Manipulating thyroid status alters endoplasmic reticulum calcium homeostasis in rat cerebellum

Thyroid-related hormones regulate the efficiency and expression of sarco-endoplasmic reticulum calcium ATPases in cardiac and skeletal muscle. However, little is known about the relationship between thyroid hormones and calcium (Ca2+) homeostasis in the brain. It is hypothesized that manipulating rat thyroid hormone levels would induce significant brain Ca2+ adaptations consistent with clinical findings. Adult male Sprague-Dawley rats were assigned to one of three treatment groups for 28 days: control, hypothyroid (6-n-propyl-2-thiouracil (PTU), an inhibitor of thyroxine (T4) synthesis), and hyperthyroid (T4). Throughout, rats were given weekly behavioral tests. Ca2+ accumulation decreased in the cerebellum in both hyper- and hypothyroid animals. This was specific to different ER pools of calcium with regional heterogeneity in the response to thyroid hormone manipulation. Behavioral tasks demonstrated sensitivity to thyroid manipulation, and corresponded to alterations in calcium homeostasis. Ca2+ accumulation heterogeneity in chronic hyper- and hypothyroid animals potentially explains clinical manifestations of altered thyroid status.     

Hippocampal Up-Regulation of Apolipoprotein D in a Rat Model of Maternal Hypo- and Hyperthyroidism: Implication of Oxidative Stress

Thyroid disorders impair various functions of the hippocampus where thyroid hormone receptors are localized in the brain. Hyper and hypothyroidism are associated with large changes in brain oxidative stress. Apolipoprotein D (APOD) is a conserved glycoprotein that increased in response to oxidative stress in the brain and has been suggested function as an antioxidant in the brain. Thus, the goal of this work was to explore the effect of maternal hypo- and hyperthyroidism on the Apod expression in the pup’s brain regarding changes in oxidative stress. For induction hypo and hyperthyroidism in adult female rats, 100 ppm propylthiouracil (PTU) and 8 ppm levothyroxine administrated 1 month before copulation to the week 3 after delivery in drinking water. The hippocampal region of rat pups was isolated and used for immunohistochemistry and quantitative RT-PCR on postnatal day (PND)5, PND10 and PND20. Results revealed that APOD over-expressed in both hypo- and hyperthyroid groups on PND5, PND10, and PND20. There was a proportional increase between the Apod expression and oxidative stress in the hyperthyroid group but not the hypothyroid in different days. Regarding the wide functions of thyroid hormones, oxidative stress does not suggest to be the only mechanism that involves Apod gene expression in thyroid disturbances.

     

Effects of neonatal hypothyroidism on rat brain gene expression.

To define at the molecular biological level the effects of thyroid hormone on brain development we have examined cDNA clones of brain mRNAs and identified several whose expression is altered in hypothyroid animals during the neonatal period. Clones were identified with probes prepared by subtractive or differential hybridization, and those corresponding to mRNAs altered in hypothyroidism were further studied by Northern blot analysis. Using RNA prepared from whole brains, no effect of hypothyroidism was found on the expression of the astroglial gene coding for glial fibrillary acidic protein. Among genes of neuronal expression, no significant alterations were found in the steady state levels of mRNAs coding for neuron-specific enolase, microtubule-associated protein-2, Tau, or nerve growth factor. N-CAM mRNA increased slightly in hypothyroid brains. In contrast a 2- to 3-fold decrease was found in the mRNA coding for a novel neuronal gene, RC3. This is the first neuronal gene known to be significantly altered at the mRNA level by thyroid hormone deprivation. The abundance of the mRNAs for the major myelin proteins proteolipid protein, myelin basic protein, and myelin-associated glycoprotein, expressed by oligodendrocytes, were also decreased in hypothyroid brains. Developmental studies on RC3 and myelin-associated glycoprotein expression indicated that the corresponding mRNAs accumulate in the brain of normal rats during the first 15-20 days of neonatal life. A similar accumulation occurred in hypothyroid brains, but at much reduced levels. The results demonstrate that thyroid hormone controls the steady state levels of particular mRNAs during brain development.     

Cystic fibrosis as a bowel cancer syndrome and the potential role of CK2

Thyroid hormones are major regulators of postnatal brain development. Thyroid hormones act through nuclear receptors to modulate the expression of specific genes in the brain. We have used microarray analysis to identify novel responsive genes in 14-day-old hypothyroid rat brains, and discovered that synaptosomal-associated protein of 25 kDa (SNAP-25) was one of the thyroid hormone-responsive genes. SNAP-25 is a presynaptic plasma membrane protein and an integral component of the vesicle docking and fusion machinery mediating secretion of neurotransmitters and is required for neuritic outgrowth and synaptogenesis. Using microarray analysis we have shown that SNAP-25 was down-regulated in the hypothyroid rat brain compared with the age-matched controls. Real-time RT-PCR and western blotting analysis confirmed that SNAP-25 mRNA and protein levels decreased significantly in the developing hypothyroid rat brain. Our data suggest that in the developing rat brain, SNAP-25 expression is regulated by thyroid hormone, and thyroid hormone deficiency can cause decreased expression of SNAP-25 and this may on some level account for the impaired brain development seen in hypothyroidism.     

Thyroid dependent maturation of β-adrenergic receptors in the rat lung

β-Adrenergic receptors were identified in membranes of fetal and postnatal rat lung with (?)-[³H]dihydroalprenolol, [³H]DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to adult levels by day 28 of postnatal life. The increase of β-adrenergic receptors occurring between postnatal days 15 and 28 was dependent on thyroxine (T4) in propylthiouracil treated pups. β-Adrenergic receptors on day 28 were identical in euthyroid (PTU + T4) as compared to normal control pups (489±31 and 491±30 femtomoles·mg^?1) however receptors were markedly reduced in 28 day hypothyroid pups (PTU alone), Bmax = 294±21.5, m±S.E. p<0.01. Treatment of the hypothyroid pups with T4 for three days on postnatal day 25 increased β-adrenergic receptors approximately two-fold by day 28. This thyroid hormone dependent increase in lung β-adrenergic receptors occurs between postnatal days 15 and 28 coincident with the known increase in thyroid gland activity in the rat pup.     

Thyroid hormone stimulates rat pro-natriodilatin mRNA levels in primary cardiocyte cultures

Pro-natriodilatin (PND) is the precursor for atrial natriuretic peptide (ANP), a hormone which plays an important role in cardiovascular homeostasis. Since the effects of thyroid hormone (T3) on the cardiovascular and renal systems appear to mimic those elicited by ANP, we studied the effect of T3 on PND gene expression using rat neonatal cardiocytes in primary cultures. Treatment of cardiocytes for 48 h with T3 (5 X 10(-9) M) results in a maximal increase in PND mRNA levels; this increase is two fold in atrial and four fold in ventricular cell cultures. These results taken together with a previous report showing decreased plasma ANP in hypothyroid and increased plasma ANP in hyperthyroid rats suggest that at least some of the cardiovascular and renal effects of T3 may be mediated by a T3-dependent increase in PND gene expression.     

Regulation of Five Tubulin Isotypes by Thyroid Hormone During Brain Development

Nucleic acid probes derived from the 3' noncoding region of five tubulin cDNAs were used to study the effects of thyroid hormone deficiency on the expression of the mRNAs encoding two alpha (alpha 1 and alpha 2)- and three beta (beta 2, beta 4, and beta 5)-tubulin isotypes in the developing cerebral hemispheres and cerebellum. The content of alpha 1, which markedly declines during development in both brain regions, is maintained at high levels in the hypothyroid cerebellum, whereas it is decreased in the cerebral hemispheres. The alpha 2 level also declines during development and is decreased in both regions by thyroid hormone deficiency, but only during the two first postnatal weeks. Thyroid hormone deficiency slightly increases at all stages the beta 2 level in the cerebellum, whereas a decrease is observed at early stages in the cerebral hemispheres. The beta 5 level seems to be independent of thyroid hormone in the cerebral hemispheres, whereas it decreases at early stages in the hypothyroid cerebellum. Finally, the expression of the brain-specific beta 4 isotype is markedly depressed by thyroid hormone deficiency, particularly in the cerebellum. These data suggest that the genes encoding the tubulin isotypes are, directly or not, differently regulated by thyroid hormone during brain development. This might contribute to abnormal neurite outgrowth seen in the hypothyroid brain and therefore to impairment in brain functions produced by thyroid hormone deficiency.     

Effects of experimental hypothyroidism on myelin sheath structural organization

A previous study using the 2′3′cyclic nucleotide 3′phosphodiesterase (CNPase), an oligodendroglial marker that also stain ensheathed fibers, showed a decrease in the number of immunoreactive fibers and a change in the pattern of CNPase immunoreactivity (CNPase+) in hypothyroid animals. CNPase+ fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls. As CNPase, in adult animals, can be found only in regions in which oligodendrocyte cytoplasm remains as internal, external and paranodal loops, it was suggested that the reduction of thyroid hormone levels leads to a failure in myelin compaction. Previous data showed a higher frequency of some abnormalities in myelin sheath as multiple cytoplasmic loops and redundant myelin profiles in mutant animals that present a failure in myelin compaction. The increase in the frequency of these abnormalities (multiple internal and external loops and redundant myelin) indicates a failure in the interrelations between the axons and the oligodendroglial processes. To verify if the thyroid hormone deficiency during CNS development disturbs these interrelations, we evaluated the frequency of the morphological abnormalities (multiple internal and external loops and redundant myelin) in myelin sheath of corpus callosum (cc) in experimental hypothyroidism. Randomic fields were kept by electron microscopy and the analysis of the frequency of morphological abnormalities showed a significant difference in hypothyroid animals at 60-day-old (PND60), with no significant differences at 90-day-old (PND90) animals. The frequency of multiple internal loops is higher in hypothyroid animals at PND60 that indicates a disturbance in the wrapping by the oligodendroglial process. These findings showed that thyroid hormone might modulate the axon-oligodendroglial relationships that are important for the adequate temporal sequence of events that occur during myelinogenesis, with possible consequences on myelin compaction.     

Suckling ability and maternal prolactin levels in hypothyroid rats

Long-Evans rats and their offspring were made hypothyroid by addition of the antithyroid goitrogen 6-N-propylthiouracil (PTU) to the drinking water (0.1%) from the day of parturition. Serum concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH) and thyroxine (T4) were determined by double radioimmunoassay (RIA). From the fifth postnatal day, body weight of PTU-treated pups was significantly lower than that of control rats, and a strikingly elevated serum TSH level and nondetectable amount of T4 were measured both in PTU-exposed mothers and their offspring at Day 10 postpartum. To test the youngs' suckling capability and the amount of maternal milk production, 10- and 15-day-old normal and PTU-treated pups were separated from their mothers for 4 hr in the morning and then reunited and allowed to suckle. Normal pups gained body weight at the end of both the first and second hour postreunion, while PTU pups gained only during the first hour and lost weight in the second hour of testing. When the pups were exchanged between normal and PTU mothers, opposite results were obtained, indicating that the reduced gain in hypothyroid rats was not due to impaired suckling capability, or insufficient sensory stimulation for milk secretion but to a decreased milk production of PTU mothers. In accordance with this, in lactating hypothyroid rats both the basal (presuckling) level and the suckling-induced rise of serum PRL were found significantly depressed.     

Osteopontin gene expression in the aorta and the heart of propylthiouracil-induced hypothyroid mice

Summary It is known that there is abnormal osteopontin (OPN) expression at the sites of atherosclerotic lesions. In the Apolipoprotein E gene knockout (ApoE-KO) mouse, a model of the atherosclerotic process, altered cholesterol metabolism with associated increase in OPN expression is evident at 12–22 weeks in the aorta and at 22 weeks in the heart. In this study, we analyzed another animal model of hypothyroid mice created by ingestion of propylthiouracil (PTU). After 2 weeks of PTU ingestion, the animals had significant decreases in thyroid hormones (T3 and T4) and immediate increases in blood lipids/cholesterol. Hypothyroid mice showed 1.3-, 1.5-, 2-fold increases in blood levels of total cholesterol, triglycerides, and low density lipoprotein-cholesterol respectively. Semi-quantitative RT-PCR analysis showed that hypothyroid mice had 1.4- to 2-fold increases of OPN mRNA expression in the aorta and 1.5-fold increases in the heart. Hypothyroid animals treated with T3 (5 μg/day for 6 days) or statin (0.2 mg/30 g for 2 weeks) reduce blood lipids and aortic OPN mRNA expression. Data obtained with ELISA analyses showed 1.5- and 1.7-fold increases in OPN protein in the aorta (10 weeks) and the heart (22 weeks), respectively. This increase is close to the mRNA expression in both tissues of hypothyroid mice. In addition, western blots showed several variants of OPN protein expressed in the aorta and the heart. The decrease in the 70 kDa OPN is accompanied by an increase in 45 kDa OPN in the aorta of hypothyroid mice. In contrast, only 45 kDa OPN is found in the heart of control and hypothyroid mice. These data indicate that the increase of OPN mRNA and protein expression occurs in cardiovascular tissues of hypothyroid mice.     

Cellular colocalization and coregulation between hypothalamic pro-TRH and prohormone convertases in hypothyroidism

The prohormone convertases (PCs), PC1/3 and PC2, are involved in the tissue-specific endoproteolytic posttranslational processing of many hormonal precursors within the secretory pathway. One important prohormone, pro-thyrotropin-releasing hormone (TRH), is expressed in both hypophysiotropic (where it regulates the secretion of thyroid-stimulating hormone) and nonhypophysiotropic regions of the brain. Pro-TRH is processed at specific sites in the secretory pathway, primarily by PC1/3 followed by PC2. We hypothesized that thyroid hormone status in specific nuclei of the brain would alter pro-TRH processing by inducing changes in PC1/3 and PC2 expression. Therefore, we examined pro-TRH, PC1/3, and PC2 coexpression and coregulation in the paraventricular nucleus (PVN), lateral hypothalamus (LH), and ventromedial nucleus (VMN) of hypothyroid and euthyroid rats. Our results show that 6-n-propyl-2-thiouracil (PTU) treatment producing hypothyroidism induced a significant increase in the expression of PC1/3, PC2, and pro-TRH in the PVN and LH, but not VMN. When confocal studies were performed, an increase in colocalization of PC1/3 or PC2 in pro-TRH was observed only in PVN, a response that was especially prominent in the ventral and medial areas of the PVN. PTU did not regulate colocalization in the VMH or LH. Regulation of colocalization of processing enzyme and prohormone expression is a novel mechanism to alter hormonal biosynthesis.     

Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart.

We have studied the influence of thyroid hormone status in vivo on expression of the genes encoding guanine nucleotide-binding regulatory protein (G protein) alpha-subunits Gs alpha, Gi alpha(2), Gi alpha(3), and both the 36-kDa form (beta 1) and the 35-kDa form (beta 2) of the beta-subunit in rat ventricle. The relative amounts of immunoactive Gi alpha(2) and Gi alpha(3) were greater in ventricular membranes from hypothyroid animals than from euthyroid animals (1.9- and 2.6-fold, respectively). A corresponding 2.3-fold increase in Gi alpha(2) mRNA was observed as well as a 1.5-fold increase in Gi alpha(3) mRNA. The relative amounts of immunoactive beta 1 and beta 2 polypeptides were also increased (2.8- and 1.8-fold, respectively) in the hypothyroid state and corresponded with comparable increases in the relative levels of beta 1 and beta 2 mRNAs. No difference was seen between the amounts of Gi alpha(2), Gi alpha(3), beta 1, and beta 2 in the euthyroid state and the hyperthyroid state. In contrast to these effects of thyroid hormone status on Gi alpha and beta, the steady-state amounts of Gs alpha protein and mRNA were not altered by thyroid hormone status. Thyroid hormone status did not alter sensitivity of adenylyl cyclase to stimulation by sodium fluoride or guanyl-5'-yl imidodiphosphate (GppNHp), nor did it influence GppNHp-induced inhibition of forskolin-stimulated enzyme activity. These results demonstrate that thyroid hormone status in vivo can regulate expression of specific G protein subunits in rat myocardium. However, the physiological consequences of these changes remain unclear.     

Modulation of rat liver mitochondrial antioxidant defence system by thyroid hormone

In the present study the effect of thyroid hormone (T(3)) on oxidative stress parameters of mitochondria of rat liver is reported. Hypothyroidism is induced in male adult rats by giving 0.05% propylthiouracil (PTU) in drinking water for 30 days and in order to know the effect of thyroid hormone, PTU-treated rats were injected with 20 microg T(3)/100 g body weight/day for 3 days. The results of the present study indicate that administration of T(3) to hypothyroid (PTU-treated) rats resulted in significant augmentation of oxidative stress parameters such as thiobarbituric acid reactive substances and protein carbonyl content of mitochondria in comparison to its control and euthyroid rats. The hydrogen peroxide content of the mitochondria of liver increased in hypothyroid rats and was brought to a normal level by T(3) treatment. Induction of hypothyroidism by PTU treatment to rats also resulted in the augmentation of total and CN-sensitive superoxide dismutase (SOD) activities of the mitochondria, which was reduced when hypothyroid rats were challenged with T(3). Although CN-resistant SOD activity of the mitochondria remained unaltered in response to hypothyroidism induced by PTU treatment, its activity decreased when hypothyroid rats were injected with T(3). The catalase activity of the mitochondria decreased significantly by PTU treatment and was restored to normal when PTU-treated rats were given T(3). Total, Se-independent and Se-dependent glutathione peroxidase activities of the mitochondria were increased following PTU treatment and reduced when T(3) was administered to PTU-treated rats. The reduced and oxidised glutathione contents of the mitochondria of liver increased significantly in hypothyroid rats and their level was restored to normal when hypothyroid rats were injected with T(3). The results of the present study suggest that the mitochondrial antioxidant defence system is considerably influenced by the thyroid states of the body.     

Perinatal exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters sex-dependent expression of hepatic CYP2C11

The cytochrome P450 (CYP) isoform CYP2C11 is specifically expressed in the liver of adult male rats, and 5alpha-reductase is specifically expressed in the liver of the adult female rats. The sexually dimorphic expressions of these hepatic enzymes are regulated by the sex-dependent profiles of the circulating growth hormone (GH). However, it is not well known whether hormonal imprinting or activation factors in the neonatal brain influence the sexually dimorphic expression patterns of hepatic enzymes. We therefore examined the effect of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on sex-dependent expressions of hepatic enzymes. Pregnant rats were treated with TCDD at a dose of 0, 200, or 800 ng/kg on gestation day 15, exposing the pups to the chemical. Although the expression of CYP2C11 protein in the livers of male pups on postnatal day (PND) 49 was significantly higher than that of the controls, but the 5alpha-reductase activities in the livers of female pups were not altered by exposure to TCDD. Focusing on perinatal periods, testosterone and estrogen levels significantly increased in the brain of male pups on PND 2. The results suggest that the alteration of testosterone and estrogen levels affect hormonal imprinting in the neonatal brain of male pups, and thus induces a change in the level of male-specific hepatic CYP2C11. We conclude that perinatal exposure to TCDD at low doses may change the sexual differentiation of the neonatal brain in male rats.     

Plasma homocysteine is decreased in the hypothyroid rat

Recent clinical studies have indicated that plasma homocysteine was significantly increased in hypothyroid patients. Since hyperhomocysteinemia is an independent risk factor for cardiovascular disease we investigated homocysteine metabolism in hypothyroid rats. Hypothyroidism was induced in one study by addition of propylthiouracil (PTU) to the drinking water for 2 weeks. In a second study, thyroidectomized and sham-operated rats were used with thyroid hormone replacement via mini-osmotic pumps. Unlike the human hypothyroid patients, both groups of hypothyroid rats exhibited decreased total plasma homocysteine (30% in PTU rats, 50% in thyroidectomized rats) versus their respective controls. Thyroid replacement normalised homocysteine levels in the thyroidectomized rat. Increased activities of the hepatic trans-sulfuration enzymes were found in both models of hypothyroidism. These results provide a possible explanation for the decreased plasma homocysteine concentrations. The hypothyroid rat cannot be used as a model to study homocysteine metabolism in hypothyroid patients.     

Thyroid hormone differentially regulates cellular development in neonatal rat heart and kidney.

The role of thyroid hormone in the control of cardiac and renal cell development was examined in neonatal rats made hyperthyroid by administration of triiodothyronine (T3, 0.1 mg/kg s.c. on postnatal days 1-5) or hypothyroid by administration of propylthiouracil (PTU, 20 mg/kg s.c. given to dams on gestational day 17 through postnatal day 5 and to pups on postnatal days 1-5). Indices of total cell number (total DNA per tissue), cell packing density (DNA per g tissue), and relative cell size (protein/DNA ratio) were evaluated from birth through young adulthood. PTU administration led to primary shortfalls in cell number that were of similar magnitude in both tissues, but persisted somewhat longer in the kidney than in the heart. Deficits in cell packing density and cell size in the hypothyroid animals were secondary to the effect on cell number, displaying smaller magnitudes of effect and a lag in appearance and disappearance of the deficits compared to that for total DNA; indeed, the phase in which tissues were restoring their cell numbers was accompanied by increased cell packing density, reflecting a more rapid restitution of cell numbers than tissue weight or cell size. In contrast to the relatively similar effects of PTU on developing cardiac and renal cells, the effects of T3 were selective for the heart. Although T3 caused general growth impairment, it evoked marked cardiac overgrowth that was accompanied by a striking increase in cell number and a small increase in cell size. The cardiac hyperplasia is unique to the developing animal, as post-replicative heart cells in adult animals show only hypertrophy in response to thyroid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid Hormone Influences Antioxidant Defense System in Adult Rat Brain

The objective of the current study was to find out whether thyroid hormone influences antioxidant defense parameters of rat brain. Several oxidative stress and antioxidant defense parameters of mitochondrial (MF) and post-mitochondrial (PMF) fractions of cerebral cortex (CC) of adult rats were compared among euthyroid (control), hypothyroid [6-n-propylthiouracil (PTU)-challenged], and hyperthyroid (T3-treatment to PTU-challenged rats) states. Oxidative stress parameters, such as thiobarbituric acid-reactive substances (TBA-RS) and protein carbonyl content (PC), in MF declined following PTU challenge in comparison to euthyroid rats. On the other hand, when PTU-challenged rats were treated with T3, a significant increase in the level of oxidative stress parameters in MF was recorded. Hydrogen peroxide content of MF as well as PMF of CC was elevated by PTU-challenge and brought to normal level by subsequent treatment of T3. Although mitochondrial glutathione (reduced or oxidized) status did not change following PTU challenge, a significant reduction in oxidized glutathione (GSSG) level was noticed in PMF following the treatment. T3 administration to PTU-challenged rats had no effect on mitochondrial glutathione status. Total and CN-resistant superoxide dismutase (SOD) activities in MF of CC augmented following PTU challenge. CN-resistant SOD activity did not change when PTU-challenged rats were treated with T3. Although CN-sensitive SOD activity of PMF remained unaltered in response to PTU challenge, its activity increased when PTU-challenged rats were treated with T3. Catalase activity in PMF of CC of PTU-challenged rats increased, whereas the activity was decreased when hypothyroid rats were treated with T3. Similarly, total and Se-dependent glutathione peroxidase (GPx) activities of MF increased following PTU challenge and reduced following administration of T3. Se-independent GPx activity of MF and PMF and glutathione reductase activity of PMF decreased following PTU challenge and did not change further when rats were treated with T3. On the other hand, glutathione S-transferase activity of MF and PMF of CC did not change following PTU challenge but decreased below detectable level following T3 treatment. Results of the current investigation suggest that antioxidant defense parameters of adult rat brain are considerably influenced by thyroid states of the body.