Stefan Zorad

Tyrosine hydroxylase mRNA levels in locus ceruleus of rats during adaptation to long-term immobilization stress exposure

The major central norepinephrinergic nucleus, locus ceruleus (LC), is thought to participate in modulation of such brain areas as cerebral cortex, septum, hippocampus, thalamus, hypothalamus, and cerebellum in animals facing various physiological challenges, including stress. Exposure of experimental animals to different stressors causes an increase in LC activity and gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. The aim of this work was to investigate the effect of a single and repeated (7 times) or long-term repeated (42 times) daily immobilization stress (IMMO) on TH mRNA levels in LC of laboratory rats by in situ hybridization method. A single IMMO caused significant elevation of LC TH mRNA levels in comparison to unstressed controls. This was found immediately and at 3 and 6 h after IMMO, and progressively increased up to 24 h after the first IMMO terminated. Further exposure to IMMO did not cause additional increases in LC TH mRNA levels, which stayed significantly elevated in comparison to unstressed rats. In animals that underwent IMMO for 42 times, the LC TH gene expression, 24 h after the last stress exposure, was significantly lower when compared to that of singly or seven times stressed rats. Thus, our results indicate a possible adaptation of catecholamine-synthesizing system at the level of TH gene expression in LC of rats exposed to long-term repeated IMMO.

Subchronic treatment of rats with oxytocin results in improved adipocyte differentiation and increased gene expression of factors involved in adipogenesis

BACKGROUND AND PURPOSE Treatment with thiazolidinediones, insulin‐sensitizing drugs, enhances adipogenesis, which may result in unwanted increase in adiposity. Based on the suggested metabolic effects of oxytocin, the aims of the present study were to: (i) determine whether chronic treatment with oxytocin exerts positive effects on white adipose tissue growth without increasing adiposity; (ii) investigate possible mechanisms of action of oxytocin by measuring the level of gene expression of adipogenic factors; and (iii) test the hypothesis that oxytocins effect on adipose tissue involves specific activation of eukaryotic elongation factor 2 (eEF2).

Adipokine Protein Expression Pattern in Growth Hormone Deficiency Predisposes to the Increased Fat Cell Size and the Whole Body Metabolic Derangements

CONTEXT GH deficiency (GHD) in adults is associated with central adiposity, dyslipidemia, and insulin resistance. OBJECTIVE The objective of the study was to test the hypothesis that GHD might change the spectrum of adipokines and thus influence the adipose tissue and the whole-body metabolic and inflammatory status leading to development of insulin resistance. DESIGN This was a single-center observational study with a cross-sectional design. PARTICIPANTS AND METHODS Protein arrays were used to characterize adipokines expressed in the sc adipose tissue obtained from young GHD adults and compared with age-, gender-, and body mass index (BMI)-matched group of healthy individuals. All subjects underwent an oral glucose tolerance test, euglycemic hyperinsulinemic clamp, and magnetic resonance imaging examination. RESULTS Presence of abdominal obesity, enlarged adipocytes, increased circulating high-sensitivity C-reactive protein, impaired glucose tolerance, and decreased insulin action were found in GHD. Changes in adipokine protein expression due to GHD were highly dependent on the obesity phenotype. Lean GHD individuals (BMI approximately 23 kg/m(2)) had decreased protein levels for stem cell factor and epithelial growth factor, indicating a possible defect in adipocyte differentiation and proliferation. Decrease of vascular endothelial growth factor, stromal cell-derived factor, angiopoietin-2, and brain-derived neurotrophic factor advocated for attenuated angiogenesis and neurogenesis. Presence of obesity (BMI approximately 31 kg/m(2)) eliminated these inhibitory effects. However, adipose tissue expansion in GHD individuals was paralleled by an elevation of adipose tissue proinflammatory cytokines (IL-1beta, interferon-gamma) and chemoattractants (interferon-inducible T cell alpha-chemoattractant, monocyte chemotactic protein-2, monocyte chemotactic protein-3, eotaxin). CONCLUSION Our data demonstrate that GHD modulates adipokine and cytokine protein expression pattern, which might influence the adipose tissue growth and differentiation and predispose to tissue hypoxia, inflammation, and a defect in the whole-body insulin action.

Melatonin reduces cardiac remodeling and improves survival in rats with isoproterenol‐induced heart failure

Melatonin was previously shown to reduce blood pressure and left ventricular (LV) remodeling in several models of experimental heart damage. This study investigated whether melatonin prevents LV remodeling and improves survival in isoproterenol‐induced heart failure. In the first experiment, four groups of 3‐month‐old male Wistar rats (12 per group) were treated for 2 wk as follows: controls, rats treated with melatonin (10 mg/kg/day) (M), rats treated with isoproterenol (5 mg/kg/day intraperitoneally the second week) (Iso), and rats treated with melatonin (2 wk) and isoproterenol (the second week) in corresponding doses (IsoM). In the second experiment, 30 rats were treated with isoproterenol and 30 rats with isoproterenol plus melatonin for a period of 28 days and their mortality was investigated. Isoproterenol‐induced heart failure with hypertrophy of the left and right ventricles (LV, RV), lowered systolic blood pressure (SBP) and elevated pulmonary congestion. Fibrotic rebuilding was accompanied by alterations of tubulin level in the LV and oxidative stress development. Melatonin failed to reduce the weight of the LV or RV; however, it curtailed the weight of the lungs and attenuated the decline in SBP. Moreover, melatonin decreased the level of oxidative stress and of insoluble and total collagen and partly prevented the beta‐tubulin alteration in the LV. Most importantly, melatonin reduced mortality and prolonged the average survival time. In conclusion, melatonin exerts cardioprotective effects and improves outcome in a model of isoproterenol‐induced heart damage. The antiremodeling effect of melatonin may be of potential benefit in patients with heart failure.

Expression of nuclear retinoic acid receptor in peripheral blood mononuclear cells (PBMC) of healthy subjects

In vertebrates, both nuclear all-trans and 9-cis retinoic acid receptors (RAR and RXR) belonging to the steroid/thyroid/retinoid nuclear receptor superfamily play a crucial role in the vitamin A action. Qualitative analysis of all known RAR or RXR subtypes in both pooled and non-pooled peripheral blood mononuclear cells (PBMC) from healthy human subjects has been performed by reverse transcription and polymerase chain reaction (RT-PCR). Our data, based on qualitative RT-PCR analysis has shown that human PBMC are capable to express RAR alpha, RAR gamma, RXR alpha, and RXR beta.

The AT2 subtype of the angiotensin II receptors has differential sensitivity to dithiothreitol in specific brain nuclei of young rats

We studied the effect of the sulfhydryl reducing agent dithiothreitol on the binding of the angiotensin II agonist [125I][Sar1]-angiotensin II to AT2 receptors in selected brain areas of young (2-week-old) rats. In the inferior olive and the hypoglossal nucleus, angiotensin II binding to AT2 receptors was insensitive to 5 mM dithiothreitol. Conversely, in the ventral and mediodorsal thalamic, medial geniculate, and oculomotor nuclei, the superior colliculus and the cerebellar cortex, incubation with 5 mM dithiothreitol significantly decreased angiotensin II binding to AT2 receptors to about 40% of control. These data suggest that brain AT2 receptors are heterogeneous with respect to their sensitivity to sulfhydryl reducing agents.

Hypooxytocinaemia in obese Zucker rats relates to oxytocin degradation in liver and adipose tissue

The metabolic action of oxytocin has recently been intensively studied to assess the ability of the peptide to regulate energy homeostasis. Despite the obvious weight-reducing effect of oxytocin observed in experimental studies, plasma oxytocin levels were found to be unchanged or even elevated in human obesity. The aim of our study was to evaluate the changes in the oxytocin system in Zucker rats, an animal model closely mirroring morbid obesity in humans. Plasma oxytocin levels were measured in obese Zucker rats and lean controls by enzyme immunoassay after plasma extraction. The expression of oxytocin and oxytocin receptor (OXTR) was assessed at the mRNA and protein levels by quantitative real-time PCR and immunoblotting respectively. Plasma and tissue activity of oxytocinase, the main enzyme involved in oxytocin degradation, were measured by fluorometric assay using an arylamide derivate as the substrate. Obese Zucker rats displayed a marked reduction in plasma oxytocin levels. Elevated liver and adipose tissue oxytocinase activity was noticed in obese Zucker rats. Hypothalamic oxytocin gene expression was not altered by the obese phenotype. OXTR mRNA and protein levels were upregulated in the adipose tissue of obese animals in contrast to the reduced OXTR protein levels in skeletal muscle. Our results show that obesity is associated with reduced plasma oxytocin due to increased peptide degradation by liver and adipose tissue rather than changes in hormone synthesis. This study highlights the importance of the oxytocin system in the pathogenesis of obesity and suggests oxytocinase inhibition as a candidate approach in the therapy of obesity.

Characterization of brain angiotensin II AT2 receptor subtype using [125I] CGP 42112A

Recently two subtypes of angiotensin receptors have been described, AT1 and AT2. Currently used radiolabeled agonists and antagonists are not able to discriminate between these receptors subtypes. Here we characterize the use of [125I] CGP 42112A, a novel, specific ligand for AT2 receptors, in a membrane binding assay and in autoradiography of brain sections of 2 week old rats. [125I] CGP 42112A bound with high affinity and autoradiography revealed binding selectively localized to areas known to express the AT2 receptor subtype only. CGP 42112A, angiotensin II, angiotensin III and PD 123177 competed for [125I] CGP 42112A binding, with potencies consistent with high affinity and specific binding to AT2 receptors. Thus [125I] CGP 42112A will be a useful new tool to study AT2 receptors.

Catecholamine Synthesizing Enzymes and Their Modulation by Immobilization Stress in Knockout Mice

Abstract: The c‐fos knockout mice (c‐fos KO) and corticotropin‐releasing hormone knockout mice (CRH KO) can serve as interesting models for studying mechanisms involved in response of the organism to stress, focused mainly on the hypothalamic‐pituitary‐adrenal (HPA) axis and sympathoadrenal system (SAS). The present study focused on the investigation of changes in gene expression of catecholamine biosynthesizing enzymes tyrosine hydroxylase (TH), dopamine‐β‐hydroxylase (DBH), and phenylethanolamine N‐methyltransferase (PNMT) in adrenal medulla of c‐fos KO and CRH KO mice stressed by immobilization. Levels of TH, DBH, and PNMT mRNA were determined by reverse transcription‐polymerase chain reaction (RT‐PCR). Single immobilization for 2 h significantly increased adrenomedullary TH, DBH, and PNMT mRNA levels in both c‐fos KO and wild‐type (WT) mice compared to unstressed controls. In CRH KO mice, PNMT gene expression was not increased to the same extent after single, but especially after repeated immobilization as in WT mice, in contrast to TH and DBH mRNA levels. Thus, our data indicate that CRH deficiency can influence the PNMT mRNA level in adrenal medulla during stress, confirming the idea that the HPA axis plays the crucial role in PNMT gene regulation in mice. On the other hand, c‐Fos protein probably does not play a crucial role in TH, DBH, and PNMT gene expression in adrenal medulla under stress conditions.

Hypertension and Cardiovascular Remodelling in Rats Exposed to Continuous Light: Protection by ACE-Inhibition and Melatonin

Exposure of rats to continuous light attenuates melatonin production and results in hypertension development. This study investigated whether hypertension induced by continuous light (24 hours/day) exposure induces heart and aorta remodelling and if these alterations are prevented by melatonin or angiotensin converting enzyme inhibitor captopril. Four groups of 3-month-old male Wistar rats (10 per group) were treated as follows for six weeks: untreated controls, exposed to continuous light, light-exposed, and treated with either captopril (100 mg/kg/day) or melatonin (10 mg/kg/day). Exposure to continuous light led to hypertension, left ventricular (LV) hypertrophy and fibrosis, and enhancement of the oxidative load in the LV and aorta. Increase in systolic blood pressure by continuous light exposure was prevented completely by captopril and partially by melatonin. Both captopril and melatonin reduced the wall thickness and cross-sectional area of the aorta and reduced the level of oxidative stress. However, only captopril reduced LV hypertrophy development and only melatonin reduced LV hydroxyproline concentration in insoluble and total collagen in rats exposed to continuous light. In conclusion, captopril prevented LV hypertrophy development in the continuous light-induced hypertension model, while only melatonin significantly reduced fibrosis. This antifibrotic action of melatonin may be protective in hypertensive heart disease.