Days Of T3 Treatment Research Articles

BNP as a New Biomarker of Cardiac Thyroid Hormone Function.

BackgroundCardiac re-expression of fetal genes in patients with heart failure (HF) suggests the presence of low cardiac tissue thyroid hormone (TH) function. However, serum concentrations of T3 and T4 are often normal or subclinically low, necessitating an alternative serum biomarker for low cardiac TH function to guide treatment of these patients. The clinical literature suggests that serum Brain Natriuretic Peptide (BNP) levels are inversely associated with serum triiodo-L-thyronine (T3) levels. The objective of this study was to investigate BNP as a potential serum biomarker for TH function in the heart.MethodsTwo animal models of thyroid hormone deficiency: (1) 8-weeks of propyl thiouracil-induced hypothyroidism (Hypo) in adult female rats were subsequently treated with oral T3 (10 μg/kg/d) for 3, 6, or 14 days; (2) HF induced by coronary artery ligation (myocardial infarction, MI) in adult female rats was treated daily with low dose oral T3 (5 μg/kg/d) for 8 or 16 wks.ResultsSix days of T3 treatment of Hypo rats normalized most cardiac functional parameters. Serum levels of BNP increased 5-fold in Hypo rats, while T3 treatment normalized BNP by day 14, showing a significant inverse relationship between serum BNP and free or total T3 concentrations. Myocardial BNP mRNA was increased 2.5-fold in Hypo rats and its expression was decreased to normal values by 14 days of T3 treatment. Measurements of hemodynamic function showed significant dysfunction in MI rats after 16 weeks, with serum BNP increased by 4.5-fold and serum free and total T3 decreased significantly. Treatment with T3 decreased serum BNP while increasing total T3 indicating an inverse correlation between these two biologic factors (r2 = 0.676, p < 0.001). Myocardial BNP mRNA was increased 5-fold in MI rats which was significantly decreased by T3 over 8 to 16 week treatment periods.ConclusionsResults from the two models of TH dysfunction confirmed an inverse relationship between tissue and serum T3 and BNP, such that the reduction in serum BNP could potentially be utilized to monitor efficacy and dosing of T3 treatment. Thus, serum BNP may serve as a reliable biomarker for cardiac TH function.

Differential Hepatic Gene Expression Profile and Histone Modification After Acute or Chronic Hyperthyroidism or During Remission.

In acute and chronic hyperthyroid patients, clinical symptoms may not always reflect their thyroid hormone status. After normalization of serum thyroid hormone concentrations in these patients, some symptoms caused by hyperthyroidism may remain unchanged (1). Even in subclinical hyperthyroid patients, functional recovery after restoration to euthyroidism is sometimes incomplete (2). The underlying mechanisms of such incomplete recovery are not yet fully understood. Indeed, functional adaptation or irreversible alteration may be induced during exposure to excess thyroid hormone. The liver is a representative target organ of thyroid hormone action, where T3 regulates various hepatic functions including cholesterol, triglyceride, and carbohydrate metabolism (3). To accomplish this, T3 regulates the expression of many rate-limiting enzymes of such metabolic pathways. In addition, thyroid hormone signaling activates autophagy, which plays a key role in regulating lipid metabolism, mitochondrial quality, and various proteins regulating metabolic pathways in hepatocytes (4). Thus, dysregulation of thyroid hormone action in liver may induce serious clinical conditions such as nonalcoholic fatty liver disease (5). To examine the role of thyroid hormone on the expression of hepatic genes, several groups have applied DNA microarray analysis to the hepatic gene expression profile (6). In these studies, it has become clear that the transcriptomic response is different between acute and chronic T3 treatment (7). However, the expression profile of T3-sensitive genes after amelioration of hyperthyroidism has not yet been studied. In this issue of Endocrinology, Ohba et al (8) examined the effects of a single T3 injection, chronic T3 injections (14 d), or chronic injections followed T3 withdrawal for 10 days on the hepatic gene expression profile of adult male mice using gene expression microarrays. Because they used a large dose of T3 (20 g/100 g body weight), all mice became hyperthyroid, which was confirmed by suppressed TSH levels. TSH levels then increased transiently 5 days after the last T3 injection, before normalizing by 10 days. They identified 680 genes that were induced by either a single or chronic injection of T3. Interestingly, only 10% of these genes were induced by both treatments, indicating that most acutely induced T3-sensitive genes became desensitized to the administration dose over time. Furthermore, 10% of genes did not return to normal levels of expression despite normalization of thyroid hormone concentrations after T3 withdrawal. Although Ohba et al (8) found many genes that were altered by T3 treatment or its withdrawal, such genes may not be directly regulated by thyroid hormone. Thus, they studied further the expression of 8 genes (B cell lymphoma 3 [Bcl3], cytochrome P450 17 -hydroxylase [Cyp17a1], Cyp 17,20-lyase [Cpt1a], fatty acid synthase [Fasn], fibroblast growth factor 21 [Fgf21], isocitrate dehydrogenase 3 [Idh3a], thyroid hormone-responsive protein [Thrsp], and type-1 iodothyronine deiodinase [Dio1]) thought to have functional thyroid hormone response elements. Interestingly, despite this, the expression pattern was not always the same. Among 6 genes that were induced by acute T3 treatment (Bcl3, Cpt1a, Dio1, Fasn, Idh3a, and Thrsp), the expression of 4 genes (Bcl3, Cpt1a, Fasn, and Thrsp) was not elevated after 14 days of T3 treatment, indicating desensitization by chronic treatment, whereas both Dio1 and Idh3a were induced by both

Microarray approach in experimental hyperthyroidism reveals two novel responsive genes in the rat heart (1148.8)

In order to get further insight upon the effects of Thyroid hormone (T3) in the heart, we have used a microarray approach to identify responsive genes in rats submitted to T3 injections for 12, 24h and 7 days. Firstly, we have compared 5 different algorithms to analyze microarray data and found MAS5 to be the best fit in our model. We have detected novel responsive genes at the intercalated disk and sarcomere M‐band levels. qRT‐PCR analysis and western blot confirmed a transient increase in cadherin‐13 expression (2.7 fold for qRT‐PCR and 2.4 for Western Blot, both p&lt;0.05) peaking at 24 hours of T3 treatment and a progressive decrease of M‐protein gene expression (0.2 fold, p &lt;0.05, at 7 days of T3 treatment). Because M‐protein is a structural sarcomeric protein and has a predicted role in sarcomere stability we decided to further investigate cellular/molecular mechanisms. M‐protein is up regulated in hypothyroidism (3.4 fold) and repressed by T3 in a dose dependent manner in vivo and also in cultured neonatal cardiomyocytes. Reporter assays revealed that the M‐protein promoter is responsive to T3. siRNA M‐protein knock down showed reduced contractility, highlighting the importance of this protein for sarcomere stability. We conclude that T3 quickly and strongly repress M‐protein at the transcriptional level, which might help to explain the long‐term deleterious effects of T3 in the heart.Grant Funding Source: FAPESP and CNPq

Muscle RING finger‐1 (MuRF1) inhibits thyroid receptorα transcriptional activity and thyroid hormone‐dependent cardiac hypertrophy

We previously identified that MuRF1 regulates PPARα transcriptional activity in cardiomyocytes by post‐translationally modifying the protein with ubiquitin. To determine if MuRF1 has a broader role in regulating nuclear receptors, we determined MuRF1's effect on thyroid receptor‐α(TRα) activity, the predominant TR subtype in the heart. Using thyroid receptor response element (TRE)‐driven luciferase assays, we found that MuRF1 significantly inhibited triiodothyronine (T3)‐dependent increases in TRα activity. To explore the physiological significance of MuRF1's inhibition of TRα activity, we challenged MuRF1 −/− and MuRF1 transgenic (with cardiac‐specific overexpression) to daily T3 treatment (1 mg/kg/day) for 14 days and followed the heart by echocardiography at baseline, 7 days, and 14 days. After 14 days of T3 treatment, MuRF1−/− animals exhibited a 90.9% increase in LV mass over baseline, while wildtype littermates showed only a 58.9% increase in LV mass over baseline. Conversely, MuRF1 Tg mice showed a 44.4% increase in LV mass over baseline compared to the 73.0% increase in LV mass over baseline exhibited by wildtype littermates. These results suggest MuRF1 inhibits thyroid hormone‐dependent cardiac growth governed, at least in part, by MuRF1's ability to directly interact with TRα, inhibiting its transcriptional activity likely by post‐translational modification with ubiquitin.

Pairing morphology with gene expression in thyroid hormone-induced intestinal remodeling and identification of a core set of TH-induced genes across tadpole tissues

Thyroid hormone (T3) plays a central role in vertebrate post-embryonic development, and amphibian metamorphosis provides a unique opportunity to examine T3-dependent developmental changes. To establish a molecular framework for understanding T3-induced morphological change, we identified a set of gene expression profiles controlled by T3 in the intestine via microarray analysis. Samples were obtained from premetamorphic Xenopus laevis tadpole intestines after 0, 1, 3, and 6 days of T3 treatment, which induces successive cell death and proliferation essential for intestinal remodeling. Using a set of 21,807 60-mer oligonucleotide probes representing > 98% of the Unigene clusters, we found that 1997 genes were differentially regulated by 1.5-fold or more during this remodeling process and were clustered into four temporal expression profiles; transiently up- or downregulated and late up- or downregulated. Gene Ontology categories most significantly associated with these clusters were proteolysis, cell cycle, development and transcription, and electron transport and metabolism, respectively. These categories are common with those found for T3-regulated genes from brain, limb, and tail, although more than 70% of T3-regulated genes are tissue-specific, likely due to the fact that not all genes are annotated into GO categories and that GO categories common to different organs also contain genes regulated by T3 tissue specifically. Finally, a core set of upregulated genes, most previously unknown to be T3-regulated, were identified and enriched in genes involved in transcription and cell signaling.

Tissue-specific regulation of cytochrome c oxidase subunit expression by thyroid hormone.

The influence of thyroid hormone (T(3)) on respiration is partly mediated via its effect on the cytochrome c oxidase (COX) enzyme, a multi-subunit complex within the mitochondrial respiratory chain. We compared the expression of COX subunits I, III, Vb, and VIc and thyroid receptors (TR)alpha1 and TRbeta1 with functional changes in COX activity in tissues that possess high oxidative capacities. In response to 5 days of T(3) treatment, TRbeta1 increased 1.6-fold in liver, whereas TRalpha1 remained unchanged. T(3) also induced concomitant increases in the protein and mRNA expression of nuclear-encoded subunit COX Vb in liver, matched by a 1.3-fold increase in binding to a putative thyroid response element (TRE) within the COX Vb promoter in liver, suggesting transcriptional regulation. In contrast, T(3) had no effect on COX Vb expression in heart. T(3) produced a significant increase in COX III mRNA in liver but decreased COX III mRNA in heart. These changes were matched by parallel alterations in mitochondrial transcription factor A expression in both tissues. In contrast, COX I protein increased in both liver and heart 1.7- and 1.5-fold (P < 0.05), respectively. These changes in COX I closely paralleled the T(3)-induced increases in COX activity observed in both of these tissues. In liver, T(3) induced a coordinated increase in the expression of the nuclear (COX Vb) and mitochondrial (COX I) genomes at the protein level. However, in heart, the main effect of T(3) was restricted to the expression of mitochondrial DNA subunits. Thus our data suggest that T(3) regulates the expression of COX subunits by both transcriptional and posttranscriptional mechanisms. The nature of this regulation differs between tissues possessing a high mitochondrial content, like liver and heart.

Different time course of changes in sarcoplasmic reticulum and myosin isoforms in rat soleus muscle at early stage of hyperthyroidism.

We studied changes in the expression of myosin and sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and in Ca2+ uptake and release rates by the SR in rat slow-twitch soleus muscles treated with thyroid hormone (3,5,3'-triiodo-l-thyronine, T3) for 1, 3, 7 and 21 days. The content of fast SR Ca2+-ATPase isoform SERCA1a protein significantly increased as early as 3 days of T3 treatment, while a rise in fast myosin heavy chain isoforms was found only 21 days after the onset of T3 administration. T3-induced slow-to-fast transitions were much less pronounced in myosin light chain than in myosin heavy chain. Moreover, in vitro measurements of SR Ca2+-handling capacity with indo-1 indicated that increases in uptake rate preceded those of release rate (7 vs. 21 days). These data suggest that changes in the myosin and SERCA expressions seem not to occur in a coordinated manner at the early stage of hyperthyroidism and that intervention with T3 may temporarily produce slow-twitch type I fibres which contain, in addition to slow SERCA2a isoform, the appreciable amounts of fast SERCA1a isoform. Moreover, it is speculated that some fibres in the hyperthyroid soleus muscles may temporarily acquire unique contractile properties distinct from normal fast and slow fibres.

Expression of the myosin heavy chain genes in the tail muscle of thyroid hormone-induced metamorphosingRana catesbeiana tadpoles

In tadpoles of the North American bullfrog, Rana catesbeiana, spontaneous and thyroid hormone (T3)-induced metamorphosis is characterized by regression of the tail, which is preceded by a decrease in total protein synthesis in tail tissues. We have demonstrated that thyroid hormone treatment of a tadpole does not affect the synthesis of all proteins equally in the tadpole tail muscle. For example, the synthesis of myosin heavy chains (MHCs) is depressed within 1 day and decreases to 45% of control values after 5 days of T3 treatment, whereas the decreased synthesis of soluble muscle proteins is transient and returns to above control levels by day 5. To determine whether the hormone-induced decrease in MHC synthesis is the result of changes in the transcription of translation of MHC mRNAs, we isolated cDNAs complementary to five different MHC mRNAs from a tail muscle cDNA library and used them to examine the levels of each MHC mRNA in the tail muscle of T3-treated tadpoles. mRNAs that recognize the cDNAs for these five different MHCs are all expressed in the tadpole tail and limb muscles, as well as in the adult leg muscles. MHC mRNAs unique to tadpole tail were not detected. Interestingly, the relative amounts of mRNA for four of the five MHCs increase in tail muscle after T3 treatment of the tadpole, suggesting that repression of MHC gene expression at the protein level does not result from a decrease in the amount of MHC mRNAs. Rather, these results support the contention that the decreased synthesis of MHCs in the tail muscle of T3-treated tadpoles is caused by this hormone, either directly or indirectly, depressing the translation of the MHC mRNAs in this tissue. These results, coupled with the observation that the synthesis of soluble muscle proteins is depressed only in a transient fashion, suggest that T3 may be initiating the expression of a gene(s) that encodes a protein(s) responsible for inhibiting the translation of the MHCs and, perhaps, other structural proteins in the tadpole tail muscle. Whatever the case, the translational regulation of MHC synthesis occurs well before any degradation of the tail tissue is evident and appears to be one of the earliest events in the hormone-induced cell death program of the tadpole tail muscle.

Response of the rat heart to catecholamines and thyroid hormones.

Catecholamines and thyroid hormones have a similar influence on heart function and metabolism, but this may occur in a differential manner and to a different extent. In this study, the effects of norepinephrine (NE) and of triiodothyronine (T3) were studied in regard to the function of the left (LV) and right ventricle (RV) and to the oxidative pentose phosphate pathway (PPP). NE was applied in rats as continuous i.v. infusion (0.2 mg/kg/h) for three days. T3 was given as daily s.c. injections (0.2 mg/kg) for the same period of time. LV and RV function was measured in the closed-chest trapanal-anesthetized animals using special Millar ultraminature catheter pressure transducers. NE induced an increase in heart rate, in mean arterial pressure, and in total peripheral resistance (TPR). The cardiac RNA/DNA and the left ventricular weight/body weight ratios were increased by about 40%. These effects were prevented by simultaneous alpha- and beta-receptor blockade with prazosin and metoprolol, respectively, but not by verapamil which abolished the hemodynamic effects. RVSP was significantly elevated by NE in a dose-dependent manner. The functional effects of T3 on the LV were not as pronounced as those induced by NE. Heart rate and LV dp/dtmax were increased by T3, and this increase was prevented by concomitant beta-receptor blockade with metoprolol. In contrast to NE, T3 induced an increase in cardiac output and a concomitant decrease in TPR. The RNA/DNA ratio was elevated and cardiac hypertrophy had developed after treatment for three days with T3. These changes were not affected by beta-receptor blockade with metoprolol. RVSP was increased by T3 to a lesser extent than with NE. In metabolic terms it turned out that only NE, but not T3 had a stimulating effect on the cardiac PPP. NE increased the mRNA and activity of glucose-6-phosphate dehydrogenase (G-6-PD), the first and regulating enzyme of this pathway. However, there was no effect of T3 on G-6-PD activity nor on 6-phosphogluconate dehydrogenase activity, one of the following enzymes in the pathway within the first 5 days of T3 treatment. These results demonstrate that the functional effects of T3 were not as pronounced as or even different from those of NE, and that T3 lacked a stimulating effect on the cardiac PPP.

Triiodothyronine reduces growth hormone secretion and pituitary growth hormone mRNA in the chicken, in vivo and in vitro.

The influence of triiodothyronine (T3) on growth hormone (GH) mRNA and GH secretion has been examined in the chicken. Initially T3 treatment in the diet for three days did not alter plasma concentrations of GH. Plasma concentrations of GH were depressed with seven and 14 days of T3 treatment (1 or 5 ppm in the diet). There was a concomitant decline in pituitary GH mRNA with T3 treatment. Pituitary GH content was reduced with 14 but not seven days of T3 treatment. No effect of T3 was observed on the percentage by somatotroph cells in the anterior pituitary gland by fluorescence flow cytometry analysis. While exposure of pituitary cells in vitro from young chickens to GHRF for 2 hr increased GH mRNA, no effect was observed with T3. The presence of T3, for 48 hr in vitro, tended to reduce GH mRNA in adenohypophyseal cells from young chickens and decreased GH mRNA with anterior pituitary cells from adult chicks. It is concluded that T3 chronic administration of T3 depresses circulating concentrations of GH, at least in part, by decreasing GH mRNA and hence GH synthesis.

Effect of L-triiodothyronine on liver microsomal delta 6 and delta 5 desaturase activity of male rats.

The effect of different doses of L-triiodothyronine (T3) on the activity of delta 6 and delta 5 desaturases and lipid fatty acid composition was studied in liver microsomes of male rats. The activity of delta 6 and delta 5 desaturases was decreased 24 and 28%, respectively, in animals administered a daily intraperitoneal dose of 1000 micrograms T3/100 g body wt. for 5 days, whereas with 500 micrograms T3/100 g body wt. only delta 6 desaturase activity was decreased. On the other hand, no enzyme activity changed at a shorter period of hormone treatment. Changes in microsomal fatty acid composition did not seem to be a direct consequence of desaturation activity, since after 1 and 5 days of T3 treatment, the concentrations of 18:2 (n-6) and 20:3 (n-6) decreased and only after 1 day that of 20:4 (n-6) increased in spite of unchanged or decreased delta 6 and delta 5 desaturase activities. Other factors than desaturation activity must be involved in fatty acid composition of thyroid hormone-treated rats, such as diet, membrane lipid synthesis and degradation, fatty acid turn-over and oxidation.

Effect of food deprivation and altered thyroid status on the hypothalamic-pituitary-thyroid axis in the rat.

Propylthiouracil (PTU) was administered to rats for different lengths of time with or without food deprivation on the last 2 days. Within 4 days of PTU treatment peripheral 3,5,3'-tri-iodothyronine (T3) fell to low levels and beta-subunit of thyroid-stimulating hormone (beta-TSH) mRNA increased significantly in the anterior pituitary. Pro-thyrotrophin-releasing hormone (pro-TRH) mRNA in the hypothalamic paraventricular nucleus (PVN) increased significantly in the control group of animals by 8 days and in the food-deprived group by day 12; the increment of pro-TRH mRNA in the food-deprived group on day 12 was significantly less than that in the control group. In a second study, animals were treated with intraperitoneal injections of T3 with or without the food deprivation. After 4 days of T3 treatment, peripheral T3 levels were markedly increased and pro-TRH mRNA in the PVN and beta-TSH mRNA in the anterior pituitary were significantly reduced. Food deprivation had no additional suppressive effect. These studies confirm that the predominant effect of food deprivation on the thyroid axis is at the hypothalamic or suprahypothalamic level and that it can, at least in part, overcome the increase in TRH mRNA due to diminished T3 feedback.

Effects of triiodothyronine in spontaneously hypertensive rats. Studies on cardiac metabolism, function, and heart weight.

We have studied the effects of triiodothyronine (T3) on heart function, on the myocardial oxidative pentose phosphate pathway, and on heart weight in spontaneously hypertensive (SHR) rats. Another aim was to examine whether these T3-effects may be reversible. T3 was administered daily (0.2 mg/kg s.c.) for 14 days. Compared to the untreated SHR controls, T3 induced an increase in heart rate (beats/min) from 357 +/- 10 (n = 17) to 553 +/- 10 (n = 17), in the pressure-rate-product (mm Hg/min) from 78 400 +/- 4500 (n = 15) to 113 700 +/- 4800 (n = 15), and in the heart weight/body weight ratio (mg/g) from 4.2 +/- 0.2 (n = 20) to 5.8 +/- 0.2 (n = 19). The activity of myocardial glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the oxidative pentose phosphate pathway (units/g protein), was elevated from 4.2 +/- 0.2 (n = 9) to 7.0 +/- 0.6 (n = 9) after 14 days of T3-treatment, while the activity of 6-phosphogluconate dehydrogenase, one of the following enzymes in the pathway, was not altered appreciably. These changes returned to the respective control values when T3-treatment was discontinued for 14 days. Our results demonstrate that T3 had a positive chronotropic effect and induced an additional heart enlargement in an animal model with already established cardiac hyperfunction and hypertrophy. The effects on heart function and weight, which were fully reversible, were not as pronounced as in normal Sprague-Dawley rats.

Effects of thyroid hormone on Na+-K+ transport in resting and stimulated rat skeletal muscle.

The effects of hypothyroidism and 3,5,3'-triiodothyronine (T3) treatment on passive Na+-K+ fluxes and Na+-K+ pump concentration were investigated in isolated rat muscle. Within 12 h after a single dose of T3 (20 micrograms/100 g body wt), K+ efflux had increased by 21% in soleus and by 20% in extensor digitorum longus muscle. In the presence of ouabain, even larger effects were observed. These changes were associated with a 12% rise in amiloride-suppressible Na+ influx but no significant increase in [3H]ouabain binding site concentration. After 3 days of T3 treatment, the stimulating effect on K+ efflux and Na+ influx in soleus reached a plateau approximately 80 and 40% above control levels, respectively, whereas the maximum increase in [3H]ouabain binding site concentration (103%) was only fully developed after 8 days. Hypothyroidism decreased 86Rb efflux by 30%. The efflux of K+ and the influx of Na+ per contraction (both approximately 7 nmol/g wet wt) as well as the net loss of K+ induced by electrical stimulation were unaffected by T3 treatment. The rise in resting K+ efflux after 12-24 h of T3 treatment could be partly blocked by dantrolene or trifluoroperazine, indicating that an increase in the cytoplasmic Ca2+ concentration may contribute to the early rise in K+ efflux. It is concluded that the early rise in the resting passive leaks of Na+ and K+ induced by T3 is a major driving force for Na+-K+ pump synthesis.

Thyroid hormone induces synthesis and accumulation of tropomyosin and myosin heavy chain in limb buds of premetamorphic tadpoles.

Myosin heavy chain (MHC) and tropomyosin (Tm) have been isolated from limb muscles of the North American bullfrog, Rana catesbeiana, and injected into rabbits to raise monospecific antibodies. These antibodies were used to study the localization and synthesis of myosin heavy chain and tropomyosin in the limb buds of premetamorphic (stage VI-VII) tadpoles treated with triiodothyronine (T3) to induce metamorphosis. Indirect immunofluorescence localization detects the accumulation of both MHC and Tm in the developing thigh region within 24 h of T3 treatment. During the subsequent 48 h, the accumulation of these proteins is enhanced in the thigh and progresses from thigh to the distal regions of the limb. Quantitative immunochemical determinations indicate that within 24 h of T3 treatment, synthesis of Tm and MHC are increased 23-fold and 6-fold, respectively. Following 5 days of T3 treatment, the synthetic rates of Tm and MHC are 266 and 70 times the control values, respectively. Both methods suggest that Tm is synthesized and accumulated at a greater rate than myosin heavy chain. These observations suggest that T3 promotes the differentiation of muscle in the limb buds of premetamorphic tadpoles and that limb development promoted by T3 in tadpoles is similar to that described during the embryonic development of higher vertebrates.

Hormonal Regulation of Thyrotropin Alpha and Beta Subunit mRNAs

We have examined the effects of 3,5 3'-triiodo-L-thyronine (T3), dexamethasone, bromocriptine, thyrotropin releasing hormone (TRH) and estrogen on the levels of pituitary alpha and TSH-beta protein and mRNA levels in hypothyroid mice. After 3 days of treatment with T3 (0.5 micrograms/100 g body weight) serum TSH, alpha and TSH-beta levels were 77%, 79% and 44% of control, respectively. Pituitary alpha and TSH-beta mRNA content was estimated by dot blot hybridization of total RNA with 32P-labelled alpha and TSH-beta plasmid probes. There was no change in alpha mRNA after 3 days of T3 treatment but TSH-beta mRNA had decreased to 60% of control. With T3 at 2 micrograms/100 g body weight for 3 days, TSH protein was 27% of control and TSH-beta was undetectable, but there was no change in alpha. TSH-beta mRNA was decreased to 40% of control at 1 day and was barely detectable at 3 days, whereas alpha mRNA was 70% of control at 1 day and 42% at 3 days. Dexamethasone and bromocriptine caused no consistent change in pituitary levels of alpha and TSH-beta mRNA. Treatment with TRH caused small increases in serum TSH and in both alpha and TSH-beta mRNA levels. Estrogen treatment increased serum TSH and subunit levels and TSH-beta mRNA, but not alpha. We conclude that thyroid hormones decrease alpha and beta subunit mRNA levels discordantly in both the hypothyroid pituitary and in thyrotropic tumors and that the suppressive effect of thyroid hormone is the major regulator of TSH.

Transcriptional regulation of the thyrotropin subunit genes by thyroid hormone.

Thyrotropin (TSH), a pituitary glycoprotein, consists of two subunits (alpha and beta), and regulates thyroid hormone production by the thyroid gland. The thyroid hormones, in turn, feedback negatively on TSH production. We have investigated the effect of triiodothyronine (T3) on the transcription of the TSH subunit genes. Hypothyroid LAF1 mice bearing TtT 97 thyrotropic tumors were injected daily with T3 (5 micrograms/100 g of body weight, intraperitoneal) and killed after 0, 0.5, 1, 4, or 120 h. Nuclei were isolated from the tumors and allowed to continue RNA synthesis in the presence of [alpha-32P]UTP. Newly synthesized mRNA sequences were quantitated by hybridization to immobilized cloned cDNA containing sequences specific for either alpha subunit or TSH-beta mRNA. Basal levels of mRNA synthesis were similar for alpha subunit and TSH-beta. After only 30 min of T3 treatment in vivo, mRNA synthesis was decreased for both alpha subunit (by 28%) and TSH-beta (by 61%). These decreases were maximum after 4 h, (75% for alpha subunit, greater than 95% for TSH-beta mRNA synthesis), and were maintained for the 5 days of T3 treatment. Decreases in mRNA synthesis precede decreases in tissue mRNA concentrations. Our studies suggest that T3 rapidly decreases the transcription of both the alpha subunit and TSH-beta genes, and that the transcription of the TSH-beta gene is affected to a greater extent than the alpha subunit gene.

Regulation of thyrotropin biosynthesis. Discordant effect of thyroid hormone on alpha and beta subunit mRNA levels.

We have studied the regulation of the biosynthesis of thyrotropin (TSH) and its alpha and beta subunits by thyroid hormone in thyrotropic tumors carried in hypothyroid mice. Treatment with 3,5,3'-triiodo-L-thyronine (T3) (20 micrograms/100 g, body weight) daily for 4 or 10 days reduced serum TSH to 3 and 0.3% of control, respectively. Serum levels of free alpha subunit were reduced to 60 and 11% of control at 4 days and 10 days, respectively, and serum free TSH-beta was undetectable at both time points. There was no significant decrease in tumor TSH content after 4 days of treatment and, after 10 days, TSH content was reduced to 15% of control levels. There was no significant effect of T3 on tumor alpha subunit levels at either 4 or 10 days. In contrast, tumor TSH-beta content was markedly reduced after 4 days and 10 days of T3 treatment, to 29 and 10% of control levels, respectively. Translation of tumor poly(A) mRNA in a rabbit reticulocyte lysate system showed that thyroid hormone decreased translatable TSH-beta mRNA to undetectable levels at both 4 and 10 days, whereas translatable alpha mRNA was reduced strikingly only at 10 days in one of two tumors. RNA blot hybridization with 32P-labeled plasmid probes containing alpha or TSH-beta cDNAs showed that TSH-beta mRNA was reduced to less than 10% of control after both 4 and 10 days of T3 treatment, whereas, again, alpha mRNA was only reduced in one of two tumors at 10 days. Our data thus show that thyroid hormone affects alpha and TSH-beta mRNA and protein levels discordantly and suggest that regulation of TSH biosynthesis may occur predominantly at the level of TSH-beta mRNA.

Regulation of hepatic lipogenesis in starved and diabetic animals by thyroid hormone.

The effects of intragastric feeding with glucose and of the administration of L-triiodothyronine (T3) on in vivo rates of hepatic lipogenesis were investigated in control (fed ad libitum on normal diet), diabetic (fed ad libitum on normal diet), fat-fed (fed ad libitum on high-fat diet), and starved (food removed for 48 h) rats. Two days of T3 treatment increased hepatic lipogenesis in control and fat-fed animals but not in the diabetic or starved animals, although increases in lipogenesis in diabetic animals were observed after 4 days of T3 treatment. Intragastric glucose feeding increased hepatic lipogenesis in the livers of control animals and T3-treated control animals. Such increases are mediated by an increase in the circulating insulin concentration, as increases are not observed in diabetic rats or T3-treated diabetic rats. Glucose feeding failed to increase hepatic lipogenesis in fat-fed rats or starved rats. Insulin injection together with glucose feeding increased lipogenesis in the fat-fed group but not the starved group; i.e., impaired insulin secretion following an oral glucose load may in part explain the lack of response in the fat-fed but not the starved animals. Marked increases in hepatic lipogenesis after glucose feeding were, however, observed if either the starved or the fat-fed animals were treated with T3. The physiological implications of these observations are discussed.

Kinetics of deoxyribonucleic acid synthesis and replication of thyrotrophs and somatotrophs during development of hypothyroidism and L-triiodothyronine treatment of hypothyroid rats.

The rates of DNA synthesis and cell replication were measured in somatotrophs (St) and thyrotrophs (T3) in vivo during the development of hypothyroidism and during T3 treatment of hypothyroid rats. Groups of rats were killed at different times after [3H]thymidine injection for 30 days after thyroidectomy and during 20 days of T3 treatment (10 μg/100 g BW day) of rats thyroidectomized for 30 days. The number of St and Tt per pituitary was determined by a method combining cell counting and DNA measurement in dispersed cells as well as the percentage distribution of cells at the electron microscopic level. [3H]Thymidine incorporation into pituitary DNA was measured, and the distribution of incorporated counts among anterior pituitary cells was assessed by electron microscopic autoradiography. [3H]Thymidine incorporation into St and Tt DNA and the relative specific activity of these cell populations were then calculated. After thyroidectomy, the number of St decreased at a constant fractional rate (1.3% day1)...