[T3], the time of appearance of T3[T4] in BAT nuclei, nor NTR maximal binding capacity (0.71 ng T3/mg DNA) was affected by hypothyroidism. Kinetic analyses indicated a maximal BAT NTR occupancy of 40% at euthyroid serum T3 concentrations if T4 is not present. Replacement with T4 normalized both serum T4 and T3, while replacement with T3 normalized serum T3; for all other doses of T4, serum T4 and T3 concentrations were predictably related to the dose. 5'D-II activity decreased with increasing doses of T4, but for each dose of T4, this activity was 2-4 times greater at 4 C than at 25 C. BAT NTR occupancy normalized with 2 micrograms T4/kg in rats maintained at 25 C and with 4 micrograms T4/kg in cold-exposed rats, although in neither condition were serum T4 and T3 normalized nor more than 30% of NTR occupied by plasma T3.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue, Brown↗

PubMed1990

Factors determining the differential tissue response of Na/K-ATPase to thyroid hormone in the neonatal rat.

The concentration of ouabain binding sites (OBS), a measure of the abundance of sodium/potassium-dependent ATPase (Na/K-ATPase), was measured in cerebral cortex (CC), cerebellum (CBL), brown adipose tissue (BAT), and heart of neonatal rats with congenital hypothyroidism. In euthyroid rats, the concentration of OBS was 50- to 100-fold greater in the nervous tissue than in the nonnervous tissue. Congenital hypothyroidism resulted in a significant reduction in the number of OBS in all four tissues. Although in absolute terms (pmol/mg protein) the reduction was greater in the nervous tissues, in relative terms it was much greater in the nonnervous tissues. The restoration of OBS concentration was much more sensitive to T4 in CC, CBL, and BAT than in heart. In contrast, the response of OBS concentration to T3 in hypothyroid rats was greater in the heart, followed by the BAT and CBL, being minimal in CC. The sensitivity to T4 replacement correlated with the degree of the stimulation of the type II 5'deiodinase (5'D-II) by hypothyroidism, whereas the response to exogenous T3 correlated with the fraction of the tissue T3 that derives from plasma T3 and inversely with the plasma T3 concentration required to saturate 50% of the nuclear receptors as reported in previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1990

Thyroid nodularity after childhood irradiation for lymphoid hyperplasia: a comparison of questionnaire and clinical findings.

Ionizing radiation is a well-established cause of thyroid cancer and nodularity, however, important questions relating to the magnitude of the risk following low-dose medical exposures remain unresolved. To address these issues, we conducted a follow-up study of 1590 individuals treated between 1938 and 1969 with X-rays for childhood lymphoid hyperplasia (av. thyroid dose = 24 cGy) and 1499 individuals treated with surgery only. Thyroid nodularity was determined from self-administered questionnaires completed by 1195 irradiated and 1063 surgically-treated subjects and from clinical examinations of 602 irradiated and 457 non-irradiated subjects. A much higher relative risk (RR) for radiation-induced thyroid nodules was estimated from the questionnaire than from the clinical examination data, 15.8 and 2.7, respectively. (The corresponding estimates of excess RR per cGy were 64 and 7%). Analysis of the examination data revealed a strong dose-response relationship, similar excess RR/cGy for males and females, and an inverse relationship with age at exposure. Although the thyroid gland is one of the most sensitive organs to the neoplastic effects of radiation, the radiation-induced risk of thyroid nodularity reported from questionnaire studies may over-estimate the true risk.

Adult↗

PubMed1990

Effects of congenital hypothyroidism on microtubule-associated protein-2 expression in the cerebellum of the rat.

In view of the defective neurotubule assembly observed in congenital hypothyroidism and the striking morphological abnormalities of the cerebellum in this condition, we have investigated the expression of microtubule-associated protein-2 (MAP2) in the cerebellum of rats with congenital hypothyroidism. Analysis included the measurement of immunoreactive MAP2 and its mRNA. In addition, the intracellular distribution of MAP2 was studied by immunostaining of the appropriate histological preparations. The results showed that the developmental increase in MAP2 is delayed in congenital hypothyroidism, but eventually the concentration of this protein reached normal levels in animals with this condition, even if untreated. These abnormalities in the immunoreactive protein are not paralleled by abnormalities in the abundance of MAP2 mRNA, which was not affected by the thyroid status of the animals. In spite of the normalization of the content of the protein, the distribution of MAP2 in the Purkinje cells of hypothyroid rats remained abnormal. Whereas in euthyroid rats the protein rapidly migrated into the dendrites, in the Purkinje cells of hypothyroid pups, MAP2 remained largely confined to the body and the most proximal part of the dendrites. These results suggest that thyroid hormone affects the expression of MAP2 at translation or posttranslational levels. The abnormality in distribution may result from some posttranslational abnormality of the protein itself or some underlying defect in the function of the neurons. These observations are probably relevant to the abnormalities in cerebellar function seen in animals and humans with untreated congenital hypothyroidism.

Animals↗

PubMed1990

Iodothyronine deiodinase activities in FRTL5 cells: predominance of type I 5'-deiodinase.

FRTL5 cells, a thyroid follicular cell line derived from normal rat thyroid, has been extensively used as a model system to study various aspects of the physiology of the thyroid epithelium. The capacity of these cells to metabolize iodothyronines and to generate T3 from T4 has not been previously examined. Here we studied the deiodination of T4, T3, and rT3 in homogenates of FRTL5 cells. By far, these homogenates were more potent catalyzing the 5'-deiodination (outer ring) of T4 and rT3 than the inner ring deiodination of T4 or T3. Both the production of rT3 and the degradation of newly formed T3 from T4 were very limited. Thus, when T4 was used as a substrate, T3 and iodide accumulated in a linear fashion with time, and initially the amounts of iodide and T3 were approximately equal. rT3 and 3,3'-diiodothyronine were rapidly deiodinated by these homogenates, with the 3'-deiodination of 3,3'-diiodothyronine occurring at a slower rate than the 5'-deiodination of rT3. The iodothyronine 5'-deiodinase activity corresponded to type I, as indicated by the following: the Km for T4 and T3 was in the micromolar range; rT3 was a better substrate than T4 (maximum velocity = 101 vs. 19 pmol/min.mg protein; Km = 0.83 vs. 3.1 microM, respectively); and the kinetics of inhibition by 6n-propyl-2-thiouracil were uncompetitive and substrate dependent, suggesting ping-pong kinetics. The type I 5'-deiodinase activity of FRTL5 cells was distinctly stimulated by TSH. This stimulation seems to be mediated by cAMP and requires serum as a permissive factor. In conclusion, 1) FRTL5 cells exhibit both inner and outer ring iodothyronine-deiodinating activities; 2) iodothyronine 5'-deiodination is by far more active; 3) the 5'-deiodination has been characterized as type I deiodinase based on substrate preference, enzyme kinetics, and inhibitors; 4) in all respect iodothyronine deiodination by FRTL5 cell homogenates proceeded with marked similarity to that in homogenates or microsomes of thyroid glands from several species; and 5) the FRTL5 type I deiodinase is more active than that reported in thyroid tissue and as active as that reported in liver and kidney, the prototype of type I deiodinase-containing tissues. The present studies indicate that FRTL5 cells are an excellent model system to study cellular and biochemical aspects of the regulation of this enzyme as well as its regulation by TSH and putative serum factors.

Animals↗

PubMed1990

Effect of total sleep deprivation on 5'-deiodinase activity of rat brown adipose tissue.

Prolonged sleep deprivation of the rat produces a progressive increase in energy expenditure and an eventual decrease in body temperature, which suggests a profound derangement in thermoregulation. Because increased thermogenic activity in brown adipose tissue (BAT) is a likely mechanism mediating the observed increase in energy expenditure, we focused our attention on the effect of total sleep deprivation on BAT type II 5'-deiodinase (5'D-II), since its activation indicates BAT stimulation and is essential for full BAT thermogenic response. Five euthyroid rats were subjected to total (92%) sleep deprivation (euD-rats). Sharing the sleep deprivation apparatus, yoked control rats (euC-rats) received the same degree of physical stimulation as the D-rats, but were only partially (25%) sleep deprived. Additional cage controls (euCC-rats) were housed in the same room. Since during sleep deprivation the animals undergo a reduction in plasma T4 concentration and inability to maintain body temperature heralds death, an identical study was performed in five trios of hyperthyroid rats (hyperD-, hyperC-, and hyper CC-rats) given daily ip injections of 15 micrograms T4/100 g BW, 10 days before and throughout the deprivation period. Experiments were carried out at an ambient temperature of 29 C, close to thermoneutrality for rats. Sleep deprivation in hyperD-rats was maintained until death seemed imminent (9-14 days), and in euD-rats for 12-15 days. Sleep deprivation induced a significant increase in BAT 5'D-II activity in both hyperD- and euD-rats compared with that in euCC-rats (P less than 0.01). BAT 5'D-II in euC-rats was also significantly higher than that in euCC-rats (P less than 0.05), probably because they were partially sleep deprived. BAT 5'D-II activity in hyperD-rats was increased compared to that in both hyperC- and hyperCC-rats (P less than 0.05), in which the activity was slightly but not significantly lower than that in euCC-rats. No significant differences were observed in liver and kidney type I 5'-D (5'D-I) and in pituitary 5'D-II among euD-rats, euC-rats, and euCC-rats. As expected, the hyperthyroid groups (hyperD-rats, hyperC-rats, and hyperCC-rats) had significantly higher kidney 5'D-I and lower pituitary 5'D-II than the euCC-rats. Liver 5'D-I was also significantly increased in the hyperC-rats and hyperCC-rats, but not in the hyperD-rats. These observations indicate that total sleep deprivation is associated with a marked increase in BAT 5'D-II activity in both euthyroid and hyperthyroid rats.(ABSTRACT TRUNCATED AT 400 WORDS)

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