Localization and role of leptin in the thyroid gland of the lizard Podarcis sicula (Reptilia, Lacertidae)

Leptin, the product of the ob gene, is a hormone secreted by adipocytes that regulates food intake and energy expenditure. The hypothalamus-pituitary-thyroid axis is markedly influenced by the metabolism status, being suppressed during food deprivation. The present study was designed to ascertain whether (1) lizard thyroid gland expresses the long form of leptin receptor (Ob-Rb) and (2) the leptin administration affects the thyroid gland activity in this species (and to verify whether leptin plays a similar role in reptiles as observed in the other vertebrates). The presence of leptin receptor in the thyroid gland of Podarcis sicula was demonstrated by immunohistochemical technique (avidin-biotin-peroxidase complex--ABC method). The role of leptin in the control of thyroid gland activity was studied in vivo using light microscopy (LM) technique coupled to a specific radioimmunoassay for thyroid-stimulating hormone (TSH) and thyroid hormones (T4 and T3). Leptin (0.1 mg/100 g body wt)/day increased T4 and T3 release for 3 days but decreased the plasma concentration of TSH; using LM clear signs of stimulation in the thyroid gland were observed. These findings suggest that systemic administration of leptin stimulates the morphophysiology of the thyroid gland in the lizard through a direct mechanism involving Ob-Rb.     

Thyroid function and polyamines. III. Changes in ornithine decarboxylase activity and polyamine contents in the rat thyroid during hyperplasia and involution

Changes in ornithine decarboxylase (ODC) activity and in polyamine contents of the rat thyroid were studied under various experimental conditions. Methylthiouracil (MTU) treatment produced several-fold increases in the thyroid ODC activity and in the content of putrescine, spermidine and spermine within a week. While serum thyrotropin (TSH) levels increased gradually up to 3 weeks, the content of both putrescine and spermidine tended to reach a plateau after 2 weeks of the goitrogen treatment; spermine content continued to increase progressively for 3 weeks. Discontinuance of MTU at 7 days resulted in a rapid decline in the elevated thyroid ODC activity, followed by a diminution of putrescine, spermidine and RNA contents. Thyroidal putrescine, spermidine and RNA responded more sensitively to both introduction and withdrawal of TSH stimulation than thyroidal spermine and DNA. Excess iodide, having no effect on the basal level of thyroid ODC, suppressed the MTU-induced increase in this enzyme activity without affecting circulating TSH, thyroxine (T4) and triiodothyronine (T3) levels. There was a significant negative correlation between the ODC activity and intrathyroidal concentration of iodine in MTU-pretreated rats. Theophylline increased the thyroid weight and ODC activity when given to rats fed with a subeffective dose of MTU. Analyses of serum TSH, T4, T3 and of thyroidal iodine revealed that TSH-induced thyroid ODC activity was suppressed by increased circulating thyroid hormones and/or intrathyroidal iodine. Furthermore, it was suggested that thyroid hormones and excess iodide acted directly on the thyroid to alter polyamine biosynthesis, possibly by changing the responsiveness of the gland to TSH.     

The effects of bromocriptine and prolactin on porphyrin biosynthesis in the Harderian gland of the male hamster,Mesocritecus auratus

Porphyrin biosynthesis was examined in the Harderian gland of the male golden hamster by fluorometric assays of gland porphyrin content and by measuring the activity of a rate-limiting enzyme for haem biosynthesis, 5-aminolaevulinic acid synthase. Both porphyrin content and enzyme activity are low in normal male glands but were greatly raised in males castrated for 6 weeks. However, porphyrin synthesis remained at basal levels in castrates given the dopamine agonist bromocriptine; this suppression could be reversed by simultaneous prolactin administration, and castrated males receiving prolactin alone exhibited very high enzyme activity and porphyrin content. Bromocriptine also prevents the morphological feminisation of the Harderian gland which would normally occur after castration; again, the simultaneous administration of prolactin permits feminisation to occur. The results support the hypothesis that, while androgens have an inhibitory effect on porphyrin synthesis within this model, other factors, including prolactin, are permissive.Abbreviations ALA 5-aminolaevulinic acid - ALA-s 5-aminolaevulinate synthase - GH growth hormone     

Production of superoxide dismutase in Streptococcus lactis by a combination of use of hyperbaric oxygen and fermentation with cross-flow filtration

The production conditions of superoxide dismutase (SOD) in the cells of Streptococcus lactis by using hyperbaric oxygen (O(2)) are described. The SOD activity of anaerobically grown cells was 5-6 U/mg protein. When the culture broth was pressurized by O(2) at 6 atm, the SOD activity was more than twice as high as that under anaerobic culture conditions. However, there is little or no significant increase in SOD activity by exogenous addition of catalase for detoxifying hydrogen peroxide accumulated in the broth and/or controlling the pH of the broth at 6.8 during the pressurization by O(2). The increase in SOD activity by hyperbaric O(2) was possible not only at the late-logarithmic growth phase but also at the initial time for the stationary growth phase. For improvement of SOD productivity, we tried a two-stage culture in which SOD activity in S. lactis cells was enhanced by pressurizing the culture broth using hyperbaric O(2) after achievement of a high-concentration cultivation in the anerobic fermentation system with a microfiltration module.     

Prooxidant activity of the superoxide dismutase (SOD)-mimetic EUK-8 in proliferating and growth-arrested Escherichia coli cells

Numerous studies have aimed to alleviate oxidative stress in a wide range of organisms by increasing superoxide dismutase (SOD) activity. However, experimental approaches have yielded contradictory evidence, and kinetics models have shown that increases in SOD activity may increase, decrease, or not change hydrogen peroxide (H2O2) production, depending on the balance of the various processes that produce and consume superoxide (O2-). In this study we tested whether administration of EUK-8, a synthetic mimetic of the SOD enzyme, can protect starving Escherichia coli cells against stasis-induced oxidative stress. Surprisingly, administration of EUK-8 to starving E. coli cells enhances the production of reactive oxygen species (ROS), resulting in a massive increase of oxidative damage and replicative death of the bacteria. Our results confirm that manipulation of ROS levels by increasing SOD activity does not necessarily result in a consequent decline of oxidative stress and can yield opposite results in a relatively simple model system such as starving E. coli cells.     

Antioxidant response system in the short-term post-wounding effect in Mesembryanthemum crystallinum leaves

Mechanical wounding of Mesembryanthemum crystallinum leaves in planta induced a fast decrease in stomatal conductance, which was related to accumulation of hydrogen peroxide (H(2)O(2)). Higher levels of H(2)O(2) were accompanied by an increase in total activity of superoxide dismutase (SOD) and a decrease in catalase (CAT) activity. Among SOD forms, manganese SOD (MnSOD) and copper/zinc SOD (Cu/ZnSOD) seem to be especially important sources of H(2)O(2) at early stages of wounding response. Moreover, NADP-malic enzyme (NADP-ME), one of the key enzymes of primary carbon metabolism, which is also involved in stress responses, showed a strong increase in activity in wounded leaves. All these symptoms: high accumulation of H(2)O(2), high activities of Cu/ZnSOD and NADP-ME, together with the decrease of CAT activity, were also observed in the major veins of unwounded leaves. The potential role of veinal tissues as an important source of H(2)O(2) during wounding response is discussed.     

Reduction in molecular synthesis or enzyme activity of superoxide dismutases and catalase contributes to oxidative stress and neurogenic hypertension in spontaneously hypertensive rats

A balance between production and elimination of reactive oxygen species such as superoxide anion (O2*-) and hydrogen peroxide (H2O2) tightly regulates the homeostasis of cellular oxidative stress, which contributes to a variety of cardiovascular diseases, including hypertension. The present study assessed the hypothesis that O2*- or H2O2 levels augmented by the reduced molecular synthesis or enzyme activity of superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPx) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that generate tonic vasomotor tone are located, contribute to the pathogenesis of hypertension. We found that copper/zinc SOD (SOD1), manganese SOD (SOD2), or CAT, but not GPx, mRNA or protein expression and enzyme activity in the RVLM of spontaneously hypertensive rats (SHR) were significantly lower than those in normotensive Wistar-Kyoto (WKY) rats, along with a significantly higher level of O2*- or H2O2. A causative relationship between these biochemical correlates of oxidative stress and neurogenic hypertension was established when gene transfer by microinjection of adenovirus encoding SOD1, SOD2, or CAT into the bilateral RVLM promoted a long-lasting reduction in arterial pressure in SHR, but not WKY rats, accompanied by an enhanced SOD1, SOD2, or CAT protein expression or enzyme activity and reduced O2*- or H2O2 level in the RVLM. These results together suggest that downregulation of gene expression and enzyme activity of the antioxidant SOD1, SOD2, or CAT may underlie the augmented levels of O2*- and H2O2 in the RVLM, leading to oxidative stress and hypertension in SHR.     

Effect of prolactin administration on ovarian and thyroidal activity in relation to gonadotropic and thyrotropic potency in a freshwater catfish, Heteropneustes fossilis (Bloch).

Radiophosphorus incorporation by ovary was the criterion used for the assessment of ovarian activity in H. fossilis. Prolactin administration at different dose levels failed to elicit any obvious change in ovarian 32P uptake. From the findings of this experiment it appears that prolactin has no influence on ovarian activity. However, it exerted an inhibitory effect on the thyroid activity in intact H. fossilis. It was evident by retarded thyroidal 131I uptake. But the declension in serum PB131I was less pronounced. TSH content of the pituitary gland in response to prolactin administration was also reduced. It seems that prolactin interferes in thyroidal iodine accumulation perhaps by retarding the synthesis of TSH, but apparently does not prevent thyroxine output to a perceptible level.     

Bicarbonate enhances the peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical.

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro-gamma-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H(2)O(2)-mediated nitration of gamma-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H(2)O(2)-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H(2)O(2)-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H(2)O(2), suggest that SOD1/H(2)O(2) oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H(2)O(2) by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226-1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H(2)O(2). However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.     

Role of superoxide dismutase in the oxidation of N-alkanes by yeasts.

Yeast microorganisms from Candida genus are investigated for their superoxide dismutase (SOD) and catalase activity during cultivation on N-alkanes. The later caused a considerable increase of Cu/Zn SOD activity of yeast cells in comparison with glucose. A correlation between SOD and catalase activity existed. It is further observed that cells of Candida lipolytica 68-72 which contain a high level of Cu/Zn SOD were more resistant to lethality of exogenous O2-. An over-production of Cu/Zn SOD during the assimilation of N-alkanes by yeasts is also connected to their considerable resistance to increased concentrations of Cu2+ and Zn2+ ions in the nutrient medium. The results are consistent with the assumption that the enhanced resistance of yeast cells to O2- and high concentrations of Cu2+ and Zn(2+)-ions are due to the increased activity of Cu/Zn SOD and that SOD is involved in the protection of some cellular components. Polyacrylamide gel electrophoresis of Candida lipolytica cell-free extracts revealed the same chromatic bands of SOD activity under growth on glucose and N-alkanes. The type of the carbon source used from yeast cells as a single source of carbon and energy had no influence on the SOD profile of the cell.     

AICAR-Induced Activation of AMPK Inhibits TSH/SREBP-2/HMGCR Pathway in Liver

Our previous study found that thyroid-stimulating hormone promoted sterol regulatory element-binding protein-2 (SREBP-2) expression and suppressed AMP-activated protein kinase (AMPK) activity in the liver, but it was unclear whether there was a direct link between TSH, AMPK and SREBP-2. Here, we demonstrate that the 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR)-induced activation of AMPK directly inhibited the expression of SREBP-2 and its target genes HMGCR and HMGCS, which are key enzymes in cholesterol biosynthesis, and suppressed the TSH-stimulated up-regulation of SREBP-2 in HepG2 cells; similar results were obtained in TSH receptor knockout mice. Furthermore, AMPK, an evolutionally conserved serine/threonine kinase, phosphorylated threonine residues in the precursor and nuclear forms of SREBP-2, and TSH interacted with AMPK to influence SREBP-2 phosphorylation. These findings may represent a molecular mechanism by which AMPK ameliorates the hepatic steatosis and hypercholesterolemia associated with high TSH levels in patients with subclinical hypothyroidism (SCH).     

Thyroid hormone regulation of messenger ribonucleic acid encoding thyrotropin (TSH)-releasing hormone is independent of the pituitary gland and TSH

Cellular levels of mRNA encoding pro TRH in the rostral paraventricular nucleus are reduced by thyroid hormones. To determine whether this regulatory effect of thyroid hormones requires a functional pituitary gland or, specifically, TSH, we examined the effect of T3 on proTRH mRNA in hypophysectomized, thyro-parathyroidectomized male rats with or without bovine TSH replacement. Hypophysectomy plus thyro-parathyroidectomy reduced serum T4 and TSH to undetectable levels in all animals and elevated TRH mRNA in the paraventricular nucleus over that of sham-operated animals. Eleven consecutive daily injections of T3 significantly reduced TRH mRNA levels in both sham controls and thyro-parathyroidectomized rats. However, 11 daily injections of bovine TSH (1 U/day) failed to alter the effect of T3 on TRH mRNA levels. These results demonstrate that the regulatory influence of thyroid hormones on the biosynthesis of TRH within the thyrotropic center of the brain is independent of the pituitary gland and of TSH.     

Decrease of TSH levels and epithelium/colloid ratio in rat thyroid glands following administration of proadrenomedullin N-terminal peptide (12-20).

Adrenomedullin (AM) exerts a potent and long-lasting hypotensive effect and is considered to be an important hormone in blood pressure control. AM is a 52-amino-acid peptide synthesized as part of a 185-amino-acid preprohormone that also contains 20-amino-acid residues in the N-terminus, which has similar biological activity. This sequence is named a proadrenomedullin N-terminal 20 peptide (PAMP). Also, proadrenomedullin N-terminal peptide (PAMP)(12-20) exerts vasodepressor response, however this response is 3-fold less potent than the effect evoked by full-sequence peptide. Both AM and PAMP controls secretory activity of the pituitary gland and adrenal cortex, however, their action on the other endocrine glands is not recognized. Therefore, the aim of the study was to examine whether PAMP(12-20) is able to affect the structure and function of the rat thyroid gland. In adult female rats, subcutaneous PAMP(12-20) administration (1 or 4 nmol/rat/day for 6 days, autopsy 60 min after the last injection) had no effect on the weight of the thyroid gland. Peptide administration however, resulted in a dose-dependent increase in the volume of thyroid colloid, and lowered epithelium/colloid ratio in the gland (3.76 +/- 0.49, 2.66 +/- 0.27, 2.38 +/- 0.26, means +/- SE, n = 6, control, 1 and 4nmol PAMP/rat, respectively). PAMP administration changed neither the length of thyroid capillaries per unit area of surface nor their diameter. Lower dose of PAMP(12-20) significantly lowered blood TSH concentration (p < 0.01) while total and free T3 and T4 concentrations remained unchanged. Collectively, these findings suggest that PAMP(12-20) exerts a mild inhibitory effect on secretory activity of the rat thyroid gland.     

Iodide modulation of Ca2+ efflux from mouse thyroid

In a preceding report, we showed evidence that thyrotropin (TSH) stimulates Ca2+ efflux from mouse thyroid gland and that TSH stimulation of Ca2+ efflux is inhibited by acute administration of excess iodide to mice fed a low iodine diet (Hashizume et al., 1984). The observations suggested that iodide inhibits Ca2+ efflux through an inhibition of TSH-sensitive adenylate cyclase activity. We found further, that iodide inhibits dibutyryl cyclic AMP (DBC)-stimulated Ca2+ efflux. The results suggested that iodide influences the step subsequent to the generation of cyclic AMP. In this report, we studied whether iodide can inhibit Ca2+ efflux by a mechanism which is distinct from adenylate cyclase inhibition. The acute administration of excess iodide to mice fed a regular diet did not decrease the basal Ca2+ efflux rate in the thyroid. TSH-induced stimulation of Ca2+ efflux in thyroids obtained from regular diet-treated mice was not modified by iodide administration. Iodide injection to mice fed a low iodide diet, however, decreased the basal Ca2+ efflux rate though the content of cyclic AMP in the thyroids was not altered by this treatment. The decreased-Ca2+ efflux rate induced by iodide in the low iodine diet-treated thyroids was not modified by TSH in vitro. The results indicate that an acute administration of excess iodide in thyroid inhibits Ca2+ efflux not only by an inhibition of adenylate cyclase but also by an inhibitory action which is distinct from the adenylate cyclase inhibiting action of iodide.     

Species and Organ Diversity in the Effects of Hydrogen Peroxide on Superoxide Dismutase Activity In Vitro

Superoxlde dlsmutase （SOD） is ubiquitous in aerobic organisms and constitutes the first link In the enzyme scavenging system of reactive oxygen species. In the present study, species and organ diversity of SOD activity In a solution and In an in-gel assay system, as well as the effects of hydrogen peroxide （H202） on SOD activity, were Investigated. In a solution assay system, SOD activity of jackfruIt root, shoot, leaves, axes, and cotyledons, of maize embryos and endosperms, of mung bean leaves and seeds, of sacred lotus axes and cotyledons, and of rice and wheat leaves was Increased by 1-15 mmol/L H2O2. However, SOD activity In rice root and seeds, maize roots and leaves, mung bean roots and shoots, and wheat seeds was decreased by 1-15 mmol/L H2O2. The SOD activity of wheat root and soybean roots, leaves, axes, and cotyledons was Increased by 1-4 mmol/L H2O2, but was decreased by concentrations of H2O2 〉4 mmol/L. The SOD activity of soybean shoots was not affected by 1-15 mmol/L H2O2. The SOD activity In crude mltochondrla of jackfruIt, maize, and upas seeds, as well as In purified mitochondria of jackfruIt, was also Increased by 1-15 mmol/L H2O2. In the In-gel assay system, the SOD In jackfruIt cotyledons was comprised of Mn-SOD, Cu/Zn-SOD, and Fe-SOD, the crude mltochondria of jackfruit seeds and maizes embryo was comprised of Mn-SOD and Cu/ Zn-SOD, and the crude mltochondria of maize seeds was comprised of Mn-SOD only. In the present study, H2O2 markedly Inhibited Cu/Zn-SOD and Fe-SOD activity.     

Antioxidant role of zinc in PCB (Aroclor 1254) exposed ventral prostate of albino rats

The ability of zinc to retard oxidative processes has been recognized for many years. Polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental toxicants. Previous study has indicated that PCBs can have deleterious effects, including oxidative stress, on various aspects of reproduction in male rats. The aim of this study was to determine the antioxidant role of zinc in PCB-exposed ventral prostate of albino rats. A group of 20 rats were treated with Aroclor 1254 (2 mg/kg body weight/day, i.p.) for 30 days. After the PCB treatment, 10 rats were treated as PCB control. The remaining 10 rats were given zinc (Zn SO(4)) (200 mg/kg body weight/day, p.o.) for 10 days. Ventral prostatic enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) were estimated in all the groups. Hydrogen peroxide (H(2)O(2)), lipid peroxidation (LPO) and ventral prostatic acid phosphatase (ACP) were also estimated. Serum hormonal profiles such as total tri-iodothyronine (T(3)), thyroxine (T(4)), thyroid stimulating hormone (TSH), testosterone, and estradiol were estimated. Ventral prostatic androgen and estrogen receptors, ventral prostatic zinc content, and serum zinc concentration were also quantified in all the groups. Antioxidant enzymes such as SOD, CAT, GPx, GST, and ACP were decreased while an increase in H(2)O(2) and LPO were observed in PCB-treated animals. Decreased serum total T(3), T(4), testosterone, estradiol and increased TSH were observed in PCB-exposed rats. Ventral prostatic androgen and estrogen receptors were also decreased significantly in PCB-exposed rats. Zinc administration restored to previous levels all parameters except ventral prostatic ACP. These results suggest that PCB induces oxidative stress in rat ventral prostate by decreasing the levels of antioxidant enzymes; the effects could be reversed by the administration of zinc. The adverse effect of PCBs (Aroclor 1254) and zinc on ventral prostate might be due to indirect action through hormonal regulation.     

Role of cytosolic superoxide dismutase as a stimulator in anthranilamide hydroxylation by a microsomal monooxygenase system in rat liver

We have isolated a protein factor from rat liver which stimulates anthranilamide hydroxylation by the microsomes in the presence of NADPH and oxygen and showed this factor to contain Cu and Zn and to have superoxide dismutase activity [Biochim. Biophys. Acta 365, 148-157 (1974)]. In the present study, this protein factor was confirmed to be a superoxide dismutase (SOD) by comparison of the recovery of SOD activity with that of anthranilamide hydroxylation-stimulating activity at each step of its purification, by inhibition of SOD activity with NaCN and hydrogen peroxide (H2O2), and by recovery of the SOD activity of the protein factor after reconstitution with Cu2+ and/or Zn2+. At a given SOD activity level, there was no difference among the rat liver SOD, Cu,Zn-SOD from bovine erythrocytes, and Mn-SOD from Serratia marcescens in their ability to stimulate anthranilamide hydroxylation not only by rat liver microsomes, but also by the reconstituted cytochrome P-450-containing monooxygenase system. Rat liver SOD stimulated anthranilamide hydroxylation by the reconstituted system in proportion to its amount below a protein concentration of 1 microgram/ml. In anthranilamide hydroxylation by the reconstituted system without SOD, only a slight hydroxylase activity was found at the initial stage of the reaction and a marked increase in the amounts of NADPH oxidized and H2O2 formed was observed after a lag time. In the presence of rat liver SOD, however, the hydroxylase activity was markedly and continuously increased almost proportionally to reaction time with a concomitant decrease in the amounts of NADPH oxidized and H2O2 formed. In addition, a trace of 3-OH anthranilamide, one of the products, not only stimulated NADPH-dependent H2O2 formation in the reconstituted system, but also inhibited the apparent reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-OH anthranilamide were diminished by rat liver SOD. When a trace of 3-OH anthranilamide were added to a system composed of NADPH-cytochrome c (P-450) reductase and NADPH, H2O2 formation and NADPH oxidation were markedly stimulated. However, on addition of 3-OH anthranilamide to the system containing rat liver SOD, no stimulation on either H2O2 formation or NADPH oxidation was found.(ABSTRACT TRUNCATED AT 400 WORDS)