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 µg/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.

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.

Interindividual glucocorticoid sensitivity in young healthy subjects: the role of glucocorticoid receptor alpha and beta isoforms ratio.

Only few studies have addressed the interindividual variation and tissue specificity of glucocorticoid (GC) sensitivity in healthy individuals, a phenomenon observed in pathological conditions. Alternative splicing of the glucocorticoid receptor (GR) produces alpha and beta isoforms. GRbeta has dominant-negative effects on hormone-induced GRalpha effects, and an increased expression of the GRbeta has been associated with glucocorticoid resistance. We determined, using a simple, rapid, and accurate Real-Time PCR assay, the individual mRNAs expression of GRalpha and GRbeta in 26 normal subjects (mean+/-SE, age 30+/-6 years; 12 males and 14 females), in order to evaluate the role of these isoforms in glucocorticoid sensitivity in health. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a housekeeper gene. GRalpha/GAPDH, GRbeta/GAPDH and GRalpha/GRbeta ratios showed a normal distribution. We observed a higher expression of GRalpha compared to GRbeta and an interindividual variability in the GRalpha, GRbeta, and GAPDH gene expressions in the young healthy population. In addition, no correlation was observed between GRalpha/GRbeta ratio and the dexamethasone (DEX) doses needed to suppress plasma cortisol, GRalpha/GRbeta ratio and the concentration of DEX that caused inhibition of Con-A stimulated cell proliferation, and GRalpha/GRbeta ratio and the affinity of GR (Kd) of each subject. Therefore, the variability of GC sensitivity observed in normal subjects can not be ascribed to the variation in the GRalpha and GRbeta expression.

Molecular cloning of the neutral trehalase gene from Kluyveromyces lactis and the distinction between neutral and acid trehalases.

We cloned the Kluyveromyces lactis KlNTH1 gene, which encodes neutral trehalase. It showed 65.2% and 68.5% identity at nucleotide and amino acid sequence level, respectively, with the Saccharomyces cerevisiae NTH1 gene. Multiple alignment of the predicted trehalase protein sequences from yeasts, bacteria, insects, and mammals revealed two major domains of conservation. Only the yeast trehalases displayed in an N-terminal extension two consensus sites for cAMP-dependent protein phosphorylation and a putative Ca2+-binding sequence. Gene disruption of the KlNTH1 gene abolished neutral trehalase activity and clearly revealed a trehalase activity with an acid pH optimum. It also resulted in a high constitutive trehalose level. Expression of the KlNTH1 gene in an S. cerevisiae nth1Delta mutant resulted in rapid activation of the heterologous trehalase upon addition of glucose to cells growing on a nonfermentable carbon source and upon addition of a nitrogen source to cells starved for nitrogen in a glucose-containing medium. In K. lactis, the same responses were observed except that rapid activation by glucose was observed only in early-exponential-phase cells. Inactivation of K. lactis neutral trehalase by alkaline phosphatase and activation by cAMP in cell extracts are consistent with control of the enzyme by cAMP-dependent protein phosphorylation.

Comparative study of two trehalase activities from Fusarium oxysporum var. lini.

Acid and neutral trehalase activities (optimum pH of 4.6 and 6.8, respectively) from Fusarium oxysporum var. lini were studied separately through partial isolation by ammonium sulfate precipitation followed by ion-exchange chromatography on DEAE-Sephacel for neutral enzyme, or using some of their differential properties. Acid activity was unaffected by 1 mM of Ca2+, Mg2+, Mn2+, Ba2+, or EDTA. Contrarily, the neutral enzyme was activated by Ca2+ with an apparent Ka of 0.15 mM; was inhibited by EDTA, Zn2+, Hg2+, or Mg(2+)-ATP; and showed an increase in activity by the raise of buffer ionic strength or by the addition of 100 mM KCl. Acid and neutral enzymes have, respectively, an apparent optimum temperature of 45 and 30 degrees C, an apparent Km for trehalose of 0.43 and 8.45 mM, and an apparent M(r) of 160,000 and 100,000 (by glycerol gradient ultracentrifugation). Acid trehalase was specifically inhibited by acetate buffer and more stable at 50 degrees C than the neutral enzyme. Neutral enzyme exhibited a pI of 6.2 by isoelectric focusing. Contrary to neutral trehalases from other fungi, the enzyme from Fusarium oxysporum var. lini was not activated in crude extract by treatment with Mg(2+)-ATP in the presence of cAMP and not inactivated by alkaline phosphatase from Escherichia coli.

Perfil hematologico e bioquimico de pacientes com tuberculose / Hematological and biochemical profile of patients carrying lung tuberculosis

Foram colhidas amostras de sangue de 30 pacientes com tuberculose pulmonar comprovada atraves do encontro do B.A.A.R. em esfregaços de escarro corados pelo Ziehl-Neelsen.. As amostras de sangue foram empregadas, atraves de metodologia adequada, para a determinaçao do perfil hematologico e bioquimico dos tuberculosos em busca de alteraçoes que pudessem ser distribuidas a infeccao. Os resultados observados revelaram uma tendencia a leucocitose (23//dos casos com global acima de 10.000/mm3) e um elevado aumento da velocidade de hemossedimentaçao (86//apresentaram elevaçao com media de 72 a 44 mm para mulheres ehomens, respectivamente). A monocitose, referida por varios autores na tuberculose ativa, nao foi observada. Quanto ao perfil bioquimico, os resultados da determinaçao de fosfatase alcalina e valores obtidos de globulinas se mostraram elevados em 46//dos casos. Esses resultados representaram dados preliminares obtidos atraves das avaliaçoes hematologicas e bioquimicas de pacientes com tuberculose,realizadas antes do emprego da terapeutica recomendada. Estes pacientes representam uma amostragem adequadamente selecionada para a primeira etapa do projeto depesquisa sobre a avaliaçao da conduta farmacoterapeutica no setor publico, com aparticipaçao integrada dos Departamentos de Farmacia Social, Produtos Farmaceuticos e Analises Clinicas e Toxicologicas da Faculdade de Farmacia da UFMG.