Tibor Bartha

Cloning and expression of the chicken type 2 iodothyronine 5'-deiodinase.

The type 2 iodothyronine deiodinase (D2) is critical for the intracellular production of 3,5,3′-triiodothyronine from thyroxine. The D2 mRNA of higher vertebrates is over 6 kilobases (kb), and no complete cDNA clones have been reported. Using 5′- and 3′-rapid amplification of cDNA ends and two cDNA libraries, we have cloned the 6094-base pair full-length chicken D2 cDNA. The deduced protein is ∼31 kDa and contains two in-frame UGA codons presumably encoding selenocysteine. One of these is in the highly conserved active catalytic center; the other is near the carboxyl terminus. Unusual features of the cDNA include a selenocysteine insertion sequence element ∼4.8 kb 3′ to the UGA codon in the active center and three short open reading frames in the 5′-untranslated region. The K m of D2 is ∼1.0 nm for thyroxine, and the reaction is insensitive to inhibition by 6-n-propylthiouracil. Chicken D2 is expressed as a single transcript of ∼6 kb in different brain regions and in the thyroid and lung. Hypothyroidism increases D2 mRNA in the telencephalon. Unlike in mammals, D2 mRNA and activity are expressed in the liver of the chicken, suggesting a role for D2 in the generation of plasma 3,5,3′-triiodothyronine in this species.

Transcriptional regulation of iodothyronine deiodinases during embryonic development

A single dose of chicken growth hormone (cGH) or dexamethasone acutely increases circulating T(3) levels in 18-day-old chicken embryos through a reduction of hepatic type III iodothyronine deiodinase (D3). The data in the present study suggest that this decrease in D3 is induced by a direct downregulation of hepatic D3 gene transcription. The lack of effect of cGH or dexamethasone on brain and kidney D3 activity, furthermore suggests that both hormones affect peripheral thyroid hormone metabolism in a tissue specific manner. Dexamethasone administration also results in an increase in brain type II iodothyronine deiodinase (D2) activity and mRNA levels that is also regulated at a transcriptional level. In contrast, however, cGH has no effect on brain D2 activity, thereby suggesting that either GH cannot pass through the blood-brain barrier in chicken or that cGH and dexamethasone regulate thyroid hormone deiodination by different mechanisms. In addition, the very short half-life of D2 and D3 (t(1/2)<1 h) in comparison with the longer half life of type I iodothyronine deiodinase (D1, t(1/2)>8 h), allows for D2 and D3 to play a more prominent role in the acute regulation of peripheral thyroid hormone metabolism than D1.

The Guanosine Monophosphate Reductase Gene Is Conserved in Rats and Its Expression Increases Rapidly in Brown Adipose Tissue during Cold Exposure

Non-shivering thermogenesis is required for survival of rodents during cold stress. Uncoupling protein-1 acts in brown adipose tissue (BAT) to transport protons, thus dissipating the proton gradient across the inner mitochondrial membrane. This permits respiration uncoupled from ATP synthesis. UCP-1 function is inhibited by the binding of purine nucleotides, with GTP/GDP being more potent than ATP/ADP. We used a cDNA subtraction analysis to identify cDNAs rapidly induced by cold exposure. One of these encodes rat guanosine monophosphate reductase (GMP-r). This was surprising in that previous data had suggested that this enzyme was absent in rodents. Rat GMP-r is 96% identical to human GMP-r, and its mRNA is increased 30-fold in BAT within 6 h of cold exposure. The gene is also expressed (but not cold-responsive) in muscle and kidney, but not in white fat. We speculate that the physiological function of the marked increase in BAT GMP-r during cold stress may be to deplete the brown adipocyte of guanine nucleotides, converting them to IMP, thus permitting enhanced UCP-1 function. This is a previously unrecognized regulatory aspect of thermogenesis, an essential physiological response of rodents to cold.

Endocrine factors in the hypothalamic regulation of food intake in females: a review of the physiological roles and interactions of ghrelin, leptin, thyroid hormones, oestrogen and insulin

Controlling energy homeostasis involves modulating the desire to eat and regulating energy expenditure. The controlling machinery includes a complex interplay of hormones secreted at various peripheral endocrine endpoints, such as the gastrointestinal tract, the adipose tissue, thyroid gland and thyroid hormone-exporting organs, the ovary and the pancreas, and, last but not least, the brain itself. The peripheral hormones that are the focus of the present review (ghrelin, leptin, thyroid hormones, oestrogen and insulin) play integrated regulatory roles in and provide feedback information on the nutritional and energetic status of the body. As peripheral signals, these hormones modulate central pathways in the brain, including the hypothalamus, to influence food intake, energy expenditure and to maintain energy homeostasis. Since the growth of the literature on the role of various hormones in the regulation of energy homeostasis shows a remarkable and dynamic expansion, it is now becoming increasingly difficult to understand the individual and interactive roles of hormonal mechanisms in their true complexity. Therefore, our goal is to review, in the context of general physiology, the roles of the five best-known peripheral trophic hormones (ghrelin, leptin, thyroid hormones, oestrogen and insulin, respectively) and discuss their interactions in the hypothalamic regulation of food intake.

Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications

BackgroundBased on its distribution in the brain, ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) may play a role in the hypothalamic regulation of homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further characterize the morphological attributes of NTPDase3-immunoreactive (IR) hypothalamic structures in the rat brain, here we investigated: 1.) The cellular and subcellular localization of NTPDase3; 2.) The effects of 17β-estradiol on the expression level of hypothalamic NTPDase3; and 3.) The effects of NTPDase inhibition in hypothalamic synaptosomal preparations.MethodsCombined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of estrogen on hypothalamic NTPDase3 expression was studied by western blot technique. Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated using a Clark-type oxygen electrode.ResultsCombined light- and electron microscopic analysis of immunostained hypothalamic slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly present in excitatory axon terminals and in distinct segments of the perikaryal plasma membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase (GAD) indicated that γ-amino-butyric-acid- (GABA) ergic hypothalamic neurons do not express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly present in excitatory neurons. We also investigated whether estrogen influences the expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of mitochondrial respiration rates with and without the inhibition of NTPDases confirmed the presence of NTPDases, including NTPDase3 in neuronal mitochondria and showed that blockade of mitochondrial NTPDase functions decreases state 3 mitochondrial respiration rate and total mitochondrial respiratory capacity.ConclusionAltogether, these results suggest the possibility that NTPDases, among them NTPDase3, may play an estrogen-dependent modulatory role in the regulation of intracellular availability of ATP needed for excitatory neuronal functions including neurotransmission.

Partial cloning and localization of leptin and leptin receptor in the mammary gland of the Egyptian water buffalo.

Originally an overall metabolic control was attributed to the leptin hormone, which is produced mainly by the adipose tissue. Recently, leptin gene expression was demonstrated in several additional peripheral tissues. Furthermore, several isoforms of leptin receptor were found both in the central nervous system and in the peripheral tissues. Using reverse transcription and polymerase chain reaction analysis we demonstrate that leptin is expressed both in the adipose tissue and in the lactating mammary gland tissue of Egyptian water buffalo. Our results show that, short and long isoforms of leptin receptor are expressed in buffalo mammary gland tissue. We have partially cloned the buffalo leptin and its short and long isoforms of receptor, which show a high sequence homology to previously published sequences of other mammalian species especially to that of other ruminants. Localization of leptin and its receptor mRNA transcripts, as determined by in situ hybridization procedure, revealed that leptin and its receptor transcripts are expressed specifically in the alveolar epithelial cells of the mammary gland. These morphological data support that leptin could also act as an autocrine and paracrine mediator for mammary gland metabolism and as a facilitator of alveolar epithelial cell activity during lactation.

Effects of energy restriction on thyroid hormone metabolism in chickens

Energy restriction induces changes in thyroid hormone economy in the form of a complex adaptation mechanism, in order to conserve energy storage and protein reserves. In the present work, thyroid hormone serum concentrations, hepatic deiodinase enzyme activities and hepatic deiodinase mRNA expression were examined after feed restriction and fasting. We demonstrate that during energy restriction, T 3 concentration is lowered due to a decreased T 4 activation and increased T 3 inactivation. We show that hepatic type-I deiodinase (D1) is not affected by energy restriction, however, hepatic D2 is decreased on both transcriptional and enzyme activity levels. Furthermore, hepatic D3 is increased after feed restriction in the liver. We also show that the hypothalamic feedback is not involved in the changes in serum T 3 and T 4 concentrations. Our data indicate that D2 enzyme contributes to the special hormone-exporting role of the chicken liver and this enzyme can be modulated by feed restriction.

Impaired local deiodination of thyroxine to triiodothyronine in dogs with symmetrical truncal alopecia

Thyroid dysfunction causes certain dermatological alterations in dogs. Insufficient delivery of thyroid hormone to the skin may originate not only from inadequate thyroid function but also from impaired local activation of thyroxine in the target organ. Thyroid parameters and deiodination were investigated in healthy dogs (group C) and in dogs with cutaneous lesions associated with hypothyroidism (group H) or with a low-T3 syndrome (group LT). The ability of the skin to convert T4 to T3 was impaired in both groups H and LT but not in the controls. It is concluded that impaired local deiodination may contribute to skin problems in dogs.

Hypothalamic Sidedness in Mitochondrial Metabolism New Perspectives

Morphofunctional changes in hypothalamic neurons are highly energy dependent and rely on mitochondrial metabolism. Therefore, mitochondrial adenosine triphosphate production plays a permissive role in hypothalamic regulatory events. Here, we demonstrated that in the female rat hypothalamus, mitochondrial metabolism and tissue oxygenation show an asymmetric lateralization during the estrous cycle. This asymmetry was not detected in males. The observed sidedness suggests that estrous cycle-linked hypothalamic functions in females are based on hemispheric distinction. The novel concept of hypothalamic asymmetry necessitates the revision of hypothalamic neural circuits, synaptic reorganization, and the role of hypothalamic sides in the regulation of integrated homeostatic functions.

LIGAND-INDUCED CHANGES IN OESTROGEN AND THYROID HORMONE RECEPTOR EXPRESSION IN THE DEVELOPING RAT CEREBELLUM: A COMPARATIVE QUANTITATIVE PCR AND WESTERN BLOT STUDY

Oestrogen (E2) and thyroid hormones (THs) are key regulators of cerebellar development. Recent reports implicate a complex mechanism through which E2 and THs influence the expression levels of each others receptors (ERs and TRs) to precisely mediate developmental signals and modulate signal strength. We examined the modulating effects of E2 and THs on the expression levels of their receptor mRNAs and proteins in cultured cerebellar cells obtained from 7-day-old rat pups. Cerebellar granule cell cultures were treated with either E2, THs or a combination of these hormones, and resulting receptor expression levels were determined by quantitative PCR and Western blot techniques. The results were compared to non-treated controls and to samples obtained from 14-day-old in situ cerebella. Additionally, we determined the glial effects on the regulation of ER-TR expression levels. The results show that (i) ER and TR expression depends on the combined presence of E2 and THs; (ii) glial cells mediate the hormonal regulation of neuronal ER-TR expression and (iii) loss of tissue integrity results in characteristic changes in ER-TR expression levels. These observations suggest that both E2 and THs, in adequate amounts, are required for the precise orchestration of cerebellar development and that alterations in the ratio of E2/THs may influence signalling mechanisms involved in neurodevelopment. Comparison of data from in vitro and in situ samples revealed a shift in receptor expression levels after loss of tissue integrity, suggesting that such adjusting/regenerative mechanisms may function after cerebellar tissue injury as well.