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BACKGROUND Both fructose consumption and chronic stress contribute to the development of metabolic disorders. The consequences of such combination are not fully understood. OBJECTIVE We investigated whether fructose supplementation and chronic stress synergistically disturb hepatic lipid and glucose metabolism. The role of energy sensing, redox, and inflammatory status during development of metabolic disturbances was investigated. METHODS Female Wistar rats, aged 2.5 mo, were divided into 4 experimental groups: control (C) fed a standard diet (commercial food and drinking water); fructose (F) fed the same food and 10% fructose solution; stress (S) fed the standard diet and subjected to chronic unpredictable stress and, stress + fructose (SF) combining conditions F and S as above. Stress included daily stressors: cold water forced swimming, physical restraint, cold room, wet bedding, rocking, switching, or tilting cages. After 9 wk, hepatic enzymes and transcription factors involved in gluconeogenesis, lipogenesis, fatty acid oxidation, antioxidative defence, energy sensing, and cytokines were assessed by qPCR, Western blotting, and spectrophotometry and analyzed by 2-factor ANOVA. RESULTS Fructose increased AMP-activated protein kinase (AMPK) phosphorylation (40%; P

Aims: CYP2C19 transgenic mouse expresses the human CYP2C19 gene in the liver and developing brain, and it exhibits altered neurodevelopment associated with impairments in emotionality and locomotion. Because the validation of new animal models is essential for the understanding of the aetiology and pathophysiology of movement disorders, the objective was to characterise motoric phenotype in CYP2C19 transgenic mice and to investigate its validity as a new animal model of ataxia. Methods: The rotarod, paw-print and beam-walking tests were utilised to characterise the motoric phenotype. The volumes of 20 brain regions in CYP2C19 transgenic and wild-type mice were quantified by 9.4T gadolinium-enhanced post-mortem structural neuroimaging. Antioxidative enzymatic activity was quantified biochemically. Dopaminergic alterations were characterised by chromatographic quantification of concentrations of dopamine and its metabolites and by subsequent immunohistochemical analyses. The beam-walking test was repeated after the treatment with dopamine receptor antagonists ecopipam and raclopride. Results: CYP2C19 transgenic mice exhibit abnormal, unilateral ataxia-like gait, clasping reflex and 5.6-fold more paw-slips in the beam-walking test; the motoric phenotype was more pronounced in youth. Transgenic mice exhibited a profound reduction of 12% in cerebellar volume and a moderate reduction of 4% in hippocampal volume; both regions exhibited an increased antioxidative enzyme activity. CYP2C19 mice were hyperdopaminergic; however, the motoric impairment was not ameliorated by dopamine receptor antagonists, and there was no alteration in the number of midbrain dopaminergic neurons in CYP2C19 mice. Conclusions: Humanised CYP2C19 transgenic mice exhibit altered gait and functional motoric impairments; this phenotype is likely caused by an aberrant cerebellar development.

Chronic use of atypical antipsychotics may produce hepatic damage. Atypical antipsychotics, including clozapine, sertindole, and ziprasidone, are extensively metabolized by the liver and this process generates toxic-free radical metabolic intermediates which may contribute to liver damage. The aim of this study was to investigate whether clozapine, sertindole, or ziprasidone affected hepatic antioxidant defense enzymes which consequently led to disturbed redox homeostasis. The expression and activity of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), and glutathione-S-transferases (GST) were measured in rat livers at doses corresponding to human antipsychotic therapy. Clozapine increased activity of SOD types 1 and 2, GR and GST, but reduced CAT activity. Sertindole elevated activities of both SODs. In ziprasidone-treated rats only decreased CAT activity was found. All three antipsychotics produced mild-to-moderate hepatic histopathological changes categorized as regenerative alterations. No apparent signs of immune cell infiltration, microvesicular or macrovesicular fatty change, or hepatocytes in mitosis were observed. In conclusion, a 4-week long daily treatment with clozapine, sertindole, or ziprasidone altered hepatic antioxidant enzyme activities and induced histopathological changes in liver. The most severe alterations were noted in clozapine-treated rats. Data indicate that redox disturbances may contribute to liver dysfunction after long-term atypical antipsychotic drug treatment.

BackgroundAnimal models are essential for understanding etiology and pathophysiology of movement disorders. Previously, we have found that mice transgenic for the human CYP2C19 gene, expressed in the liver and developing brain, exhibit altered neurodevelopment associated with impairments of their motor function and emotionality.ObjectivesTo characterize motoric phenotype of the CYP2C19 transgenic mice and validate its usefulness as an animal model of ataxia.MethodsThe rotarod and beam-walking tests were utilized to quantify the functional alterations induced by motoric phenotype. Dopaminergic system was assessed by tyrosine hydroxylase immunohistochemistry and by chromatographic quantification of the whole-brain dopamine levels. Beam-walking test was also repeated after the treatment with the dopamine receptor antagonists, ecopipam and raclopride. The volumes of 20 brain regions in the CYP2C19 transgenic mice and controls were quantified by 9.4T gadolinium-enhanced postmortem structural neuroimaging.ResultsCYP2C19 transgenic mice were found to exhibit abnormal, unilateral ataxia-like gait, clasping reflex and 5.6-fold more paw-slips using the beam-walking test (pCYP2C19 mice; however, the motoric impairment was not ameliorated by dopamine receptor antagonists and there was also no midbrain dopamine neuron loss in CYP2C19 mice. However, in these mice, cerebellar volume was drastically decreased (11.8% [95%CI: −14.7, −9.0], qq=0.015, n=59).ConclusionsHumanized CYP2C19 transgenic mice exhibit altered motoric function and functional motoric impairments; this phenotype is likely caused by an aberrant cerebellar development.

Introduction: Obesity and related metabolic disturbances are frequently related to modern lifestyle and are characterized by excessive fructose intake. Visceral adipose tissue (VAT) inflammation has a central role in the development of insulin resistance, type 2 diabetes (T2D), and metabolic syndrome. Since sex-related differences in susceptibility and progression of metabolic disorders are not yet fully understood, our aim was to examine inflammation and insulin signaling in VAT of fructose-fed female and male adult rats.Methods: We analyzed effects of 9-week 10% fructose-enriched diet on energy intake, VAT mass and histology, and systemic insulin sensitivity. VAT insulin signaling and markers of VAT inflammation, and antioxidative defense status were also evaluated.Results: The fructose diet had no effect on VAT mass and systemic insulin signaling in the female and male rats, while it raised plasma uric acid, increased PPARγ level in the VAT, and initiated the development of a distinctive population of small adipocytes in the females. Also, adipose tissue insulin resistance, evidenced by increased PTP1B and insulin receptor substrate 1 (IRS1) inhibitory phosphorylation and decreased Akt activity, was detected. In addition, fructose stimulated the nuclear accumulation of NFκB, increased expression of proinflammatory cytokines (IL-1β, IL-6, and TNFα), and protein level of macrophage marker F4/80, superoxide dismutase 1, and glutathione reductase. In contrast to the females, the fructose diet had no effect on plasma uric acid and VAT inflammation in the male rats, but less prominent alterations in VAT insulin signaling were observed.Conclusion: Even though dietary fructose did not elicit changes in energy intake and led to obesity in the females, it initiated the proliferation of small-sized adipocytes capable of storing fats further. In contrast to the males, this state of VAT was accompanied with enhanced inflammation, which most likely contributed to the development of insulin resistance. The observed distinction could possibly originate from sex-related differences in uric acid metabolism. Our results suggest that VAT inflammation could precede obesity and start even before the measurable increase in VAT mass, making it a silent risk factor for the development of T2D. Our results emphasize that adipose tissue dysfunction, rather than its simple enlargement, could significantly contribute to the onset and development of obesity and related metabolic disorders.

The modern lifestyle brings both excessive fructose consumption and daily exposure to stress which could lead to metabolic disturbances and type 2 diabetes. Muscles are important points of glucose and lipid metabolism, with a crucial role in the maintenance of systemic energy homeostasis. We investigated whether 9-week fructose-enriched diet, with and without exposure to 4-week unpredictable stress, disturbs insulin signaling in the skeletal muscle of male rats and evaluated potential contributory roles of muscle lipid metabolism, glucocorticoid signaling and inflammation. The combination of fructose-enriched diet and stress increased peroxisome proliferator-activated receptors-α and -δ and stimulated lipid uptake, lipolysis and β-oxidation in the muscle of fructose-fed stressed rats. Combination of treatment also decreased systemic insulin sensitivity judged by lower R-QUICKI, and lowered muscle protein content and stimulatory phosphorylations of insulin receptor supstrate-1 and Akt, as well as the level of 11β-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor. At the same time, increased levels of protein tyrosine phosphatase-1B, nuclear factor-κB, tumor necrosis factor-α, were observed in the muscle of fructose-fed stressed rats. Based on these results, we propose that decreased glucocorticoid signaling in the skeletal muscle can make a setting for lipid-induced inflammation and the development of insulin resistance in fructose-fed stressed rats.

Novel polyurethane nanocomposite (PUN) materials containing different surface-functionalized mesoporous silica nanoparticles (MSNs) were prepared by in situ polymerization methodology. Polyurethane network was formed from poly(dimethylsiloxane)-based macrodiol (PDMS), 4,4′-methylenediphenyldiisocyanate (MDI), and hyperbranched polyester of the second pseudo-generation (BH-20; used as crosslinking agent). PU and PU/MSN nanocomposites contained equal ratios of soft PDMS and hard MDI-BH-20 segments. Non-functionalized and surface-functionalized (with 3-(trihydroxysilyl)propyl methylphosphonate (FOMSN) and 2-[methoxy(polyethyleneoxy)6−9propyl]trimethoxysilane (PEGMSN)) MSNs were used as the nanofillers at a concentration of 1 wt%. Prepared materials were characterized by Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), dynamic mechanical thermal analyses (DMTA), nanoindentation, equilibrium swelling and water absorption measurements. Characteristics of the prepared PUNs when in contact with a biological environment were assessed through testing their biocompatibility, protein adsorption and adhesion of endothelial cells. The favourable influence of MSNs on the physico-chemical and biological characteristics of these novel PUN materials was identified, which evidences their vast applicability potential as coatings for medical devices and implants.

The effects of early-life fructose consumption on hepatic signaling pathways and their relation to the development of metabolic disorders in later life are not fully understood. To investigate whether fructose overconsumption at a young age induces alterations in glucocorticoid signaling that might contribute to development of metabolic disturbances, we analysed glucocorticoid receptor hormone-binding parameters and expression of its target genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) and lipid metabolism (lipin-1), as well as redox and inflammatory status in the liver of female rats subjected to a fructose-rich diet immediately after weaning. The fructose diet increased hepatic corticosterone concentration, 11&beta, hydroxysteroid dehydrogenase type 1 level, glucocorticoid receptor protein level and hormone-binding activity, as well as lipin-1 level. The expression of glucose-6-phosphatase was reduced in fructose-fed rats, while phosphoenolpyruvate carboxykinase remained unaltered. The fructose-rich diet increased the level of fructose transporter GLUT2, while the expression of fructolytic enzymes fructokinase and aldolase B remained unaltered. The diet also affected pro-inflammatory pathways, but had no effect on the antioxidant defence system. In conclusion, a fructose-rich diet applied immediately after weaning promoted lipogenesis and enhanced hepatic glucocorticoid signaling, possibly to protect against inflammatory damage, but without an effect on gluconeogenesis and antioxidant enzymes. Yet, prolonged treatment might ultimately lead to more pronounced metabolic disturbances.

Polycystic ovary syndrome (PCOS) is a complex reproductive disorder often associated with obesity, insulin resistance, and dyslipidemia. Hormonal changes in PCOS may also include altered glucocorticoid signaling. Our aim was to examine whether alterations in hepatic glucocorticoid signaling are associated with disturbances of glucose and lipid metabolism in animal model of PCOS. Female rats, 3 weeks old, were subcutaneously implanted with 5α-dihydrotestosterone (DHT) or placebo pellets for 90 days to induce PCOS. Expression of 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) and A-ring reductases (5α and 5β), as well as intracellular distribution of glucocorticoid receptor (GR) and expression of its regulated genes were examined in the liver. Proteins of hepatic lipid and carbohydrate metabolism and markers of inflammation were also assessed. DHT treatment induced increase in body and liver mass, as well as in triglycerides and free fatty acids levels in plasma. Elevation of 11βHSD1 and reduction of 5α-reductase expression was observed together with increased hepatic corticosterone concentration and nuclear GR activation. Induced expression of Kruppel-like factor 15 and decreased expression of genes for proinflammatory cytokines and de novo lipogenesis (DNL) were detected in the liver of DHT-treated rats, while DNL regulators and proinflammatory markers were not changed. However, increased mRNA levels of stearoyl-CoA desaturase and apolipoprotein B were observed in DHT animals. DHT treatment stimulated hepatic glucocorticoid prereceptor metabolism through increased corticosterone availability which is associated with enhanced GR activation. This does not affect gluconeogenesis and DNL, but could be linked to stimulated triglyceride synthesis and hypertriglyceridemia.

SCOPE Intake of fructose-sweetened beverages and chronic stress (CS) both increase risk of cardiometabolic diseases. The aim is to investigate whether these factors synergistically perturb lipid metabolism in rat liver and kidney. METHODS AND RESULTS Fractional de novo lipogenesis (fDNL), intrahepatic- and intrarenal-triglycerides (IHTG and IRTG), de novo palmitate (DNPalm) content, FA composition, VLDL-TGs kinetics, and key metabolic gene expression at the end of the feeding and non-feeding phases in rats exposed to standard chow diet, chow diet + CS, 20% liquid high-fructose supplementation (HFr), or HFr+CS are measured. HFr induces hypertriglyceridemia, up-regulates fructose-metabolism and gluconeogenic enzymes, increases IHTG and DNPalm content in IHTG and IRTG, and augments fDNL at the end of the feeding phase. These changes are diminished after the non-feeding phase. CS does not exert such effects, but when combined with HFr, it reduces IHTG and visceral adiposity, enhances lipogenic gene expression and fDNL, and increases VLDL-DNPalm secretion. CONCLUSION Liquid high-fructose supplementation increases IHTG and VLDL-TG secretion after the feeding phase, the latter being the result of stimulated hepatic and renal DNL. Chronic stress potentiates the effects of high fructose on fDNL and export of newly synthesized VLDL-TGs, and decreases fructose-induced intrahepatic TG accumulation after the feeding phase. This is the peer reviewed version of the following article: Vojnović Milutinović D, Brkljačić J, Teofilović A, Bursać B, Nikolić M, Gligorovska L, Kovačević S, Djordjevic A, Preitner F, Tappy L, Matić G, Veličković N. Chronic Stress Potentiates High Fructose-Induced Lipogenesis in Rat Liver and Kidney. Mol Nutr Food Res. 2020;e1901141, which has been published in final form at [http://dx.doi.org/10.1002/mnfr.201901141]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

Overconsumption of fructose-enriched beverages and everyday stress are involved in the pathogenesis of metabolic disorders through modulation of hepatic glucose metabolism. The aim of the study was to investigate whether interaction of high-fructose diet and chronic stress alter insulin and glucocorticoid signalling thus affecting hepatic glucose homeostasis. High-fructose diet led to hyperinsulinemia, increased glucose transporter 2 level, elevated protein kinase B (Akt) phosphorylation, increased glucokinase mRNA and phospho-to-total glycogen synthase kinase 3 ratio and decreased expression of gluconeogenic genes. Fructose diet also led to stimulated glucocorticoid prereceptor metabolism, but downstream signalling remained unchanged due to increased glucocorticoid clearance. Stress did not affect hepatic insulin and glucocorticoid signalling nor glucose metabolism, while the interaction of the factors was observed only for glucokinase expression. The results suggest that, under conditions of fructose-induced hyperinsulinemia, suppression of gluconeogenesis and glycogen synthase activation contribute to the maintenance of glucose homeostasis. The increased glucocorticoid inactivation may represent an adaptive mechanism to prevent hyperglycaemia.

OBJECTIVES/GOALS: The aim of this study was to define the transcription profiles of the molecular markers of mitochondrial biogenesis and fusion/architecture, and the markers of mtDNA copy numbers in the peripheral blood mononuclear cells (PBMCs) from war veterans with/without post-traumatic stress disorder (PTSD). METHODS/STUDY POPULATION: The peripheral blood mononuclear cells (PBMCs) from war veterans with/without post-traumatic stress disorder (PTSD) were used to monitor transcription profile of the molecular markers of mitochondrial biogenesis and fusion/architecture, as well as the markers of mtDNA copy numbers. The human male immortalized monocytes were exposed in vitro to hormonal markers of PTSD in order to monitor the effects of each particular hormonal marker on the molecular markers of mitochondrial biogenesis and fusion/architecture, as well as the markers of mtDNA copy numbers. RQ-PCR analyses were used to define transcriptional profile of above mentioned markers. RESULTS/ANTICIPATED RESULTS: The transcription profiles of above mentioned markers were disturbed, with high individual variability within the groups. A significant increase in the expression of the PPARGC1A transcript was observed in a group of subjects with current PTSD, as well as in the subjects with “life-time” PTSD, compared to healthy controls. PPARGC1B, NRF2 and MFN2 transcripts increased only in PBMCs of “life-time"-PTSD, while the level of transcripts for other investigated genes and the ratio of markers of mtDNA copy numbers showed no significant difference between groups. The in vitro results showed parallelism with the results obtained using the PBMCs from the subjects of the PTSD study. DISCUSSION/SIGNIFICANCE OF IMPACT: Although preliminary (the analysis require a larger number of subjects), the results are first findings and a solid base for further extensive multidisciplinary research in order to clarify the molecular mechanisms for the prevention and treatment of trauma-induced pathological conditions.

A bstract: An increase in fructose consumption coincides with a rising incidence of metabolic disorders. Dietary fructose has been shown to affect hepatic lipid metabolism in a way that may lead to lipid deposition in the liver. In this study, we tested the hypothesis that the effects of fructose overconsumption on hepatic lipid metabolism differ between sexes. To that end we examined the effects of a high-fructose diet on the expression of key enzymes and transcription factors involved in the regulation of fatty acid oxidation and de novo lipogenesis in the liver of 12-week-old male and female Wistar rats. Immediately after weaning, the rats were subjected to a standard diet and 10% fructose solution or drinking water for 9 weeks. The fructose-enriched diet induced hypertriglyceridemia and increased hepatic de novo lipogenesis in both sexes, without lipid deposition in the liver. At the same time, visceral adiposity was observed only in female rats, while in males the treatment stimulated hepatic fatty acid oxidation. The fructose-enriched diet induced sex-specific effects on hepatic lipid metabolism in young rats. These results imply that male and female rats employ different strategies to cope with dietary fructose-related energy overload and to avoid lipid accumulation in the liver. https://doi.org/10.2298/ABS190306023N Received: March 6, 2019; Revised: April 2, 2019; Accepted: April 2, 2019; Published online: April 9, 2019 How to cite this article: Brkljacic J, Velickovic N, Elakovic I, Teofilovic A, Vojnovic-Milutinovic D, Đorđevic A, Matic G. Fructose-enriched diet affects hepatic lipid metabolism in young male and female rats in different ways. Arch Biol Sci. 2019;71(3):417-24.

Gender-related differences in susceptibility to stress and stress-related disorders such as depression, and in response to treatment with antidepressants have been observed, but the underlying molecular mechanisms are still unknown. Considering the role of glucocorticoid hormones in the systemic reaction against stress and in pathogenesis of depression, the aim of the present work was to study gender-related differences in glucocorticoid signaling and in response of this system to a typical antidepressant drug, imipramine. Gender-related differences in glucocorticoid receptor functional properties were assessed using hepatic and renal whole cell extracts of female and male rats before and after long-term imipramine treatment. The receptor's hormone-binding parameters, B(max) and K(D), were determined by radioligand binding assay, the glucocorticoid receptor and heat shock proteins (Hsp70 and Hsp90) levels by quantitative immunoblotting, and the interaction of these proteins within glucocorticoid receptor heterocomplex by co-immunoprecipitation. Glucocorticoid receptor binding potency (B(max)/K(D) ratio) was significantly higher in males than females both before and after treatment with imipramine. Gender-specific changes in the glucocorticoid receptor binding parameters in the examined tissues were observed in response to imipramine, and were found to be associated with alterations in the receptor interaction with Hsp70 and Hsp90. The results of the study point to sexual dimorphism in the glucocorticoid signaling and imply that glucocorticoid receptor functional alterations contribute to gender-related differences in vulnerability to stress and stress-related disorders, and in response to antidepressant drugs. (c) 2009 Elsevier B.V. All rights reserved. Ministry of Science of the Republic of Serbia [143003]

The effects of mercury (Hg) on basal and dexamethasone-induced tyrosine aminotransferase (TAT) activity in rat liver were studied. Comparison of TAT activity after in vitro and in vivo mercury application revealed the influence of the metal only when applied in vivo, suggesting that the effects are expressed at the level of TAT gene transcription. Intraperitoneal administration of mercury at 1, 2 or 3 mg Hg kg−1 b.w. 4 h before decapitation was shown to stimulate the basal activity of TAT. The most prominent increase was observed 4 h after the metal administration. When applied at 1 and 2 mg Hg kg−1 b.w. mercury was also shown to reduce partially the extent of the enzyme induction by dexamethasone, which was injected intraperitoneally at 5 mg kg−1 b.w. 5 h before death. The highest dose of mercury (3 mg Hg kg−1 b.w.) almost completely abolished the dexamethasone effect. The finding that mercury increases basal activity of the enzyme while decreasing its induction by dexamethasone suggests that stimulatory effects of this metal on TAT activity are probably mediated by factors other than glucocorticoids. Copyright © 2005 John Wiley & Sons, Ltd.

Background: Selection of appropriate endogenous control is a critical step in gene expression analysis. The aim of this study was to evaluate expression stability of four frequently used endogenous controls: beta-actin, glyceraldehyde-3-phosphate dehydrogenase, beta(2)-microglobulin and RNA polymerase II polypeptide A in peripheral blood mononuclear cells from war veterans with and without posttraumatic stress disorder (PTSD). The study was designed as to identify suitable reference gene(s) for normalization of gene expression in peripheral blood mononuclear cells in response to war trauma and/or PTSD. Results: The variability in expression of the four endogenous controls was assessed by TaqMan Real-time RT-PCR in peripheral blood mononuclear cells from: war veterans with current PTSD, those with lifetime PTSD, trauma controls and healthy subjects. Expression stability was analyzed by GeNorm and NormFinder software packages, and by direct comparison of Ct values. Both, GeNorm and NormFinder identified beta-actin and glyceraldehyde-3-phosphate dehydrogenase as a pair of genes with the lowest stability value. Conclusions: The combination of beta-actin and glyceraldehyde-3-phosphate dehydrogenase appeared to be the most suitable reference for studying alterations in gene expression in peripheral blood mononuclear cells related to vulnerability and resilience to PTSD, as well as to trauma-provoked developing of this disorder and recovery from it. Using glyceraldehyde-3-phosphate dehydrogenase, beta-actin and beta(2)-microglobulin as individual endogenous controls would provide satisfactory data, while RNA polymerase II polypeptide A could not be recommended. Ministry of Science of Serbia [143003]; European Commission [INCO-CT-2004-509213]

Glucocorticoid receptor (GR) transduces the glucocorticoid (GC) signal that could lead to metabolic derangements depending on the tissue responsiveness to GC. We aimed to investigate possible causative relation of the GR functional properties in peripheral blood mononuclear cells of women with polycystic ovary syndrome (PCOS), with their clinical and biochemical characteristics. Thirty women with PCOS [mean age: 26.5 ± 5.1 years, mean body mass index (BMI) 24.5 ± 5 kg/m(2)], and thirty respective controls were analyzed for the number of GR sites per cell (B (max)), apparent equilibrium dissociation constant (K (d)), and binding potency (GR potency). A strong association between B (max) and K (d) (r = 0.70, P < 0.0001), and GR potency with age (r = 0.49, P = 0.009) was observed in PCOS women. The multiple regression analyses within the PCOS group revealed that independent predictors for K (d) were BMI, total cholesterol, and dehydroepiandrosterone-sulfate (DHEA-S) (r = 0.58, P = 0.038), while for GR potency (r = 0.687, P = 0.013) were age, BMI, DHEA-S, and basal cortisol concentration. The results suggest that PCOS pathophysiology may be related to alterations of a cross stalk between glucocorticoid signaling, age, and metabolic parameters. These findings should be further explored in studies on the role of GR in PCOS-related metabolic derangements.

The heavy metal cadmium has been recognized as an extreme-ly hazardous environmental contaminant that can be highlyphytotoxic. Excessive amounts of cadmium in the environmentcan affect the performance of plants at various levels of biolog-ical organization, from the cellular up to the ecosystem level.Exposure to this harmful heavy metal can cause disturbance ofseveral essential physiological processes in plants, includingphotosynthesis, respiration and transpiration (SereginandIvanov, 2001; Ciscatoet al., 2002). At the cellular lev-el, toxic effects of cadmium may result from binding of the met-al to sulfhydryl groups of proteins, leading to disruption of pro-tein structure and function (Hall, 2002). Plants have a range of potential protective mechanisms atthe cellular level that are involved in detoxification and thus indevelopment of tolerance to heavy metal-induced stress. Thecellular stress response is a general defense mechanism com-monly seen in all organisms, from bacteria to higher organisms,including plants. This response is characterized by rapid andtransient synthesis of heat shock proteins (HSPs) (Nover,1991; Sanita di Toppiand Gabbrielli, 1999). Ap-pearance of damaged proteins in the cell after cadmium treat-ment serves as a signal that triggers the activation of HSPgenes, whose products (HSPs) prevent and repair protein dam-aged (Hightoweret al., 1994). As a member of the HSPfamily, HSP70 plays an important role under stressful condi-tions, serving to minimize injury and assist in cellular recovery(Pelham, 1984). Several different HSP70 isoforms havebeen described in plants; some of them are predominantly con-stitutively expressed, while others are inducible. There are onlya few reports indicating that cadmium intox ication provoked in-duction of HSP70 in kidney bean (Leitaet al., 1991,1993).The kidney bean (

The influence of mercury on the association of rat kidney glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70 was investigated. The GR heterocomplexes with Hsp90 and Hsp70 were immunopurified from the renal cytosol of rats administered different doses of mercury (1, 2 and 3 mg Hg kg−1 b.w.). A quantitative immunoblotting procedure was applied to determine the levels of GR, Hsp90 and two nucleocytoplasmic Hsp70 isoforms (constitutive Hsp73 and inducible Hsp72) in the renal cytosol, as well as the amounts of these proteins within GR heterocomplexes immunoprecipitated by anti-GR antibody. Mercury was found to stimulate GR association with all the examined Hsps. The most prominent effect of the metal was stimulation of Hsp72 interaction with GR. On the other hand, the metal administration led to an increase of Hsp90 level in the cytosol, while the cytosolic levels of Hsp70 isoforms remained unaltered. These findings suggest that association of Hsps, at least Hsp70, with the GR might be ascribed to changes in the affinity of their interaction rather than to changes in the Hsp availability in the cytosol. Therefore, GR heterocomplex assembly seems to be a controlled process enabling chaperoning and functioning of the GR to be in concert with physiological demands. Copyright © 2006 John Wiley & Sons, Ltd.

Gender-related differences in the effects of imipramine, on the protein levels of glucocorticoid receptor (GR), and heat shock proteins Hsp90 and Hsp70, as well as on dexamethasone binding to corticosteroid receptors (CRs) in the pituitary, hypothalamus, hippocampus and brain cortex of non-depressed rats were studied. Differences between female and male animals in the GR protein level in the tissues of untreated animals were not noticed. However, imipramine led to opposite changes in the cellular level of GR protein in the brain of female and male rats, as well as to gender- and tissue-specific changes in in vitro dexamethasone binding to GR and mineralocorticoid receptor (MR) in the hippocampus and brain cortex. Gender-related differences in the expression of Hsp90 and Hsp70 were noticed mainly in the hippocampus, only after imipramine treatment. The observed changes in the response of GR to imipramine suggest that this antidepressant may affect both the level of the receptor protein and the mechanisms regulating its binding ability in a gender-related manner. null

The subject of the present study is the influence of mercury on association of rat liver glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70. The glucocorticoid receptor heterocomplexes with Hsp90 and Hsp70 were immunopurified from the liver cytosol of rats administered with different doses of mercury. The amounts of co-immunopurified apo-receptor, Hsp90 and Hsp70 were then determined by quantitative Western blotting. The ratio between the amount of heat shock protein Hsp90 or Hsp70 and the amount of apo-receptor within immunopurified heterocomplexes was found to increase in response to mercury administration. On the other hand, the levels of Hsp90 and Hsp70 in hepatic cytosol remained unaltered. The finding that mercury stimulates association of the two heat shock proteins with the glucocorticoid receptor, rendering the cytosolic heat shock protein levels unchanged, suggests that mercury affects the mechanisms controlling the assembly of the receptor heterocomplexes. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:257–260, 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20032

The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined after in vivo and in vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when applied in vitro at 0 degrees C, mercury produced reduction of the receptor binding activity similar to that observed in vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.