Short-term effects of thyroid hormones during development: Focus on signal transduction

Extranuclear or nongenomic effects of thyroid hormones are mediated by receptors located at the plasma membrane or inside cells, and are independent of protein synthesis. Recently the αVβ3 integrin was identified as a cell membrane receptor for thyroid hormones, and a wide variety of nongenomic effects have now been shown to be induced through binding of thyroid hormones to this receptor. However, also other thyroid hormone receptors can produce nongenomic effects, including the cytoplasmic TRα and TRβ receptors and probably also a G protein-coupled membrane receptor, and increasing importance is now given to thyroid hormone metabolites like 3,5-diiodothyronine and reverse T₃ that can mimick some nongenomic effects of T₃ and T₄. Signal transduction from the αVβ3 integrin may proceed through at least three independent pathways (protein kinase C, Src or mitogen-activated kinases) but the details are still unknown. Thyroid hormones induce nongenomic effects on at least three important Na⁺-dependent transport systems, the Na⁺/K⁺-ATPase, the Na⁺/H⁺ exchanger, and amino acid transport System A, leading to a mitogenic response in embryo cells; but modulation of the same transport systems may have different roles in other cells and at different developmental stages. It seems that thyroid hormones in many cases can modulate nongenomically the same targets affected by the nuclear receptors through long-term mechanisms. Recent results on nongenomic effects confirm the old theory that the primary role of thyroid hormones is to keep the steady-state level of functioning of the cell, but more and more mechanisms are discovered by which this goal can be achieved.     

Thyroxine signal transduction in liver cells involves phospholipase C and phospholipase D activation. Genomic independent action of thyroid hormone

Background  

Numerous investigations demonstrate a novel role of thyroid hormone as a modulator of signal transduction. Protein kinase C (PKC) is critical to the mechanism by which thyroid hormones potentiate both the antiviral and immunomodulatory actions of IFNγ in different cells and regulate the exchange of signalling phospholipids in hepatocytes. Because nothing is known about accumulation of PKC modulator - diacylglycerol in cells treated with T₄, we examined the nongenomic effect of thyroid hormones on DAG formation and phospholipase activation in liver cells.     

Direct actions of prostaglandins E1, A1, and F2α on myocardial contraction

The inotropic and chronotropic actions of prostaglandin (PG) types PGE₁, PGA₁, and PGF_2α were studied in isolated guinea pig right and left atria, and papillary muscles; rabbit atria; and toad ventricular strips in order to more completely define the cardiac contractile properties of PG. All three prostaglandins, in muscle bath concentrations of 10μg/ml, exerted positive inotropic and chronotropic actions on guinea pig atrium. These contractile effects were persistent after removal of PG from the muscle bath and appeared to limit the relative response to a subsequent dose of PG. The inotropic action of PGE₁ was present over a wide range of bath calcium concentrations (1.1 to 4.4 mM/L). Beta adrenergic receptor blockade, histamine blockade, and pretreatment with reserpine failed to significantly affect the inotropic actions of PG. Norepinephrine and histamine produced more potent inotropic and chronotropic effects on guinea pig atria than did PG and these contractile effects did not exhibit persistence or tachyphylaxis. The prostaglandins did not significantly affect dose response curves for norepinephrine inotropic and chronotropic actions. The prostaglandins had no effect on the force or frequency of contraction in rabbit atria. PGE₁ exerted a positive inotropic effect on toad ventricular myocardium whereas PGA₁ had no effect and PGF_2α had a negative inotropic action.     

Leptin,NPY, Melatonin and Zinc Levels in Experimental Hypothyroidism and Hyperthyroidism: The Relation to Zinc

Since zinc mediates the effects of many hormones or is found in the structure of numerous hormone receptors, zinc deficiency leads to various functional impairments in the hormone balance. And also thyroid hormones have important activity on metabolism and feeding. NPY and leptin are affective on food intake and regulation of appetite. The present study is conducted to determine how zinc supplementation and deficiency affect thyroid hormones (free and total T3 and T4), melatonin, leptin, and NPY levels in thyroid dysfunction in rats. The experiment groups in the study were formed as follows: Control (C); Hypothyroidism (PTU); Hypothyroidism+Zinc (PTU+Zn); Hypothyroidism+Zinc deficient; Hyperthyroidism (H); Hyperthyroidism+Zinc (H+Zn); and Hyperthyroidism+Zinc deficient. Thyroid hormone parameters (FT₃, FT₄, TT₃, and TT₄) were found to be reduced in hypothyroidism groups and elevated in the hyperthyroidism groups. Melatonin values increased in hyperthyroidism and decreased in hypothyroidism. Leptin and NPY levels both increased in hypo- and hyperthyroidism. Zinc levels, on the other hand, decreased in hypothyroidism and increased in hyperthyroidism. Zinc supplementation, particularly when thyroid function is impaired, has been demonstrated to markedly prevent these changes.     

Free and total thyroid hormones in humans at extreme altitude

Alterations in circulatory levels of total T₄ (TT₄), total T₃ (TT₃), free T₄ (FT₄), free T₃ (FT₃), thyrotropin (TSH) and T₃ uptake (T₃U) were studied in male and female sea-level residents (SLR) at sea level, in Armed forces personnel staying at high altitude (3750 m) for prolonged duration (acclimatized lowlanders, ALL) and in high-altitude natives (HAN). Identical studies were also performed on male ALL who trekked to an extreme altitude of 5080 m and stayed at an altitude of more than 6300 m for about 6 months. The total as well as free thyroid hormones were found to be significantly higher in ALL and HAN as compared to SLR values. Both male as well as female HAN had higher levels of thyroid hormones. The rise in hormone levels in different ALL ethnic groups drawn from amongst the southern and northern parts of the country was more or less identical. In both HAN and ALL a decline in FT₃ and FT₄ occurred when these subjects trekked at subzero temperatures to extreme altitude of 5080 m but the levels were found to be higher in ALL who stayed at 6300 m for a prolonged duration. Plasma TSH did not show any appreciable change at lower altitudes but was found to be decreased at extreme altitude. The increase in thyroid hormones at high altitude was not due to an increase in hormone binding proteins, since T₃U was found to be higher at high altitudes. A decline in TSH and hormone binding proteins and an increase in the free moiety of the hormones is indicative of a subtle degree of tissue hyperthyroidism which may be playing an important role in combating the extreme cold and hypoxic environment of high altitudes.     

Activity and expression of ecto-5′-nucleotidase/CD73 are increased by thyroid hormones in vascular smooth muscle cells

Extracellular nucleotides ATP, ADP, AMP and adenosine are well known signaling molecules of the cardiovascular system that are involved in several physiological processes: cell proliferation, platelet aggregation, inflammatory processes and vascular tonus. The levels of these molecules are controlled by ecto-NTPDases and ecto-5′-nucleotidase/CD73 (ecto-5′-NT/CD73) actions, which are responsible for the complete ATP degradation to adenosine. The thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), play important roles in the vascular system promoting vasodilatation. Here we investigated the influence of thyroid hormones on the enzyme cascade that catalyzes the interconversion of purine nucleotides in vascular smooth muscle cells (VSMC). Exposure of VSMCs to 50nM T₃ or T₄ did not change ATP and ADP hydrolysis significantly. However, the same treatment caused an increase of 75% in AMP hydrolysis, which was time-dependent but dose-independent. Moreover, T₃ treatment significantly increased ecto-5′-NT/CD73 mRNA expression, which suggests a genomic effect of this hormone upon ecto-5′-NT/CD73. In addition to the importance of the ecto-5′-NT in cell proliferation and differentiation, its overexpression could result in higher extracellular levels of adenosine, an important local vasodilatator molecule.     

Membrane-initiated actions of thyroid hormones on the male reproductive system

The presence of specific nuclear receptors to thyroid hormones, described in prepubertal Sertoli cells, implies the existence of an early and critical influence of these hormones on testis development. Although the mechanism of action thyroid hormones has been classically established as a genomic action regulating testis development, our research group has demonstrated that these hormones exert several effects in Sertoli cells lacking nuclear receptor activation. These findings led to the identification of non-classical thyroid hormone binding elements in the plasma membrane of testicular cells. Through binding to these sites, thyroid hormones could exert nongenomic effects, including those on ion fluxes at the plasma membrane, on signal transduction via kinase pathways, on amino acid accumulation, on modulation of extracellular nucleotide levels and on vimentin cytoskeleton. The evidence of the participation of different K(+), Ca(2+) and Cl(-) channels in the mechanism of action of thyroid hormones, characterizes the plasma membrane as an important microenvironment able to coordinate strategic signal transduction pathways in rat testis. The physiological responses of the Sertoli cells to hormones are dependent on continuous cross-talking of different signal transduction pathways. Apparently, the choice of the signaling pathways to be activated after the interaction of the hormone with cell surface binding sites is directly related to the physiological action to be accomplished. Yet, the enormous complexity of the nongenomic actions of thyroid hormones implies that different specific binding sites located on the plasma membrane or in the cytosol are believed to initiate specific cell responses.     

Increased Oxidative Metabolism and Neurotransmitter Cycling in the Brain of Mice Lacking the Thyroid Hormone Transporter Slc16a2 (Mct8)

Mutations of the monocarboxylate transporter 8 (MCT8) cause a severe X-linked intellectual deficit and neurological impairment. MCT8 is a specific thyroid hormone (T₄ and T₃) transporter and the patients also present unusual abnormalities in the serum profile of thyroid hormone concentrations due to altered secretion and metabolism of T₄ and T₃. Given the role of thyroid hormones in brain development, it is thought that the neurological impairment is due to restricted transport of thyroid hormones to the target neurons. In this work we have investigated cerebral metabolism in mice with Mct8 deficiency. Adult male mice were infused for 30 minutes with (1-¹³C) glucose and brain extracts prepared and analyzed by ¹³C nuclear magnetic resonance spectroscopy. Genetic inactivation of Mct8 resulted in increased oxidative metabolism as reflected by increased glutamate C4 enrichment, and of glutamatergic and GABAergic neurotransmissions as observed by the increases in glutamine C4 and GABA C2 enrichments, respectively. These changes were distinct to those produced by hypothyroidism or hyperthyroidism. Similar increments in glutamate C4 enrichment and GABAergic neurotransmission were observed in the combined inactivation of Mct8 and D2, indicating that the increased neurotransmission and metabolic activity were not due to increased production of cerebral T₃ by the D2-encoded type 2 deiodinase. In conclusion, Mct8 deficiency has important metabolic consequences in the brain that could not be correlated with deficiency or excess of thyroid hormone supply to the brain during adulthood.     

Thyroid hormones induce cell proliferation and survival in ovarian granulosa cells COV434

Numerous evidences indicate that thyroid hormones exert an important role in the regulation of the reproductive system in the adult female. Although a clear demonstration of the thyroid–ovarian interaction is still lacking, it is conceivable that thyroid hormones might have a direct role in ovarian physiology via receptors in granulosa cells. In this study we analyzed if thyroid hormone treatment could affect cell proliferation and survival of COV434 cells. To this aim cell growth experiments and cell cycle analyses by flow cytometry were performed. Secondly the T₃ survival action was tested by TUNEL assay and MD30 cleavage analysis. We showed that T₃, and not T₄, can protect ovarian granulosa cells COV434 from apoptosis, regulating cell cycle and growth in the same cells. The increase in cell growth resulted in an augmented percentage of the cells in the S phase and, in a reduction of the doubling time (18%). Subsequently apoptotic pathway induced by serum deprivation has been evaluated in the cells exposed or not to thyroid hormone treatment. The T₃ treatment was able to remarkably counteract the apoptotic process. Even at the ultrastructural level there was an evident protective effect of T₃ in the cells that, besides the maintenance of the original morphology and, the absence of basophilic cytoplasm, conserved normal junctional areas. Furthermore, the protective T₃ effect evaluated by FACS analysis in the presence of a PI3K inhibitor revealed, as also confirmed by Western Blot on pAkt, that the PI3K pathway is crucial in T₃ survival action. J. Cell. Physiol. 221: 242–253, 2009. © 2009 Wiley‐Liss, Inc     

Nongenomic actions of aldosterone: physiological or pathophysiological role?

The actions of aldosterone are usually divided into persistent genomic mediated by the classical mineralocorticoid receptor versus acute nongenomic actions. Rapid, nongenomic effects of aldosterone have been shown in a variety of tissues, although the physiological relevance of these nongenomic actions remains to be established. There is now growing evidence that both the nongenomic and genomic actions of aldosterone, are mediated via the same classical mineralocorticoid receptor, and there is cross talk between the nongenomic and classical actions of steroid hormones. Activation of tissue-specific, second messenger pathways may contribute to integration of nongenomic and classical actions of aldosterone. Further studies are required to determine the physiological or pathophysiological role of these nongenomic actions of aldosterone and whether they might amplify pathophysiological effects of aldosterone.     

Thyroid-induced changes in the growth of the Liver,Kidney, and Diaphragm of Neonatal Rats

Eu-, hypo- and hyper-thyroid rats were studied 12 days postpartum. Hypothyroidism was induced by administering propylthiouracil (PTU) via the mother's drinking water beteen late gestation and throughout lactation. This procedure effectively blocked the normal early postnatal surge of T₃ and T₄. In contrast, hyperthyroidism was induced in the young pups by daily injections of T₄ from day 3 postpartum. The effects of these experimental manipulations of thyroid status on the rates of protein turnover and growth of the liver, kidney, and diaphragm were studied and compared with measurements made on appropriate euthyroid control tissues. Tissue rates of protein synthesis were decreased in response to hypothyroidism with consequent growth retardation of all three tissues and the whole animal. In contrast, the three body tissues responded very differently to the induction of hyperthyroidism. Hepatic rates of protein synthesis and growth were completely unaffected by thyroid excess. The response of the diaphragm was essentially the reverse of that seen with hypothyroidism, i.e., the enhanced rates of protein synthesis and protein degradation leading to muscle hypertrophy. The rates of protein turnover in the kidney were also increased, but unlike the diaphragm the net result was renal atrophy. Clearly, thyroid hormones influence the normal rapid growth of the neonate and its individual tissues. However, beyond a certain concentration the threshold of responsiveness to these hormones seems to vary between individual tissues. © 1994 Wiley-Liss, Inc.     

Thyroid hormones increase liver and muscle thermogenic capacity in the little buntings (Emberiza pusilla)

Thyroid hormones can increase energy expenditure and stimulate basal thermogenesis by lowering metabolic efficiency. In the present study, we examined the effects of thyroid hormones on basal heat production as well as on several physiological and biochemical measures indicative of thermogenic capacity to test our hypothesis that thyroid hormones stimulate increases in thermogenesis in little buntings. Little buntings that fed on thyroxine (T₄)–laced poultry food of 3 and 5 ppm concentrations showed increases in basal metabolic rate (BMR) during the 3-week acclimation. At the end, these buntings had lower body weights, higher levels of contents of mitochondrial protein, state 4 respiration and cytochrome c oxidase activity in liver and muscle, and higher concentrations of serum triiodothyronine (T₃) and T₄ compared to control buntings. These results support the argument that thyroid hormones play an important role in the regulation of thermogenic ability in buntings by stimulating mitochondrial respiration and enzyme activities associated with aerobic metabolism.     

Selenium,zinc, and thyroid hormones in healthy subjects

Iodothyronine 5′ deiodinase, which is mainly responsible for peripheral T₃ production, has recently been demonstrated to be a selenium (Se)-containing enzyme. The structure of nuclear thyroid hormone receptors contains Zinc (Zn) ions, crucial for the functional properties of the protein. In the elderly, reduced peripheral conversion of T₄ to T₃ with a lower T₃/T₄ ratio and overt hypothyroidism are frequently observed. We measured serum Se and RBC GSH-Px (as indices of Se status), circulating and RBC Zinc (as indices of Zn status), thyroid hormones and TSH in 109 healthy euthyroid subjects (52 women, 57 men), carefully selected to avoid abnormally low thyroid hormone levels induced by acute or chronic diseases or calorie restriction. The subjects were subdivided into three age groups. To avoid under- or malnutrition conditions, dietary records were obtained for a sample of 24 subjects, randomly selected and representative of the whole population for age and sex. Low T₃/T₄ ratios and reduced Se and RBC GSH-Px activity were observed only in the older group. A highly significant linear correlation between the T₃/T₄ ratio and indices of Se status was observed in the older group of subjects (r=0.54;p<0.002, for Se;r=0.50;p<0.002, for RBC GSH-Px). Indices of Zn status did not correlate with thyroid hormones, but RBC Zn was decreased in older as compared with younger subjects. We concluded that reduced peripheral T₄ conversion is related to impaired Se status in the elderly.     

Dynamics of Thyroid Hormones at Early Stages of Development of the Sturgeon Acipenser güldenstadti

Using radioimmunological method, the level and dynamics of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃) were studied in eggs, embryos, and pre-larvae of the Russian sturgeon Acipenser güldenstadti L. The level of hormones in eggs was higher than in the individuals at the stage of hatching. In embryogenesis the content of T₄ and T₃ hormones changed simultaneously, while in pre-larvae, synchronization was absent. The maximal T₄ values in sturgeon pre-larvae were recorded not long before transition to active nutrition. The T₄ level was statistically significantly higher in eggs and larvae developed from the eggs with a high degree of fertility. The problem of participation of thyroid hormones in processes of development and formation of adaptive mechanisms in early ontogenesis of sturgeons is discussed.     

Pyrroloquinoline quinone ameliorates l‐thyroxine‐induced hyperthyroidism and associated problems in rats

Pyrroloquinoline quinone (PQQ) is believed to be a strong antioxidant. In this study, we have evaluated its hitherto unknown role in l ‐thyroxin (L‐T₄)‐induced hyperthyroidism considering laboratory rat as a model. Alterations in the serum concentration of thyroxin (T₄) and triiodothyronine (T₃); lipid peroxidation (LPO) of liver, kidney, heart, muscles and brain; in the endogenous antioxidants such as superoxide dismutase, catalase and glutathione and in serum total cholesterol, high‐density lipoprotien, triglycerides, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and urea were evaluated. Administration of l ‐T₄ (500‐µg kg^?1 body weight) enhanced not only the serum T₃ and T₄ levels but also the tissue LPO, serum SGOT, SGPT and urea with a parallel decrease in the levels of antioxidants and serum lipids. However, on simultaneous administration of PQQ (5 mg kg^?1 for 6 days), all these adverse effects were ameliorated, indicating the potential of PQQ in the amelioration of hyperthyroidism and associated problems. Possibly, the curative effects were mediated through inhibition of oxidative stress. We suggest that PQQ may be considered for therapeutic use for hyperthyroidism after dose standardization. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of excess thyroid hormone on cell death,cell proliferation,and new neuron incorporation in the adult zebra finch telencephalon

Widespread telencephalic neuronal replacement occurs throughout life in birds. We explored the potential relationship between thyroxine (T₄) and cell turnover in the adult male zebra finch. We found that many cells in the zebra finch brain, including long‐projection neurons in the high vocal center (HVC), stained positively with an antibody to thyroid hormone receptors (TR). Labeling was generally weak in the ventricular zone (VZ) that gives rise to new neurons but some proliferative VZ cells and/or their progeny, identified by [³H]‐thymidine labeling, co‐labeled with anti‐TR antibody. Acute T₄ treatment dramatically increased the number of pyknotic and TUNEL‐positive cells in HVC and other telencephalic regions. In contrast, degenerating cells were never observed in the archistriatum or sub‐telencephalic regions, suggesting that excess T₄ augments cell death selectively in regions that show naturally occurring neuronal turnover. VZ mitotic activity was not altered shortly after acute T₄ treatment at a dosage that stimulated cell death, although [³H]‐labeling intensity per cell was slightly reduced. Moreover, the incorporation rates for neurons formed shortly before or after acute hormone treatment were no different from control values. Chronic T₄ treatment resulted in a reduction in the total number of HVC neurons. Thus, hyperthyroidism augmented neuronal death, which was not compensated for by neuronal replacement. Collectively, these results indicate that excess T₄ affects adult neuronal turnover in birds, and raises the possibility that thyroxine plays an important role in the postnatal development of the avian brain and vocal behavior. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 323–341, 2002     

Are thyroid hormones mediators of incubation temperature-induced phenotypes in birds?

Incubation temperature influences a suite of traits in avian offspring. However, the mechanisms underlying expression of these phenotypes are unknown. Given the importance of thyroid hormones in orchestrating developmental processes, we hypothesized that they may act as an upstream mechanism mediating the effects of temperature on hatchling phenotypic traits such as growth and thermoregulation. We found that plasma T₃, but not T₄ concentrations, differed among newly hatched wood ducks (Aix sponsa) from different embryonic incubation temperatures. T₄ at hatching correlated with time spent hatching, and T₃ correlated with hatchling body condition, tarsus length, time spent hatching and incubation period. In addition, the T₃ : T₄ ratio differed among incubation temperatures at hatch. Our findings are consistent with the hypothesis that incubation temperature modulates plasma thyroid hormones which in turn influences multiple aspects of duckling phenotype.