Thyroid hormone and growth: relationships with growth hormone effects and regulation

For some years, research in the field of growth endocrinology has been mainly focused on growth hormone (GH). However, it appears that GH does not always control growth rate. For instance, it does not clearly influence intra-uterine growth: moreover, although the results of GRF or GH administration appear convincing in rats, pigs or heifers, this is not the case in chickens and lambs. In addition, GH does not always clearly stimulate somatomedin production, particularly diring food restriction and fetal life, and in hypothyroid animals or sex-linked dwarf chickens. In such situations, this phenomenon is associated with a reduced T3 production, suggesting a significant influence of thyroid function on GH action, and more generally, on body growth. In fact, numerous data demonstrate that thyroid hormone is strongly involved in the regulation of body growth. In species with low maturity at birth, such as the rat. T4 and T3 affect postnatal growth eleven days earlier than the appearance of GH influence. In contrast to GH, thyroid hormone significantly influences fetal growth in sheep. Moreover, the body growth rate is clearly stimulated by T3 in dwarf animals. In addition to its complex metabolic effects involved in the general mechanisms of body growth, thyroid hormone stimulates the production of growth factors, particularly EGF and NGF. Moreover, it affects GH and somatomedin production and also their tissue activity. All these results strongly suggest that it would be difficult to study GH regulation and physiological effects without taking thyroid function into account.     

Nerve growth factor concentration in a congenitally hypothyroid mouse model (hyt/hyt) and its responsivity to thyroxine treatment

Earlier our laboratory reported the ontogenic profiles of serum thyroxine (T4) and triiodothyronine (T3) concentrations and nerve growth factor levels in the submandibular gland of Swiss-Webster mice. Further, we demonstrated a responsivity of submandibular gland-nerve growth factor concentrations to exogenously administered T4. To further our understanding of the interactions between thyroid hormones and submandibular gland-nerve growth factor we utilized a congenitally hypothyroid mouse model to examine submandibular gland-nerve growth factor in euthyroid, hypothyroid and hypothyroid-T4 replaced mouse pups. Serum T4 values in the congenitally hypothyroid (hyt/hyt) mice were unmeasurable and their growth in body weight, their incisor eruption, and their eyelid opening were significant delayed. Submandibular gland/body weight ratios were significantly reduced relative to control or heterozygous (+/hyt) animals through 40 days. The increase in submandibular gland-nerve growth factor concentrations observed in normal animals before 20 days was delayed to 35 days in the (hyt/hyt) animals. T4 treatment of (hyt/hyt) animals from 11 or 18 days of age significantly increased mean 40 day submandibular gland/body weight ratios and submandibular gland-nerve growth factor concentrations. However, the 40 day submandibular gland-nerve growth factor levels in treated animals remained significantly below the level of control euthyroid mice. Thus, the possibility of critical time of thyroid hormone replacement for normal submandibular gland maturation has not been shown but must be further explored in this model.     

Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle.

It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiation of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.     

Avian muscular dystrophy: Thyroidal influence on pectoralis muscle growth and glucose-6-phosphate dehydrogenase activity

The pectoralis muscles of dystrophic chickens (line 413) were hypertrophic on the basis of fresh weight and fat-free dry weight. They also had greater DNA content and greater glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities. Of the parameters measured, the largest differences between pectoralis muscles from dystrophic and normal (line 412) chickens were for DNA content and G6PD activity. These parameters were 4.3- and 6.7-fold, respectively, the values for control pectoralis at 5 wk of age. The average number of nuclei per unit length of isolated muscle fiber was also greater (approximately 3-fold) for the dystrophic pectoralis. Body weight and pectoralis fresh weight, fat-free dry weight, DNA content, G6PD activity and 6PGD activity were reduced significantly in propylthiouracil (PTU)-treated normal and dystrophic chickens. Moreover, the effects of PTU were more pronounced in the dystrophic strain. Thyroid deprivation significantly improved the righting ability of the dystrophic chickens, in addition to its influence on muscle hypertrophy and body growth. Thyroxine (T₄) replacement reversed the PTU effects in both strains. Of all the variables measured, total G6PD activity was the most affected by PTU treatment of dystrophic chickens and was only 16% of the control dystrophic value.In addition to the effects of thyroid deprivation on the expression of avian muscular dystrophy, we observed significant differences in thyroid-related variables in the two strains. The average thyroid weight at 4 wk and serum triiodothyronine level at 5 wk for dystrophic chickens were 65 and 76%, respectively, of the normal values. The results that we report here indicate that altered thyroid function affects the expression of avian muscular dystrophy.     

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.     

Mitochondrial respiration in muscle and liver from cold-acclimated hypothyroid rats.

The effects of long-term cold exposure on muscle and liver mitochondrial oxygen consumption in hypothyroid and normal rats were examined. Thyroid ablation was performed after 8-wk acclimation to 4 degrees C. Hypothyroid and normal controls remained in the cold for an additional 8 wk. At the end of 16-wk cold exposure, all hypothyroid rats were alive and normothermic and had normal body weight. At ambient temperature (24 degrees C), thyroid ablation induced a 65% fall in muscle mitochondrial oxygen consumption, which was reversed by thyroxine but not by norepinephrine administration. After cold acclimation was reached, suppression of thyroid function reduced muscle mitochondrial respiration by 30%, but the hypothyroid values remained about threefold higher than those in hypothyroid muscle in the warm. Blockade of beta- and alpha1-adrenergic receptors in both hypothyroid and normal rats produced hypothermia in vivo and a fall in muscle, liver, and brown adipose tissue mitochondria respiration in vitro. In normal rats, cold acclimation enhanced muscle respiration by 35%, in liver 18%, and in brown adipose tissue 450% over values in the warm. The results demonstrate that thyroid hormones, in the presence of norepinephrine, are major determinants of thermogenic activity in muscle and liver of cold-acclimated rats. After thyroid ablation, cold-induced nonshivering thermogenesis replaced 3,5,3'-triiodothyronine-induced thermogenesis, and normal body temperature was maintained.     

Proliferation of bone marrow pro-B cells is dependent on stimulation by the pituitary/thyroid axis

The frequency and absolute number of pro-B, pre-B, and B cells in the bone marrow of the hypothyroid strain of mice are significantly reduced compared with those of their normal littermates. To investigate why this is the case, various B cell developmental processes were examined in the thyroid hormone-deficient mice. These studies revealed that the frequency of pro-B cells in the S-G2/M phase of the cell cycle was significantly reduced in hypothyroid mice. That thyroid hormone deficiency was responsible for this proliferation defect was established by demonstrating that treatment of hypothyroid mice with thyroxine resulted in a specific increase in the frequency and total number of cycling pro-B cells. The latter effect was paralleled by increases in the frequency and number of bone marrow B lineage cells. Additional in vitro experiments revealed that at least some thyroid hormone effects were directly mediated on the bone marrow. Taken together, these data demonstrate that thyroid hormones are required for normal B cell production in the bone marrow through regulation of pro-B cell proliferation and establish a role for the pituitary/thyroid axis in B cell development.     

Effects of thyroid hormone deficiency on lipolysis in chicken fat cells

Effect of lack of thyroid hormones on lipolysis in chicken fat cells was studied. Isolated fat cells from hypothyroid chickens in contrast to normal animals, have an impaired ability to give lipolytic response to glucagon. However activation of triglyceride lipolysis was induced to the same level by theophylline in hypothyroid and normal chickens.     

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.     

Effect of mammalian growth hormone and prolactin on the growth of hypophysectomized chickens

Body weight gain and shank-toe growth during a 26-day treatment period following hypophysectomy were 55 and 46%, respectively, of control values, but the body weight gain was unaffected and bone growth only slightly reduced when the hypophysectomized chickens were fed a low dose of corticosterone (5 ppm). Bovine growth hormone (0.5 mg GH/kg body wt/day for 18 days) enhanced body weight gain and shank-toe length increase (an estimate of bone growth) by 46 and 33%, respectively, compared to the growth of hypophysectomized chickens receiving only corticosterone. These same endpoints were increased approximately 24% after ovine growth hormone treatment in hypophysectomized chickens not receiving corticosterone. Body weight gain during 18 days of treatment with bovine prolactin (0.5 mg PRL/kg/day) was 27% greater than the value for corticosterone-treated hypophysectomized chickens, but bone growth was unaffected. The mammalian GH preparations increased heart weight of the hypophysectomized chickens (25-29%), but pectoralis muscle weight was unaffected. GH treatment enhanced thymal weights by 71% in corticosterone-treated hypophysectomized chickens, and by 93% in hypophysectomized animals not receiving corticosterone. GH had no significant effect on bursal weights, and PRL had no effect on either of these lymphoid organ weights in corticosterone-treated hypophysectomized chickens. GH increased liver and adipose tissue weights considerably more than the large increases that followed treatment of hypophysectomized chickens with corticosterone alone (69 and 126% greater, respectively), but had no effect on these endpoints in hypophysectomized chickens not receiving corticosterone. PRL also greatly increased liver and adipose tissue weights in corticosterone-treated hypophysectomized chickens (79 and 75%, respectively). These results provide evidence that mammalian GH enhances body weight gain, bone growth, and the growth of several organs in the hypophysectomized chicken. Mammalian PRL increased body weight gain, liver weight, and adipose tissue weight in corticosterone-treated hypophysectomized chickens, but did not influence bone growth or the weights of the heart, pectoralis, thymi, or bursa.     

A thyrotoxic skeletal phenotype of advanced bone formation in mice with resistance to thyroid hormone

Thyroid hormone (T3) regulates bone turnover and mineralization in adults and is essential for skeletal development during childhood. Hyperthyroidism is an established risk factor for osteoporosis. Nevertheless, T3 actions in bone remain poorly understood. Patients with resistance to thyroid hormone, due to mutations of the T3-receptor beta (TRbeta) gene, display variable phenotypic abnormalities, particularly in the skeleton. To investigate the actions of T3 during bone development, we characterized the skeleton in TRbetaPV mutant mice. TRbetaPV mice harbor a targeted resistance to thyroid hormone mutation in TRbeta and recapitulate the human condition. A severe phenotype, which includes shortened body length, was evident in homozygous TRbetaPV/PV animals. Accelerated growth in utero was associated with advanced endochondral and intramembranous ossification. Advanced bone formation resulted in postnatal growth retardation, premature quiescence of the growth plates, and shortened bone length, together with increased bone mineralization and craniosynostosis. In situ hybridization demonstrated increased expression of fibroblast growth factor receptor-1, a T3-regulated gene in bone, in TRbetaPV/PV perichondrium, growth plate chondrocytes, and osteoblasts. Thus, the skeleton in TRbetaPV/PV mice is thyrotoxic and displays phenotypic features typical of juvenile hyperthyroidism.     

Growth hormone stimulates the peripheral conversion of thyroxine into triiodothyronine by increasing the liver 5'-monodeiodinase activity in the fasted and normal fed chicken

Normal fed and 2 days fasted Warren chickens were injected intravenously with 100 micrograms of ovine growth hormone (GH) and ovine prolactin and plasma concentrations of thyroid hormones were assayed prior and up to 2 h after injection. Fasting alone decreases T3, but increases T4. An injection of GH resulted in increases of plasma T3 concentrations in two fasting experiments by 40% (after 3/4 h) and 104% (after 1 h). In normal fed animals no increase is observed in the first experiment, whereas a 35% increase occurs in the second one. An injection of 100 micrograms prolactin does not influence T3 in normal fed or fasting animals. Both GH and prolactin, however, may decrease plasma concentrations of T4. In a separate experiment 50 micrograms and 200 micrograms of GH raised the decreased T3 levels after fasting by 39% and 60% respectively 1 h after injection and by 24 and 61% respectively in normal fed chicken, whereas prolactin was ineffective in this regard. Using Hisex chickens, the influence of an injection of 100 micrograms GH on plasma concentrations of thyroid hormones could be confirmed. At the same time GH increases the liver 5'-monodeiodinase activity by 330% after 1 h and by 147% after 2 h. The peroxidase activity is not influenced in normal fed chickens, but GH decreases this activity in food deprived animals after 1 h and 2 h. It is concluded that ovine GH, but not prolactin, stimulates the peripheral conversion of T4 into T3 in both normal fed and food deprived chicken and that this effect is dose dependent.     

Alterations in thyroid hormone physiology induced by temperature and feeding in newly hatched chickens

In the chicken the transition of a poikilotherm to a homeotherm reaction upon cold exposure takes place in the perinatal period between pipping and hatching. However, newly hatched chicks cannot maintain their body temperature within narrow limits after cold exposure. The fact that relatively little attention was payed on the role of thyroid hormones in the thermoregulatory reaction to cold of young chicks was probably due to the hypothetically long latention time that was thought to be necessary to bring about changes in secretory activity by cold stimulation. However, more recently, rapid changes (within hours) of thyroid hormone concentrations upon cold exposure were described in the chickens and the quail. In this study, changes in circulating T3 and T4 concentrations upon cold exposure of young chicks during the first two weeks were followed, that means during the period wherein NST (non-shivering thermogenesis), if it exists at all, should be progressively replaced by ST (shivering thermogenesis). Because of the importance of feeding condition on thyroid hormone levels, the experiments were carried out with and without a preceeding fasting period. In all experiments a short-term cold exposure of young chickens (1-11 days) fed ad lib decreased T3 but increased T4 levels while a reversed picture was found after short cold exposure of the fasted animals. However, after prolonged cold stimulus (15 degrees C) of young chickens fed ad lib, plasma T3 was also significantly elevated over that of controls whereas T4 levels returned to normal values. A prolonged warm treatment (37 degrees C) of young chickens fed ad lib resulted in significantly lower T3 and higher T4 concentrations. After a prolonged cold treatment no differences in T4 or T3 response upon TRH were found whereas the warm treatment abolished these responses upon TRH. However, a cold treatment at the stage of incubation during which the hypothalamo-hypophyseal control of thyroid function is established (dag 10-14) enhanced the T4 response to TRH with a long lasting effect extending to the posthatch period. Since T3 is thought to be the active form of thyroid hormones with regard to thermopoiesis we have studied more specifically the effect of blocking peripheral conversion of T4 on thermoregulatory abilities in young chicks and the influence of temperature treatment on monodeiodination capacity. The lower rectal temperatures following the interference with the peripheral monodeiodination of T4, the effect being more pronounced at the lower ambient temperature, are indicative for a preponderant role of T3 on thermogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

3,5-Diiodo-L-Thyronine Activates Brown Adipose Tissue Thermogenesis in Hypothyroid Rats

3,5-diiodo-l-thyronine (T2), a thyroid hormone derivative, is capable of increasing energy expenditure, as well as preventing high fat diet-induced overweight and related metabolic dysfunction. Most studies to date on T2 have been carried out on liver and skeletal muscle. Considering the role of brown adipose tissue (BAT) in energy and metabolic homeostasis, we explored whether T2 could activate BAT thermogenesis. Using euthyroid, hypothyroid, and T2-treated hypothyroid rats (all maintained at thermoneutrality) in morphological and functional studies, we found that hypothyroidism suppresses the maximal oxidative capacity of BAT and thermogenesis, as revealed by reduced mitochondrial content and respiration, enlarged cells and lipid droplets, and increased number of unilocular cells within the tissue. In vivo administration of T2 to hypothyroid rats activated BAT thermogenesis and increased the sympathetic innervation and vascularization of tissue. Likewise, T2 increased BAT oxidative capacity in vitro when added to BAT homogenates from hypothyroid rats. In vivo administration of T2 to hypothyroid rats enhanced mitochondrial respiration. Moreover, UCP1 seems to be a molecular determinant underlying the effect of T2 on mitochondrial thermogenesis. In fact, inhibition of mitochondrial respiration by GDP and its reactivation by fatty acids were greater in mitochondria from T2-treated hypothyroid rats than untreated hypothyroid rats. In vivo administration of T2 led to an increase in PGC-1α protein levels in nuclei (transient) and mitochondria (longer lasting), suggesting a coordinate effect of T2 in these organelles that ultimately promotes net activation of mitochondrial biogenesis and BAT thermogenesis. The effect of T2 on PGC-1α is similar to that elicited by triiodothyronine. As a whole, the data reported here indicate T2 is a thyroid hormone derivative able to activate BAT thermogenesis.     

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.