Influence of cold exposure and thyroid hormones on regulation of adrenal catecholamines.

Since thyroid hormones influence urinary excretion of catecholamines after exposure to cold, the effects of hyper- and hypo-thyroidism on adrenal tyrosine hydroxylase (TH) (EC 1.14.16.2), phenylethanolamine-N-methyl transferase (PNMT) (EC 2.1.1.28), and serum dopamine-beta-hydroxylase (DbetaH) (EC 1.14.17.1) of rats of 23 and 4 degrees C were studied. TH changes resembled the urinary excretion pattern at 4 degrees C in being higher after 8 days than after 1 day of exposure, and in declining as acclimation occurred. At 23 degrees C, TH activity of hypothyroid rats was significantly higher than in euthyroid or hyperthyroid animals, and after 1 day at 4 degrees C the value increased even more. While in the hypothyroid animals at 4 degrees C the concentration of adrenal catecholamines was less, the epinephrine to norepinephrine ratio was higher than at 23 degrees C. Very high TH activity with a decline in catecholamine concentration suggests that the capacity of TH had been exceeded. PNMT activity was significantly elevated in this group. TH activity was not decreased in the hyperthyroid group at 23 degrees C, and was increased after 8 days at 4 degrees C, suggesting that circulating thyroid hormones have no direct inhibitory effect on TH. Serum DbetaH was elevated after exposure to 4 degrees C, regardless of thyroid hormonal status. The activation of adrenal TH in hypothyroid rats at 23 degrees C and of TH, PNMT, and serum DbetaH at 4 degrees C is probably the result of increased activity of the sympathetic nervous system.     

Upregulation of Slc39a10 gene expression in response to thyroid hormones in intestine and kidney

A novel zinc transporter has been purified and cloned from rat renal brush border membrane. This transporter was designated as Zip10 encoded by Slc39a10 gene and characterized as zinc importer. Present study documents the impact of thyroid hormones on the expression of Zip10 encoded by Slc39a10 gene in rat model of hypo and hyperthyroidism. Serum T(3) and T(4) levels were reduced significantly in hypothyroid rats whereas these levels were significantly elevated in hyperthyroid rats as compared to euthyroid rats thereby confirming the validity of the model. Kinetic studies revealed a significant increase in the initial and equilibrium uptake of Zn(++) in both intestinal and renal BBMV of hyperthyroid rats in comparison to hypothyroid and euthyroid rats. By RT-PCR, Slc39a10 mRNA expression was found to be significantly decreased in hypothyroid and increased in hyperthyroid as compared to euthyroid rats. These findings are in conformity with the immunofluorescence studies that revealed markedly higher fluorescence intensity at periphery of both intestinal and renal cells isolated from hyperthyroid rats as compared to hypothyroid and euthyroid rats. Higher expression of Zip10 protein in hyperthyroid group was also confirmed by western blot. These findings suggest that expression of zinc transporter protein Zip10 (Slc39a10) in intestine and kidney is positively regulated by thyroid hormones.     

Thyroid hormone regulates tubulin expression in mammalian liver. Effects of deleting thyroid hormone receptor-alpha or -beta

Microtubules are made from polymers of alpha/beta dimers. We have observed in rat liver that, on the first day after birth, alpha-subunit is relatively high and beta-subunit low with respect to adult values. In the hypothyroid neonate, both subunits were found to be low, therefore indicating that thyroid hormone (TH) regulates these developmental changes. TH was also found to activate tubulin expression in adult liver, especially beta-subunit. To investigate the role of TH receptors (TRs) in tubulin expression, we analyzed mice lacking TRalpha or TRbeta compared with the wild type in both normal and TH-deprived adult animals. The results suggest that, in vivo, beta-tubulin protein expression in the liver is primarily under TRbeta positive control. In euthyroid mice lacking TRbeta, beta-tubulin expression was low. However, in the corresponding hypothyroid animals, it was found increased, therefore suggesting that the unliganded TRalpha might also upregulate beta-tubulin expression. Accordingly, TH administration to hypothyroid TRbeta-deprived mice reduced their high beta-tubulin expression. In parallel, the relatively high messenger level observed with these hypothyroid animals was reduced to the euthyroid level after T(3) treatment. The microtubular network of the mutant livers appeared, by immunofluorescence confocal microscopy, generally disorganized and drastically reduced in beta-tubulin in mice lacking TRbeta. In conclusion, our results indicate that beta-tubulin is critically controlled by TRbeta in the liver and that both TRs are probably needed to maintain the microtubular network organization of the liver.     

Poly(ADP-ribose) polymerase is affected early by thyroid state during liver regeneration in rats

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA synthesis, DNA repair, and cell replication and transformation, also plays a role in the early steps of liver regeneration induced by partial hepatectomy (PH). PARP and DNA topoisomerase I (Topo I) activities and de novo DNA synthesis were studied during liver regeneration in rats with altered thyroid state. Hepatic PARP activity, evaluated as [(32)P]NAD incorporated into isolated liver nuclei, was inhibited in hyperthyroid rats and increased in hypothyroid animals. In both euthyroid and hyperthyroid rats PARP activity was rapidly stimulated, peaking 6 h after PH. In hypothyroid animals, an early decrease in activity was found, at a minimum of 6 h after PH, followed by an early onset of DNA synthesis. An inverse relationship between PARP and Topo I activities was a shared feature among euthyroid, hypothyroid, and hyperthyroid rats. Together these data show that, in replicating hepatocytes, thyroid hormones exert a regulatory role on PARP activity, which reflects the control of a number of nuclear proteins involved in DNA metabolism.     

Differential regulation of Na,K-ATPase alpha 1, alpha 2, and beta subunit mRNA and protein levels by thyroid hormone

The purpose of this study was to determine the effect of thyroid status on the Na,K-ATPase alpha isoforms and beta in rat heart, skeletal muscle, kidney, and brain at the levels of mRNA, protein abundance, and enzymatic activity. Northern and dot-blot analysis of RNA (euthyroid, hypothyroid, and triiodothyronine-injected hypothyroids = hyperthyroids) and immunoblot analysis of protein (euthyroid and hypothyroid) revealed isoform-specific regulation of Na,K-ATPase by thyroid status in kidney, heart, and skeletal muscle and no regulation of sodium pump subunit levels in the brain. In general, in the transition from euthyroid to hypothyroid alpha 1 mRNA and protein levels are unchanged in kidney and skeletal muscle and slightly decreased in heart, while alpha 2 mRNA and protein are decreased significantly in heart and skeletal muscle. In hypothyroid heart and skeletal muscle, the decrease in alpha 2 protein levels was much greater than the decrease in alpha 2 mRNA levels relative to euthyroid indicating translational or post-translational regulation of alpha 2 protein abundance by triiodothyronine status in these tissues. The regulation of beta subunit by thyroid status is tissue-dependent. In hypothyroid kidney beta mRNA levels do not change, but immunodetectable beta protein levels decrease relative to euthyroid, and the decrease parallels the decrease in Na,K-ATPase activity. In hypothyroid heart and skeletal muscle beta mRNA levels decrease; beta protein decreases in heart and was not detected in the skeletal muscle. These findings demonstrate that the euthyroid levels of expression of alpha 1 in heart, alpha 2 in heart and skeletal muscle, and beta in kidney, heart, and skeletal muscle are dependent on the presence of thyroid hormone.     

Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.     

Thyroid hormones and muscle phenotype: involvement of new signaling pathways

Thyroid hormones (TH) are known to control development, body and muscle growth, as well as to determine muscle phenotype in the adult. TH affect muscle properties through nuclear receptors; they act either by a positive or a negative control on target genes that encode proteins accounting for contractile or metabolic phenotypes. Contractile activity and muscle load also affect muscle phenotype; several intracellular signaling pathways are involved in the transduction of signals related to contractile activity, including the calcineurin/NFAT pathway. Calcineurin activity is negatively controlled by MCIP-1 protein (modulatory calcineurin-interacting protein-1). We recently performed an experiment aimed at examining the specific and combined effects of the pharmacological calcineurin inhibition (using cyclosporin-A CsA administration) and thyroid hormone deficiency. The expected effects of CsA administration were only observed if TH were available, while thyroid deficiency totally blunted the muscle responses to calcineurin inhibition. In conditions of thyroid hormone deficiency, there was no response to the pharmacological inhibition of calcineurin, usually known to induce a slow-to-fast IIA transition associated with an enhancement of mitochondrial biogenesis in normothyroid rats. Moreover, thyroid deficiency markedly decreased the expression of MCIP-1 and MCIP-2 mRNA and proteins, two endogenous calcineurin inhibitors; such results clearly suggest that thyroid hormone and calcineurin pathways are interconnected.     

Acute effects of leptin on 5'-deiodinases are modulated by thyroid state of fed rats.

Leptin has been shown to modulate deiodinase type 1 (D1) and type 2 (D2) enzymes responsible for thyroxine (T4) to triiodothyronine (T3) conversion. Previously, it was demonstrated that a single injection of leptin in euthyroid fed rats rapidly increased liver, pituitary, and thyroid D1 activity, and simultaneously decreased brown adipose tissue (BAT) and hypothalamic D2 activity. We have now examined D1 and D2 activities, two hours after a single subcutaneous injection of leptin (8 microg/100 g BW) into hypo- and hyperthyroid rats. In hypothyroid rats, leptin did not modify pituitary, liver and thyroid D1, and thyroid D2 activity, while pituitary D2 was decreased by 41% (p<0.05) and hypothalamic D2 showed a 1.5-fold increase. In hyperthyroid rats, thyroid and pituitary D1, and pituitary and hypothalamic D2 were not affected by leptin injection, while liver D1 showed a 42% decrease (p<0.05). BAT D2 was decreased by leptin injection both in hypo- and hyperthyroid states (42 and 48% reduction, p<0.001). Serum TH and TSH showed the expected variations of hypo- and hyperthyroid state, and leptin had no effect. Serum insulin was lower in hypothyroid than in hyperthyroid rats and remained unchanged after leptin. Therefore, acute effects of leptin on D1 and D2 activity, expect for BAT D2, were abolished or modified by altered thyroid state, in a tissue-specific manner, showing an IN VIVO interplay of thyroid hormones and leptin in deiodinase regulation.     

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.     

Influence of thyroid states on stress gastric ulcer formation

Thyroid hormones exert a critical developmental and regulatory role on the morphology and biochemistry of gastrointestinal mucosal cells. However, the relationship between thyroid function and stress gastric lesion formation remains undetermined. This study was designed to test the hypothesis that thyroid states may affect the acute development of gastric lesions induced by cold-restraint stress. Normal (euthyroid), hyperthyroid (200 micrograms of T4 i.p. x 7 days) and hypothyroid (thyroidectomized) rats were used. Gastric lesion incidence and severity was significantly (p less than 0.05) increased in hypothyroid rats, whereas in contrast hyperthyroid rats developed significantly less gastric lesions. As anticipated, plasma levels of thyroxin (T4) were significantly (p less than 0.01) elevated in hyperthyroid rats, and undetectable in hypothyroid rats. Acute pretreatment with i.p. cimetidine (100 mg/Kg), but not T4 (200 micrograms/Kg) 1 h prior to stress completely prevented gastric lesions formation in hypothyroid rats. Finally, binding of 3H-dihydroalprenolol to beta-adrenergic receptors on brain membranes prepared from frontal cortex was reduced by 20% in hypothyroid rats after 3 h of stress. These and other data contained herein suggest that thyroid hormones contribute to modulate the responsiveness of the gastric mucosa to stress. The increased rate of ulcerogenesis observed in hypothyroid rats appears to be mediated by gastric acid secretion. The central mechanism of this response may involve decreased brain nonadrenergic receptor function.     

Involvement of L-triiodothyronine in acetylcholine metabolism in adult rat cerebrocortical synaptosomes.

Despite the recently emerging notion of thyroid-hormone involvement in neurotransmission in the adult mammalian brain, adequate evidence for a cellular basis of the process is still lacking. The present study indicates the involvement of thyroid hormones in cholinergic system of the adult rat cerebral cortex. Administration of L-triiodothyronine (T3, 0.025 to 4 microg/g) in single doses increased the synaptosomal acetylcholinesterase (AchE) and Mg2+-ATPase activity maximally at 24 hours in a dose-dependent way. Propylthiouracil (PTU)-treated hypothyroid rats showed a significant increase in AchE and Mg2+-ATPase activity compared to euthyroid rats. T3-treatment on hypothyroid rats decreased AchE activity in synaptosomes compared to the hypothyroid synaptosomal values. Mg2+-ATPase activity found in (PTU + T3)-treated group and T3-treated group remained high. These results predict that T3 stimulates acetylcholine (Ach) metabolism by increasing AchE activity as well as uptake of the released Ach through an increase in synaptosomal Mg2+-ATPase activity. This indicates a positive impact of T3 on the cholinergic system in the adult mammalian brain.     

Thyroid hormones regulate G-protein beta-subunit mRNA expression in vivo

Thyroid hormones exert "permissive effects" on the hormone-sensitive adenylate cyclase. Regulation of the expression of Gi (Gi alpha 2) and Gs by thyroid hormones in vivo was investigated at the level of mRNA. Steady-state levels of the mRNA for Gi alpha 2 and Gs alpha, as well as the G beta-subunits, were quantified using DNA excess solution hybridization analysis. Regulation of protein and mRNA expression in adipose tissue was investigated in hypothyroid, euthyroid, and hyperthyroid rats. In euthyroid animals, steady-state levels of mRNA (amol/microgram RNA) were 13.8, 5.9, and 5.7 for Gs alpha, Gi alpha 2, and G beta 1,2, respectively. Activation of adenylate cyclase by Gs is unaffected by thyroid status. Both Gs alpha and Gs alpha mRNA levels in hypothyroid rats were the same as those of controls (euthyroid). The inhibitory control of adenylate cyclase, in contrast, is markedly potentiated in hypothyroid rats. The expression of G1 alpha s and G beta-subunits was increased in hypothyroidism. Whereas Gi alpha 2 mRNA levels remained essentially unchanged, G beta 1,2 mRNA levels were observed to increase 45% in the hypothyroid state. In the hyperthyroid state G beta 1,2 mRNA levels were observed to decline by 35%. Regulation of G-protein subunit expression, at the level of mRNA, appears to be one component of permissive hormone action on transmembrane signalling.     

Influence of chronically altered thyroid status on the activity of liver mitochondrial glycerol-3-phosphate dehydrogenase in female inbred lewis rats.

The activity of liver mitochondrial flavoprotein-dependent glycerol-3-phosphate dehydrogenase (GPDH) is considered a reliable marker of thyroid status in acute and short-lasting experiments. The aim of this study was to ascertain whether GPDH activity could also be used as an index of thyroid status during chronic experiments over several months. We therefore analyzed GPDH activity in liver mitochondria of female inbred Lewis rats with thyroid status altered for 2 to 12 months. Hyperthyroid state was maintained by triiodothyronine (T (3)) or thyroxine (T (4)) administration, while methimazole was employed for inducing hypothyroidism. We found a seven- and three-fold increase of GPDH activity in female rats after T (3) or T (4) administration, respectively, compared to euthyroid females (8.9 +/- 2.3 nmol/min/mg protein), whereas administration of methimazole reduced the enzyme activity almost to one-third of the euthyroid values. These changes were not significantly influenced by the duration of hyperthyroid or hypothyroid treatment. We conclude that the level of the rat liver GPDH activity could serve as a useful marker for evaluation of hyperthyroid and hypothyroid status in chronic long-lasting experiments on female inbred Lewis rats.     

Impaired serum thyrotropin response to hypothyroidism in mice with disruption of neuromedin B receptor

Neuromedin B (NB), a neuropeptide highly concentrated in pituitary, has been proposed to be an inhibitor of thyrotropin (TSH) secretion. Previous study showed that mice with disruption of neuromedin B receptor (NBR-KO) have higher TSH release in response to thyrotropin-releasing hormone (TRH), although TSH seems to have decreased bioactivity. Here we examined in NBR-KO mice the response of TSH to thyroid hormone (TH) deprivation, obtained by methimazole treatment, or excess, obtained by acute and chronic TH administration. In response to hypothyroidism NBR-KO mice exhibited a lower magnitude increase in serum TSH compared to wild-type (WT) mice (1.7 vs. 3.3-times increase compared to euthyroid values, respectively, P<0.001). One hour after a single T4 injection (0.4 microg/100 g BW), WT and NBR-KO hypothyroid mice presented similar degree of serum TSH reduction (54%, P<0.05). However, 3 h after T4 administration, WT mice presented serum TSH similar to hypothyroid baseline, while NBR-KO mice still had decreased serum TSH (30% reduced in comparison to hypothyroid baseline P<0.05). T3 treatment of euthyroid mice for 21 days, with progressively increasing doses, significantly reduced serum TSH similarly in WT and NBR-KO mice. Also, serum T4 exhibited the same degree of suppression in WT and NBR-KO. In conclusion, disruption of neuromedin B receptor did not interfere with the sensitivity of thyroid hormone-mediated suppression of TSH release, but impaired the ability of thyrotroph to increase serum TSH in hypothyroidism, which highlights the importance of NB in modulating the set point of the hypothalamus-pituitary-thyroid axis at hypothyroidism.