Effects of the Pineal and Melatonin,With or Without Inhibitors of Thyroid Hormone/Testosterone,on Total Protein Contents of Serum,Liver and Muscle Tissues of Clarias batrachus during Different Phases of its Annual Gonadal Cycle

Studies were planned to elucidate if the pineal plays any part in the regulation of protein contents in the serum and tissues of an Indian freshwater catfish, Clarias batrachus. In addition, an attempt has been made to investigate the modulatory roles of the status of thyroid and testis by using two potent inhibitors, i.e. iopanoic acid (thyroid inhibitor) and cyproterone acetate (androgen inhibitor). The results suggest that in general, the effects of the pineal and melatonin on protein content of serum and muscle and liver tissues are mediated with the involvement of the gonads. In addition, the thyroid status modulated the effects of pinealectomy on liver and muscle protein.     

The expression of the 35- and 67-kDa calcimedins is dependent on thyroid hormone

We have investigated the expression of the 35- and 67-kDa calcimedins and calmodulin during fetal and neonatal periods and in adulthood in rat liver, muscle, and brain. The 35- and 67-kDa calcimedin expression in liver and muscle increased during the perinatal period and correlated with the thyroid status of the developing rat. In fact, animals treated with thyroxine demonstrated a precocious appearance of the 35- and 67-kDa calcimedin in liver and muscle. Animals treated with methylthiouracil, an inhibitor of T4 and T3 synthesis, strongly suppressed the synthesis of the calcimedins in these tissues. Neither treatment influenced the levels of either the 35- and 67-kDa calcimedins in brain. In contrast, each tissue examined produced a unique pattern of calmodulin expression during development. None of the tissue calmodulin concentrations changed during hyper- or hypothyroid states. Collectively, these data support the concept that the intracellular calcium signal possesses multiple, independent molecular pathways of mediation. In addition, the variety of these pathways is influenced by hormonal preconditioning in that the cellular response to elevated cytosolic calcium is dependent upon the thyroid status of a tissue.     

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.     

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.     

The effect of tri-iodothyronine administration on protein synthesis in the diabetic rat.

In young male rats diabetes caused decreases in circulating free tri-iodothyronine and RNA concentration in liver and muscle, and in the rate of protein synthesis per unit of RNA (RNA activity) in muscle. Tri-iodothyronine treatment significantly increased RNA concentrations, but not RNA activity, in these tissues. Thus: (1) impaired thyroid status is a component of the diabetic condition; (2) tri-iodothyronine cannot stimulate the translational phase of protein synthesis in the diabetic rat.     

Regulation of Ca2+-dependent protein turnover in skeletal muscle by thyroxine.

Dantrolene, an agent that inhibits Ca2+ mobilization, improved protein balance in skeletal muscle, as thyroid status was increased, by altering rates of protein synthesis and degradation. Thyroxine (T4) caused increases in protein degradation that were blocked by leupeptin, a proteinase inhibitor previously shown to inhibit Ca2+-dependent non-lysosomal proteolysis in these muscles. In addition, T4 abolished sensitivity to the lysosomotropic agent methylamine and the autophagy inhibitor 3-methyladenine, suggesting that T4 inhibits autophagic/lysosomal proteolysis.     

Effects of Selenium on Liver and Muscle Contents and Urinary Excretion of Zinc,Copper, Iron and Manganese

Selenium is a main component of glutathione peroxidase (GPX), a key antioxidant enzyme. Other elements, such as zinc, copper, manganese and iron, are also involved in the pathogenesis of oxidative damage as well as in other important metabolic pathways. The effects of selenium supplementation on the metabolism of these elements have yield controversial results .The aim of this study is to analyse the effects of selenium supplementation on liver, muscle and urinary excretion of zinc, copper, iron and manganese in a situation of oxidative stress, such as protein deficiency. The experimental design included four groups of adult male Sprague–Dawley rats, which received the Lieber–DeCarli control diet, an isocaloric 2 % protein-containing diet and another similar two groups to which selenomethionine (6 mg/l liquid diet) was added. After sacrifice (5 weeks later), muscle, liver and serum selenium were determined, as well as muscle, liver and urinary zinc, copper, manganese and iron and liver GPX activity and liver malondialdehyde. Selenium addition led to decreased liver copper, increased muscle copper, increased copper excretion and increased liver iron, whereas zinc and manganese parameters were essentially unaltered. Muscle, liver and serum selenium were all significantly correlated with liver GPX activity.     

On the regulation of sex-hormone-binding globulin--a challenge of an old dogma and outlines of an alternative mechanism

In this review, the different factors known to affect SHBG levels are discussed with respect to their possible significance in the physiological regulation of this protein: Sex steroids, puberty, nutritional status, thyroid hormones and liver disease. It is concluded that the serum levels of SHBG are related to general metabolic factors, nutritional status, growth and ageing than to the estrogen/androgen balance. The authors suggest that SHBG is regulated primarily by growth hormone, somatomedin-C and possibly other growth factors. Growth hormone may promote SHBG synthesis in the liver while somatomedin-C may stimulate its extravasation and uptake in target tissues. It is suggested that sex steroids merely have an indirect, modulating influence.     

Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats.

Levels of retinol-binding (RBP), the plasma transport protein for vitamin A, were measured by radioimmunoassay in sera and in a large number of tissues from both normal and vitamin A-deficient rats. The tissues included liver, kidney, fat, muscle, brain, eye, salivary gland, thymus, lung, heart, intestine, spleen, adrenal, testes, thyroid, and red blood cells. The RBP levels in tissues other than serum, liver, and kidneys varied from 12 mug/g of tissue for normal spleen to an undetectable level in red blood cells. Much of the RBP in the tissues with low levels may have been due to residual serum in the samples. In general, except for liver, RBP levels were lower in tissues from vitamin A-deficient rats than in those from normal rats. In normal rats, the liver, kidney, and serum levels were 30 plus or minus 4 (mean plus orminus SEM), 151 plus or minus 22, and 44 plus or minus 3 mug/g, respectively. In vitamin A-deficient rats, the liver RBP level was about three times the normal level whereas the kidney and serum levels were about one-fifth the normal values. When normal liver homogenates were fractionated by centrifugation, 67% of the RBP was recovered in the microsomal fraction and only 9% was found in the soluble 105,000 g supernate. In contrast, 76% of the RBP in homogenates of normal kidneys was in the soluble fraction. Similar results were obtained with deficient livers and kidneys. Incubation with deoxycholate released the liver RBP into the soluble fraction. RBP is produced in the liver and removed from the blood by the kidneys. The levels of RBP in normal and deficient liver, serum, and kidney appear to reflect the relative rates of RBP secretion and turnover.     

Effect of electrostimulation of the hypothalamus on protein biosynthesis in organs of adult and aged rats

The effect of hypothalamus electrical stimulation on total protein biosynthesis was studied in skeletal muscle, heart, liver, adrenal cortex and thyroid gland of adult rats. In adult animals hypothalamus stimulation provokes a pronounced increase in 3H-leucine incorporation into total protein of all tissues, as well as into liver chromatin proteins. No significant changes were observed in protein biosynthesis when hypothalamus of old rats was stimulated. This can serve as evidence of age-related decrease in the ability of the hypothalamus to stimulate protein synthesis in peripheral tissues.     

Endothelial lipase is localized to follicular epithelial cells in the thyroid gland and is moderately expressed in adipocytes

Endothelial lipase (EL), a member of the triglyceride lipase gene family, has been shown to be a key player in HDL metabolism. Northern blots revealed that EL was highly expressed in endothelium, thyroid, lung, placenta, liver, and testis. In liver and adrenal gland, EL protein was localized with vascular endothelial cells but not parenchymal cells. EL was shown to be upregulated in tissues such as atherosclerotic plaque where it was located in macrophages, endothelial cells, and medial smooth muscle cells. The purpose of this study was to investigate the cellular localization of EL in thyroid and other tissues where EL is known to be expressed. Besides its presence in vascular endothelial and smooth muscle cells, EL protein was detected in the epithelial cells that line the follicles within the thyroid gland. EL-specific immunostaining was also found near the cell surface as well as in the cytoplasm of adipocytes. Using immunoblots, EL expression was confirmed in cultured human omental and subcutaneous adipocytes. EL expression, however, was not found in preadipocytes. These findings suggest that EL plays a role in thyroid and adipocyte biology in addition to its well-known role in endothelial function and HDL metabolism.     

Effects of Pinealectomy and Exogenous Melatonin on the Thyroid Gland Activity Varies in Relation to Reproductive Status of Male Spotted Munia (Lonchura punctulata; Aves,Passeriformes)

The purpose of this investigation was to explore whether the pineal organ and its hormone melatonin has any influence on the activity of thyroid glands, if so, how that relates to the reproductive status of a hitherto unstudied seasonally breeding wild bird. Accordingly, an identical experimental regimen was followed with adult male spotted munia (Lonchura punctulata; Passeriformes) during both its gametogenically active (August-September) and inactive (March-April) phases of the annual reproductive cycle. In either case, the levels of circulating thyroid hormones (both T₃ and T₄) and cellular characteristics of thyroid glands in groups of birds were studied following surgical removal of the pineal gland and/or daily afternoon administration of melatonin (10 μg/ 100 g body weight/ day for 30 days). The results of the same experimental schedule were found to be different depending on the sexual status of the concerned birds. During the breeding phase, pinealectomy (Px) induced significantly decreased values of T₃ and increased for T₄ along with hypertrophy of the thyroid follicular cells (TFC). The changes were reversed in melatonin treated Px birds. An increased amount of T₃ and decreased concentration of serum T₄ were also observed in melatonin injected intact birds. Conversely, the responses of TFC and of thyroid hormones in blood to either Px, or Px with melatonin, or to melatonin alone in intact munias during their inactive reproductive phase were just opposite to those noted during the breeding phase. The results of the present study suggest an influence of the pineal upon the thyroid in spotted munia. Moreover, it appears that this influence is carried out by melatonin, the action of which is reversible in relation with the gametogenic status of the concerned avian species.     

Impact of monocrotophos on protein and carbohydrate metabolism in different tissues of albino rats.

The impact of monocrotophos on protein and carbohydrate metabolism in different tissues of albino rats was investigated. The monocrotophos (0.25 mg/ml) was orally intubated into an experimental group of rats. In another group, the same amount of water was orally intubated (control group) for 29 days. The protein content was increased in liver, serum and spleen of albino rats after treatment with monocrotophos. The protein content decreased in muscle and kidney, and overall the free sugar level decreased in all tissues. The glycogen content increased in muscle, serum and kidney after treatment with monocrotophos, and the glycogen content and reducing sugar level decreased in liver and spleen. The significance of these results is discussed.     

Perchloric acid-soluble proteins and protein-bound hexoses in serum and tissues of normal, hypo-and hyperthyroid rats.

The total content of perchloric acid-soluble proteins (PCA-SP) and protein-bound hexoses (PBH) were studied in blood serum and tissues (liver, kidney, lung, brain) of normal, hypo- and hyperthyroid rats. The results obtained indicate that the hyperthyroid state induced an increase in both PCA-SP and PBH in blood serum and studied tissue homogenates, and hypothyreosis causes a decrease of total glycoproteins content in all studied materials. The results were counted per 1 g of protein and in this way the nonspecific effect of thyroid hormones on the protein metabolism had no influence on the measurements.     

Leptin acute modulation of the 5'-deiodinase activities in hypothalamus, pituitary and brown adipose tissue of fed rats.

Leptin and thyroid hormones (TH) have the ability to increase energy expenditure. Biological effects of TH are dependent on thyroxine (T4) to triiodothyronine (T3) conversion by deiodinase type 1 (D1) and type 2 (D2). Leptin has been shown to stimulate the hypothalamus-pituitary-thyroid axis and, also, to modulate 5'-deiodinases in different tissues, depending on energetic status of animals. Here, we examined the acute effects of leptin on hypothalamic, pituitary and BAT D2 and pituitary D1 activities. Male fed rats received a single subcutaneous injection of saline or leptin (8 microg/100 g BW) and sacrificed 2 hours later. Leptin promoted an important decrease in hypothalamic D2 (55% reduction, p <0.001) with no changes in pituitary D2, in concomitance with a 2-fold rise in serum TSH, suggesting that leptin acted at hypothalamus in order to stimulate TRH-TSH axis. In addition, BAT D2 was decreased by 25% (p<0.05). In contrast, pituitary D1 showed a 2-fold increase (p<0.001), indicating that, as demonstrated before for liver and thyroid D1, the pituitary enzyme is also acutely up-regulated by leptin. Serum concentrations of insulin and TH of leptin-injected animals remained unchanged. Regulation of 5'-deiodinases directing the local T3 production, is a mechanism by which leptin may alter hypothalamic, pituitary and BAT functions.     

Reproductive Phase-Dependent Annual Variation in the Effects of Melatonin and Pinealectomy, with or without Iopanoic Acid/Cyproterone Acetate, in the Regulation of Serum Cholesterol in Clarias batrachus

The present study was aimed at examining the putative thyroid or androgen dependency of the roles of melatonin/the pineal in the regulation of serum cholesterol in a freshwater catfish, Clarias batrachus . During four different phases of the annual gonadal cycle melatonin-treated or pinealectomized fishes received either iopanoic acid or cyproterone acetate or vehicle in the early morning or late afternoon. The results indicate that the effects of melatonin on serum cholesterol appear to be thyroid and testis independent during the preparatory and late postspawning phases. In contrast, the effect of pinealectomy is brought about mainly by way of the thyroid and/or the testis.     

Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid

In order to gain insight into the biological significance of a collagenase inhibitor secreted by human skin fibroblasts, we examined various human connective tissues and body fluids for such a protein. The inhibitors found in these tissues were compared immunologically to skin fibroblast inhibitor by Ouchterlony analysis and by the development of a highly specific enzyme-linked immunosorbent assay (ELISA). Using this ELISA, cell cultures of human skin fibroblasts, corneal fibroblasts, gingival fibroblasts, and adult and fetal lung fibroblasts secreted similar amounts of immunoreactive inhibitor protein. Each culture medium displayed a reaction of immunologic identity with skin fibroblast inhibitor when examined in Ouchterlony gel diffusion. In testing for functional inhibitory activity, the same 1:1 stoichiometry of collagenase inhibition was observed in each culture medium that characterizes the human skin inhibitor. Other mesodermally derived human cell types, including human fetal osteoblasts, uterine smooth muscle cells, fibrosarcoma cells, and explants of tendon and articular cartilage behaved in the same manner as the fibroblast cultures. Because collagenase inhibitors with biochemical similarities to skin fibroblast inhibitor have been described in serum and in amniotic fluid, we also examined these sources of inhibitory proteins. The data indicate that both serum and amniotic fluid contain collagenase inhibitors which are immunologically and functionally identical with the skin fibroblast inhibitor. The concentration of inhibitor in serum, as measured by ELISA assay, is 1.03 +/- 0.27 micrograms/ml. The results suggest that collagenase inhibitors which are functionally equivalent and immunologically identical with human skin fibroblast collagenase inhibitor are synthesized by many, if not all, fetal and adult mesodermal tissues in the human organism. The inhibitor apparently gains access to certain body fluids such as serum and amniotic fluid. This inhibitor protein may, therefore, function in the regulation of collagen degradation in most human connective tissues.     

Reproductive Phase-Dependent Annual Variation in the Effects of Melatonin and Pinealectomy,with or without Iopanoic Acid/Cyproterone Acetate,in the Regulation of Serum Cholesterol in Clarias batrachus

The present study was aimed at examining the putative thyroid or androgen dependency of the roles of melatonin/the pineal in the regulation of serum cholesterol in a freshwater catfish, Clarias batrachus. During four different phases of the annual gonadal cycle melatonin-treated or pinealectomized fishes received either iopanoic acid or cyproterone acetate or vehicle in the early morning or late afternoon. The results indicate that the effects of melatonin on serum cholesterol appear to be thyroid and testis independent during the preparatory and late postspawning phases. In contrast, the effect of pinealectomy is brought about mainly by way of the thyroid and/or the testis.     

Altered gene expression of hydrogen sulfide-producing enzymes in the liver and muscles tissues of hyperthyroid rats

Thyroid hormones have a role in the regulation of hydrogen sulfide (H₂S) biosynthesis. In this study, we determined the effects of hyperthyroidism on H₂S levels in various tissues and messenger RNA (mRNA) expression of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in the liver and muscles of the rat. Sixteen male Wistar rats were divided into the hyperthyroid and the control groups. Hyperthyroidism was induced by adding l -thyroxine (12 mg/L) to drinking water for a period of 21 days. H₂S concentrations in serum, liver, aorta, heart, and soleus muscles, as well as mRNA expressions of CBS, CSE, and 3-MST in these tissues were measured at Day 21. Hyperthyroid rats had lower H₂S levels in the serum compared with controls (14.7 ± 1.4 vs. 25.7 ± 1.6 µmol/L, p < 0.001). Compared with controls, hyperthyroid rats had lower levels of H₂S in the aorta (89%), heart (80%), and soleus (103%) muscles, but higher levels in the liver (35%). Hyperthyroidism decreased the ratio of CBS/CSE mRNA expression in the liver and the CSE/CBS mRNA expression in the muscles by decreasing CBS levels in liver (34% cf. controls) and CSE levels in the aorta, heart, and soleus muscles (respectively, 51%, 7%, and 52% cf.). In addition, hyperthyroidism decreased the mRNA expression of 3-MST in the liver (51%) and aorta (33%), and increased it in the heart (300%) and soleus muscle (182%). In conclusion, hyperthyroidism increased H₂S levels in the liver and decreased it in muscles; these effects are at least in part due to increases and decreases in expression of CSE in the liver and muscles, respectively. These data indicate an association between thyroid hormone status and gene expression of the H₂S-producing enzymes in the rat.     

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.