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.     

Thyroid-stimulating hormone receptor in brown adipose tissue is involved in the regulation of thermogenesis

C.RF- Tshr(hyt/hyt) mice have a mutated thyroid-stimulating hormone receptor (TSHR), and, without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr(hyt/hyt) mice were exposed to cold (4 degrees C), rectal temperature rapidly dropped to 23.9 +/- 0.40 degrees C at 90 min, whereas the wild-type mice temperatures were 37.0 +/- 0.15 degrees C. When we carried out functional rat TSHR gene transfer in the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of thyroxine, although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 +/- 0.34 degrees C, which was significantly higher than that of Tshr(hyt/hyt) mice. Transfection of TSHR cDNA increased mRNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and the weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr(hyt/hyt) mice restored rectal temperature 90 min after exposure to cold (36.8 +/- 0.10 degrees C). These results indicate that not only thyroid hormone but also thyroid-stimulating hormone (TSH)/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.     

Expression of functional TSH receptor in white adipose tissues of hyt/hyt mice induces lipolysis in vivo

To determine the relative importance of TSH in white adipose tissue, we compared the adipose phenotypes of two distinct mouse models of hypothyroidism. These models differed in that the normal reciprocal relationship between thyroid hormone and TSH was intact in one and disrupted in the other. One model, thyroidectomized (THYx) mice, had a 100-fold increase in TSH and a normal TSH receptor (TSHR); in contrast, the other model, hyt/hyt mice, had a 120-fold elevation of TSH but a nonfunctional TSHR. Although both THYx and hyt/hyt mice were in a severe hypothyroid state, the epididymal fat (mg)/body wt (g) (F/B) ratio of THYx mice was much smaller than that of hyt/hyt mice (8.2 ± 0.43 vs. 14.4 ± 0.40, respectively, P < 0.001). The fat cell diameter in THYx mice was also smaller than that in hyt/hyt mice (79 ± 2.8 vs. 105 ± 2.2 μm, respectively, P < 0.001), suggesting that TSH induced lipolysis in adipose tissues. When we transferred a functional mouse TSHR gene and a control plasmid into opposite sides of epididymal fat of hyt/hyt mice by plasmid injection combined with electroporation, fat weight of the TSHR side was decreased to 60% of that of the control side. Messenger RNA levels of hormone-sensitive lipase in epididymal fat containing the transferred TSHR gene were twofold higher than those in tissue from the control side. These results indicated that TSH worked as a lipolytic factor in white adipose tissues, especially in mice in a hypothyroid state.     

Subcellular distribution of carbonic anhydrase and Na+,K+-ATPase in the brain of the hyt/hyt hypothyroid mice

Activities of carbonic anhydrase and Na⁺,K⁺-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na⁺,K⁺-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na⁺,K⁺-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na⁺,K⁺-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.     

Retarded cerebral growth of hormone-deficient mice.

1. Both the Snell dwarf mouse (dw) and the growth hormone-deficient Little mouse (lit) exhibit a microcephalic cerebrum with hypomyelination, retarded neuronal growth, and underdevelopment of axons and dendrites. 2. The hypomyelination is due to arrested glial proliferation, suggesting that the action of growth hormone on the proliferation and maturation of both glial and neuronal cells is a necessary precondition of myelin formation, apart from the complementary or synergistic actions of thyroxine. 3. In contrast, the cerebral hypomyelination present in the inherited hypothyroid mouse (hyt) is not related to arrested glial proliferation, demonstrating that thyroid hormones can act independently on myelinogenesis.     

On the thyroid hormone-induced increase in respiratory capacity of isolated rat hepatocytes.

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.     

Hypothalamic CRF immunoreactivity in genetically hypothyroid (hyt/hyt) mice

The induction of hypothyroidism in young rats by feeding thiouracil to their mothers during pregnancy has been shown to depress hypothalamic content of bioactive and immunoactive corticotropin-releasing factor (CRF). The present study was done to determine whether genetically hypothyroid young mice (hyt/hyt) born to euthyroid mothers (+/hyt) exhibited a similar depression in hypothalamic CRF immunoreactivity. Young euthyroid and hypothyroid littermate mice were examined by radioimmunoassay for hypothalamic CRF content at 15, 20, 25, or 30 days of age. Mean CRF content was depressed insignificantly (to about 80% of normal) by hypothyroidism, at 15-25 days of age. However, after weaning by the mother, 30-day-old hypothyroid pups demonstrated significantly depressed hypothalamic CRF levels (71%). It is suggested that maternal factors may be assisting in the maintenance of hypothalamic CRF until after weaning. Furthermore, genetic hypothyroidism does not appear to have nearly as marked an influence as thiouracil feeding on hypothalamic CRF levels.     

Differential hormonal response of epidermal growth factor concentration in the developing mouse: synergism of triiodothyronine and dexamethasone in epidermal maturation

The hormonal regulation of epidermal growth factor concentration in neonatal mouse skin and submandibular gland is examined by radioimmunoassay. The results show triiodothyronine but not dexamethasone effects an unequivocal elevation in skin EGF content. In contrast, the neonatal submandibular gland is hormonally unresponsive during this period. Combined therapy with T3 and dexamethasone produces precocious incisor eruption compared to the effect of either hormone alone. The mechanism of this synergistic acceleration of an EGF-related developmental event is unknown.     

Effects of estradiol and progesterone on epidermal growth factor concentration in plasma, bile, urine, submandibular gland and kidney of the mouse

Testosterone is known to increase epidermal growth factor (EGF) concentrations in mouse plasma and submandibular salivary gland. We tested in adult sialoadenectomized (sx) and sham-operated female and male mice our hypothesis that female sex steroids also affect EGF concentrations in fluids and tissues. In 10-day treatment estradiol-17 beta increased the EGF concentration in male urine and in (sx) female plasma. Progesterone increased the concentration in both sexes in plasma (sx mice) and in the kidneys. In contrast, progesterone decreased it in female urine.     

Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA⁺RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T₃-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T₄ intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)     

Human epidermal growth factor concentrations in urine, but not in saliva and serum, depend on thyroid state

To evaluate the effects of thyroid hormones on the concentration of epidermal growth factor (EGF), we determined values for the immunoreactive EGF concentration in the urine (U-irEGF) of newborn infants with congenital hypothyroidism (N = 19), and in urine, saliva and serum of adult patients with hypothyroidism (N = 11) and hyperthyroidism (N = 8). The values were expressed as SD score (SDS), i.e. deviation in SD units from their mean value of healthy subjects of the same age and sex. The SDS of relative U-irEGF (ng/mg creatinine) was lower (P less than 0.01) in newborn infants with congenital hypothyroidism (-0.8 +/- 0.2; mean +/- SEM) than in healthy infants. Their relative U-irEGF correlated with their serum T4 concentrations (r = 0.59, P less than 0.01). The SDS of relative U-irEGF was lower (P less than 0.01) in adult hypothyroid patients (-1.2 +/- 0.5) and higher (P greater than 0.05) in adult hypothyroid patients (0.9 +/- 0.6) than in healthy adult subjects. When subsequently euthyroid, their SDS of relative U-irEGF increased to -0.5 +/- 0.3 (P less than 0.01), and decreased to -0.7 +/- 1.1 (P less than 0.05), respectively. The irEGF concentrations in saliva and serum were not significantly different between the hypothyroid and hyperthyroid patients. Our results indicate that urinary excretion of irEGF in man is dependent on thyroid hormone.     

Influence of thyroid hormone on the in vitro translational activity of specific mRNAs in the rat heart

The influence of thyroid hormone on the translational activity of specific cardiac mRNA was determined by in vitro translation of RNA isolated from the heart of normal, hypothyroid, and 3,3',5-triiodo-L-thyronine-injected hypothyroid rats. Proteins synthesized in vitro in the presence of [35S]methionine were separated by two-dimensional gel electrophoresis and quantitated by a novel scanning procedure using digital matrix photometry. A total of 421 translational products were detected by fluorography and changes in the predominance of 12 of these were influenced by the thyroid state of the animals. The relative predominance of 8 species was increased in euthyroid animals, whereas 4 translational products were increased in hypothyroid animals. The majority of these thyroid hormone-related alterations occurred in spot pairs of similar molecular weights, but slightly different isoelectric points. In contrast, the relative predominance of mRNAs coding for the major contractile proteins, light chain 1, light chain 2, tropomyosin, actin, and myosin heavy chain was not altered by the thyroid status of the animals. The relative levels of these abundant mRNA species remained unaltered in spite of a thyroid hormone-related increase in total RNA levels. In vivo effects of thyroid hormone on cardiac RNA levels are complex. In addition to a general increase in total RNA and mRNA levels, increases or attenuations in the predominance of a small number of specific mRNA species are observed when euthyroid and hypothyroid animals are compared.     

Hypomyelination in the Cerebrum of the Congenitally Hypothyroid Mouse (hyt)

2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the cerebrum of the inherited primary hypothyroid mouse (hyt/hyt) is reduced in comparison with the normal heterozygate (hyt/+). No differences were observed with regard to DNA and RNA content and the RNA/DNA ratio. The results of this study indicate that hypomyelination in the hypothyroid mouse is restricted to the cerebrum, and is not related to arrested glial proliferation.     

The effects of hypothyroidism on nerve growth factor and norepinephrine concentrations in weight-bearing and non-weight-bearing bones of rats

Thyroid hormones affect bone remodelling directly via receptors in osteoblasts. Previously, however, we have shown that the euthyroid and hyperthyroid states significantly influence the concentrations of both nerve growth factor (NGF) and norepinephrine (NE) in particular bones. Both NGF and NE directly affect bone metabolism and therefore it is possible that thyroid hormone action on bone may also be indirect via its actions on these two neural-related substances. In light of previous studies, the current experiments aimed to investigate whether hypothyroidism also influenced NGF and NE concentrations in weight-bearing and non-weight-bearing rat bones. Hypothyroidism was induced by oral ingestion of propylthiouricil (PTU; 3.8+/-0.2 mg/kg/day) for 21 days. Histological examination on distal femurs and microparticle enzyme immunoassayed plasma concentrations of T3 and T4 verified the hypothyroid status in treated rats. NGF concentrations were assayed via enzyme-linked immunosorbent assay (ELISA) and NE concentrations were measured via high performance liquid chromatography (HPLC) with electrochemical detection (ECD). NGF concentrations: Femoral NGF concentrations were 207% higher in hypothyroid rats (674.9+/-88.3 ng/g) than in euthyroid rats (326.7+/-63.6 ng/g; p < 0.05). Rib NGF concentrations in hypothyroid rats (3125.1+/-450.2 ng/g) were increased by 342% compared to euthyroid ribs (914.5+/-128.6 ng/g; p < 0.01). Rib NGF concentrations in hypothyroid rats were 463% higher than in femurs of hypothyroid rats (p < 0.001). NE concentrations: In hypothyroid rats, NE concentrations were reduced by approximately 50% in both ribs (38.9 ng/g) and calvaria (41.5 ng/g) compared to euthyroid rats (74.7 ng/g and 87.4 ng/g respectively; p < 0.05 for both). These findings on hypothyroid rats may be taken in conjunction with our companion work on hyperthyroid rats (Yao et al., 2002, JMNI 2:327-334) and put in context with other reports, to indicate that (i) there are several sources of NGF in bone, some of which are stimulated by hypothyroidism and others by hyperthyroidism and (ii) the concentrations of both NGF and NE in bone are sensitive to weight-bearing and thyroid hormone status.     

Neonatal hyperthyroidism alters submandibular gland epidermal growth factor response to thyroxine in the adult mouse

Neonatal hyperthyroidism (NH) in the rat is associated with permanent reductions in serum thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations in the adult, changes suggestive of a hypothyroid state. In the adult NH rat, the thyrotroph appears to be more sensitive to the feedback effects of thyroid hormones. To determine whether thyroid hormone sensitive tissues retain their responsiveness to thyroid hormones, the long-term effects of NH on mouse submandibular gland (SMG) epidermal growth factor (EGF) content were examined. NH was induced in female mice by 20 daily subcultaneous injections of 0.4 microgram of T4 per gram of body weight. Control female mice received daily injections of vehicle alone. At 21 days of age, NH and control mice were sacrificed and SMG EGF content was measured by specific radioimmunoassay, SMG EGF content and concentration in 21-day-old NH mice exceeded that of control mice by 2400- and 1500-fold, respectively (P less than 0.001). SMG EGF content and concentration in adult (90-day-old) NH mice were slightly, but not significantly, lower than those of control mice. Mean SMG weight, however, was significantly decreased in adult NH mice (P less than 0.01). Interestingly, SMG content and concentration of EGF in adult NH mice were lower than in 21-day-old NH mice. After 5 days T4 treatment (16 micrograms/d) of adult mice, SMG weight in NH mice increased significantly (P less than 0.01) but was unchanged in control mice. SMG EGF content and concentration increased significantly in both adult NH and control mice (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Methionine-alpha-naphthyl ester, a useful chromogenic substrate for esterproteases of the mouse submandibular gland.

The two aminoacid esters, N-acetyl-l-methionine α-naphthyl ester and N-acetyl-l-alanine α-naphthyl ester have been found to be suitable chromogenic substrates for the demonstration of mouse submandibular esterproteases. Using these substrates, a complex banding pattern of esterproteases was demonstrated by disc electrophoresis of mouse submandibular gland. Of these, protease A, epidermal growth factor binding protein, and the γ-subunits of 7 S nerve growth factor could be identified.