Lilian Eslaine Costa Mendes da Silva

Estradiol-induced hypophagia is associated with the differential mRNA expression of hypothalamic neuropeptides

Estradiol participates in the control of energy homeostasis, as demonstrated by an increase in food intake and in body weight gain after ovariectomy in rats. In the present study, female Wistar rats (200-230 g, N = 5-15 per group), with free access to chow, were individually housed in metabolic cages. We investigated food intake, body weight, plasma leptin levels, measured by specific radioimmunoassay, and the hypothalamic mRNA expression of orexigenic and anorexigenic neuropeptides, determined by real-time PCR, in ovariectomized rats with (OVX+E) and without (OVX) estradiol cypionate treatment (10 microg/kg body weight, sc, for 8 days). Hormonal and mRNA expression were determined at pre-feeding and 4 h after food intake. OVX+E rats showed lower food intake, less body weight gain and lower plasma leptin levels. In the OVX+E group, we also observed a reduction of neuropeptide Y (NPY), agouti-related protein (AgRP) and cocaine- and amphetamine-regulated transcript (CART) mRNA expression in the arcuate nucleus and a decrease in orexin A in the lateral hypothalamic area (LHA). There was an increase in leptin receptor (LepRb), melanocortin-4 receptor (MC4-R), CART, and mainly corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus and LepRb and CART mRNA in the LHA. These data show that hypophagia induced by estradiol treatment is associated with reduced hypothalamic expression of orexigenic peptides such as NPY, AgRP and orexin A, and increased expression of the anorexigenic mediators MC4-R, LepRb and CRH. In conclusion, estradiol decreases food intake, and this effect seems to be mediated by peripheral factors such as leptin and the differential mRNA expression of neuropeptides in the hypothalamus.

Creatine supplementation prevents fatty liver in rats fed choline-deficient diet: a burden of one-carbon and fatty acid metabolism.

AIM To examine the effects of creatine (Cr) supplementation on liver fat accumulation in rats fed a choline-deficient diet. METHODS Twenty-four rats were divided into 3 groups of 8 based on 4 weeks of feeding an AIN-93 control diet (C), a choline-deficient diet (CDD) or a CDD supplemented with 2% Cr. The CDD diet was AIN-93 without choline. RESULTS The CDD significantly increased plasma homocysteine and TNFα concentration, as well as ALT activity. In liver, the CDD enhanced concentrations of total fat (55%), cholesterol (25%), triglycerides (87%), MDA (30%), TNFα (241%) and decreased SAM concentrations (25%) and the SAM/SAH ratio (33%). Cr supplementation prevented all these metabolic changes, except for hepatic SAM and the SAM/SAH ratio. However, no changes in PEMT gene expression or liver phosphatidylcholine levels were observed among the three experimental groups, and there were no changes in hepatic triglyceride transfer protein (MTP) mRNA level. On the contrary, Cr supplementation normalized expression of the transcription factors PPARα and PPARγ that were altered by the CDD. Further, the downstream targets and fatty acids metabolism genes, UCP2, LCAD and CPT1a, were also normalized in the Cr group as compared to CDD-fed rats. CONCLUSION Cr supplementation prevented fat liver accumulation and hepatic injures in rats fed with a CDD for 4 weeks. Our results demonstrated that one-carbon metabolism may have a small role in mitigating hepatic fat accumulation by Cr supplementation. The modulation of key genes related to fatty acid oxidation pathway suggests a new mechanism by which Cr prevents liver fat accumulation.

Hypothalamic oxytocin neurons modulate hypophagic effect induced by adrenalectomy.

Glucocorticoids have major effects on food intake, as demonstrated by the decrease of food intake following adrenalectomy (ADX); however, the mechanisms leading to these effects are not well understood. Oxytocin (OT) has been shown to reduce food intake. We evaluated the effects of glucocorticoids on OT neuron activation and OT mRNA expression in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei induced by feeding. We also evaluated the effect of pretreatment with OT-receptor antagonist ([d(CH2)5,Tyr(Me)2,Orn8]-vasotocin, OVT) on food intake in ADX rats. Fos/OT neurons in the posterior parvocellular subdivision of the PVN were increased after refeeding, with a higher number in the ADX group, compared with sham and ADX+corticosterone (B) groups, with no difference in the medial parvocellular and magnocellular subdivisions of the PVN. ADX increased OT mRNA expression in the PVN both in fasting and refeeding condition, compared with sham and ADX+B groups. In the SON, refeeding increased the number of Fos/OT neurons, with a higher number in the ADX+B group. In fasted condition, OT mRNA expression in the SON was increased in ADX and ADX+B, compared with sham group. Pretreatment with OVT reversed the ADX-induced hypophagia, with no difference between sham and ADX+B animals. The present results show that glucocorticoid withdrawal induces a higher activation of PVN OT neurons in response to feeding, and an increase of OT mRNA expression in the PVN and OT-receptor antagonist reverses the anorexigenic effect induced by ADX. These data indicate that PVN OT neurons might mediate the hypophagic effect induced by adrenalectomy.

Corticotrophin-releasing factor mediates hypophagia after adrenalectomy, increasing meal-related satiety responses

Adrenalectomy-induced hypophagia is associated with increased satiety-related responses, which involve neuronal activation of the nucleus of the solitary tract (NTS). Besides its effects on the pituitary-adrenal axis, corticotrophin-releasing factor (CRF) has been shown to play an important role in feeding behaviour, as it possesses anorexigenic effects. We evaluated feeding-induced CRF mRNA expression in the paraventricular nucleus (PVN) and the effects of pretreatment with CRF(2) receptor antagonist (Antisauvagine-30, AS30) on food intake and activation of NTS neurons in response to feeding in adrenalectomised (ADX) rats. Compared to the sham group, ADX increased CRF mRNA levels in the PVN of fasted animals, which was further augmented by refeeding. AS30 treatment did not affect food intake in the sham and ADX+corticosterone (B) groups; however, it reversed hypophagia in the ADX group. In vehicle-pretreated animals, refeeding increased the number of Fos and Fos/TH-immunoreactive neurons in the NTS in the sham, ADX and ADX+B groups, with the highest number of neurons in the ADX animals. Similarly to its effect on food intake, pretreatment with AS30 in the ADX group also reversed the increased activation of NTS neurons induced by refeeding while having no effect in the sham and ADX+B animals. The present results show that adrenalectomy induces an increase in CRF mRNA expression in the PVN potentiated by feeding and that CRF(2) receptor antagonist abolishes the anorexigenic effect and the increased activation of NTS induced by feeding in the ADX animals. These data indicate that increased activity of PVN CRF neurons modulates brainstem satiety-related responses, contributing to hypophagia after adrenalectomy.

Caldesmon: New Insights for Diagnosing Endometriosis

ABSTRACT Considerable effort has been invested in searching for less invasive methods of diagnosing endometriosis. Previous studies have indicated altered levels of the CALD1 gene (encoding the protein caldesmon) in endometriosis. The aims of our study were to investigate whether average CALD1 expression and caldesmon protein levels are differentially altered in the endometrium and endometriotic lesions and to evaluate the performance of the CALD1 gene and caldesmon protein as potential biomarkers for endometriosis. Paired biopsies of endometrial tissue (eutopic endometrium) and endometriotic lesions (ectopic endometrium) were obtained from patients with endometriosis to evaluate CALD1 gene expression and caldesmon protein levels by real-time PCR and Western blot analysis, respectively. In addition, immunostaining for caldesmon to determine cellular localization was also performed. Endometrium from women without endometriosis was used as a control. Increased CALD1 expression and caldesmon levels were detected in the endometriotic lesions. The electrophoretic profile of caldesmon by Western blot analysis was clearly different between the control group (endometrium of women without endometriosis) and the group of women with endometriosis (eutopic endometrium and endometriotic lesions). Caldesmon expression as determined by immunostaining showed no variation among the cell types in endometriotic lesions and eutopic endometrium. Stromal cells marked positively in eutopic endometrium from control patients and in the endometriotic lesions. The presence of caldesmon in the endometrium of patients with and without endometriosis permitted diagnoses with 95% sensitivity (specificity 100%) and 100% sensitivity (specificity 100%) for the disease and for minimal to mild endometriosis in the proliferative phase of the menstrual cycle, respectively. In the secretory phase, minimal to mild endometriosis was detected with 90% sensitivity and 93.3% specificity. Caldesmon is a possible predictor of endometrial dysregulation in patients with endometriosis. A potential limitation of our study is the fact that other endometrial diseases were not excluded, and therefore prospective studies are needed to confirm the potential of caldesmon as a biomarker exclusively for endometriosis.

Lacrimal osmolarity and ocular surface in experimental model of dry eye caused by toxicity

Objetivo: Descrever um modelo animal de olho seco induzido pela aplicacao topica de cloreto de benzalconio (BAC) e avaliar marcadores de integridade da superficie ocular e os achados histologicos. Metodos: Foram utilizados ratos wistar machos adultos. Foi realizada a administracao topica de colirio de BAC 0,2% no olho direito de cada animal duas vezes por dia, durante 7 dias, sendo o olho contralateral tido como controle. Apos o tratamento foi realizada a avaliacao da osmolaridade do filme lacrimal, o teste de fenol vermelho e a coloracao com fluoresceina e lisamina verde. Os animais foram sacrificados e os tecidos extraidos para o estudo histologico da cornea, por microscopia optica, corada com hematoxilina eosina (H&E). Resultados: Comparados com os controles nao tratados o grupo BAC apresentou diminuicao significativa na secrecao lacrimal, defeitos na integridade epitelial da superficie ocular marcada por corantes vitais, fluoresceina e lisamina verde alem do aumento da osmolaridade do filme lacrimal (p < 0,05). A avaliacao histologica observou-se diminuicao da espessura do epitelio e edema estromal induzidos pela aplicacao de BAC. Conclusao: O modelo animal de olho seco por toxicidade induzido pela aplicacao topica de cloreto de benzalconio apresentou alteracoes estruturais da cornea e da superficie ocular, diminuicao do volume lacrimal e hiperosmolaridade da lagrima caracteristicas dessa condicao.

Taurine supplementation does not decrease homocysteine levels and liver injury induced by a choline-deficient diet.

AIMS The aim of this study is to examine the effects of taurine supplementation on homocysteine (Hcy) metabolism and liver injury in rats fed a choline-deficient diet. MAIN METHODS Thirty rats were divided into three groups (n=10), to receive one of the following diets for 4 weeks: control diet (C), choline-deficient diet (CDD), or choline-deficient diet supplemented with taurine (CDDT). The CDD and the CDDT consisted of AIN-93 without the recommended choline content of 2.5%, and the CDDT was supplemented by the addition of 2.5% taurine. KEY FINDINGS Four weeks of ingesting a CDD resulted in a significant increase in plasma Hcy (50%) as well as a decrease in liver S-adenosylmethionine (SAM) concentration and S-adenosylmethionine/S-adenosylhomocysteine ratio. No changes were found in plasma methionine and cysteine plasma levels compared to control group. Four weeks of ingesting a CDD also caused a significant (P<0.05) increase in hepatic total fat, hepatic malondialdehyde (MDA), and plasma alanine aminotransferase (ALT) levels. In addition, reduced hepatic glutathione (GSH) levels and reduced/oxidized glutathione ratios (GSH/GSSG) were found in rats fed a CDD compared to controls. Taurine supplementation of the CDD normalized genes involved in the remethylation pathway, BHMT and CHDH, which were impaired by CDD alone. However, taurine supplementation failed to prevent CDD-induced Hcy metabolism disturbances and hepatic injury. Also, taurine added to CDD caused decreased expression of PEMT, CHKa, and CHKb, key genes involved in phosphatidylcholine (PC) synthesis and liver fat accumulation. SIGNIFICANCE Taurine supplementation failed to ameliorate impaired Hcy metabolism and liver injury caused by CDD intake.

Specific immediate early gene expression induced by high doses of salicylate in the cochlear nucleus and inferior colliculus of the rat

INTRODUCTION Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus. OBJECTIVES Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats. METHODS Rats were treated with doses known to induce tinnitus in rats (300mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry. RESULTS After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas. CONCLUSION Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.

Acute exercise alters homocysteine plasma concentration in an intensity-dependent manner due increased methyl flux in liver of rats

Purpose: We aimed to determine the effects of different intensities of acute exercise on Hcy plasma levels, and the exercise‐induced changes in Hcy liver metabolism. Method: First, thirty‐two Wistar rats were randomly submitted to an acute bout of swimming exercise carrying a load of 2% (n = 8), 4% (n = 8) and 6% (n = 8) of their total body weight attached in their tail. Control rats remained rested (n = 8). Blood samples were taken from tail vein for plasma S‐containing amino acids determination before (Rest) and post, 1, 2, 3, 4, 6, and 10 h after acute swimming exercise. Second, 56 exercised rats (4% loads) were euthanized before (Rest) and1, 2, 3, 4, 6, and 10 h after acute swimming exercise. Blood and liver samples were collected for amino acids and keys genes involved in the Hcy metabolism assay. Results: Acute exercise increases (P < 0.05) plasma Hcy concentration in an intensity‐dependent manner (rest 7.7 ± 0.8; 6% load 13.8 ± 3.6; 4% load 12.2 ± 2.9 ± and 2% load 10.1 ± 2.6, &mgr;mol/L); this increase is transient and does not promote hyperhomocysteinemia (< 15 &mgr;mol/L).Exercise‐induced increased plasma Hcywas accompanied by the decreased liver S‐adenosylmethionine/S‐adenosylhomocysteine ratio and elevated MAT1a mRNA content. Acute exercise also caused elevated mRNA of key enzymes of transsulfuration (CBS) and remethylation (BHMT and the MTRR). Conclusion: Our data provided evidence that acute exercise increases plasma Hcy concentration due to the augmented requirement for methylated compounds that increases liver SAM consumption. Also, Hcy remethylation and transsulfuration are coordinately regulated to maintain methyl balance.

Resistance exercise attenuates skeletal muscle oxidative stress, systemic pro-inflammatory state, and cachexia in Walker-256 tumor-bearing rats

The aim of this study was to investigate the effects of resistance exercise training (RET) on oxidative stress, systemic inflammatory markers, and muscle wasting in Walker-256 tumor-bearing rats. Male (Wistar) rats were divided into 4 groups: sedentary controls (n = 9), tumor-bearing (n = 9), exercised (n = 9), and tumor-bearing exercised (n = 10). Exercised and tumor-bearing exercised rats were exposed to resistance exercise of climbing a ladder apparatus with weights tied to their tails for 6 weeks. The physical activity of control and tumor-bearing rats was confined to the space of the cage. After this period, tumor-bearing and tumor-bearing exercised animals were inoculated subcutaneously with Walker-256 tumor cells (11.0 × 107 cells in 0.5 mL of phosphate-buffered saline) while control and exercised rats were injected with vehicle. Following inoculation, rats maintained resistance exercise training (exercised and tumor-bearing exercised) or sedentary behavior (control and tumor-bearing) for 12 more days, after which they were euthanized. Results showed muscle wasting in the tumor-bearing group, with body weight loss, increased systemic leukocytes, and inflammatory interleukins as well as muscular oxidative stress and reduced mTOR signaling. In contrast, RET in the tumor-bearing exercised group was able to mitigate the reduced body weight and muscle wasting with the attenuation of muscle oxidative stress and systemic inflammatory markers. RET also prevented loss of muscle strength associated with tumor development. RET, however, did not prevent the muscle proteolysis signaling via FBXO32 gene messenger RNA expression in the tumor-bearing group. In conclusion, RET performed prior tumor implantation prevents cachexia development by attenuating tumor-induced systemic pro-inflammatory condition with muscle oxidative stress and muscle damage.