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1.
PLoS One ; 8(1): e54851, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23349977

RESUMO

Stress, the physiological reaction to a stressor, is initiated in teleost fish by hormone cascades along the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol is the major stress hormone and contributes to the appropriate stress response by regulating gene expression after binding to the glucocorticoid receptor. Cortisol is inactivated when 11ß-hydroxysteroid dehydrogenase (HSD) type 2 catalyzes its oxidation to cortisone. In zebrafish, Danio rerio, cortisone can be further reduced to 20ß-hydroxycortisone. This reaction is catalyzed by 20ß-HSD type 2, recently discovered by us. Here, we substantiate the hypothesis that 20ß-HSD type 2 is involved in cortisol catabolism and stress response. We found that hsd11b2 and hsd20b2 transcripts were up-regulated upon cortisol treatment. Moreover, a cortisol-independent, short-term physical stressor led to the up-regulation of hsd11b2 and hsd20b2 along with several HPI axis genes. The morpholino-induced knock down of hsd20b2 in zebrafish embryos revealed no developmental phenotype under normal culture conditions, but prominent effects were observed after a cortisol challenge. Reporter gene experiments demonstrated that 20ß-hydroxycortisone was not a physiological ligand for the zebrafish glucocorticoid or mineralocorticoid receptor but was excreted into the fish holding water. Our experiments show that 20ß-HSD type 2, together with 11ß-HSD type 2, represents a short pathway in zebrafish to rapidly inactivate and excrete cortisol. Therefore, 20ß-HSD type 2 is an important enzyme in stress response.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Cortisona Redutase , Cortisona/metabolismo , Hidrocortisona/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Animais , Cortisona/genética , Cortisona Redutase/genética , Cortisona Redutase/metabolismo , Técnicas de Silenciamento de Genes , Hidrocortisona/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Estresse Fisiológico/genética , Regulação para Cima , Peixe-Zebra/genética , Peixe-Zebra/fisiologia
2.
Gene ; 509(1): 68-76, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22835697

RESUMO

Teleostean 20ß-hydroxysteroid dehydrogenase (20ß-HSD) is involved in final oocyte maturation and steroid hormone metabolism. It has structural and functional similarities to mammalian carbonyl reductases that are involved in the metabolism of endogenous carbonyl and xenobiotic compounds. To understand the transcriptional regulation of 20ß-HSD, here we report the cloning of 20ß-HSD promoter from two fish species, rainbow trout and air-breathing catfish. Analysis of the promoter motifs, in silico identified the presence of several sites for transcription factor binding including cAMP, xenobiotic and steroid hormone responsive elements. Luciferase reporter assays with progressive deletion constructs demonstrated that 20ß-HSD type B of trout has no promoter activity while 20ß-HSD type A of trout and catfish 20ß-HSD promoters showed basal promoter activity. A TATA box flanked by a CAAT box is important for basal transcription. Deletion of cAMP responsive element in the promoter decreased basal promoter activity significantly. Reporter assays with forskolin and IBMX, drugs that increase intracellular cAMP induced the promoter activity over the basal level. Intriguingly, ß-nafthoflavone, an arylhydrocarbon receptor ligand, induced the 20ß-HSD promoter activity and is further evidenced by the induction of 20ß-HSD expression in the livers of catfish, in vivo. These results demonstrate for the first time that 20ß-HSD expression is not only modulated by cAMP but also by xenobiotics and further studies may provide significance to the ubiquitous distribution and broad substrate specificity of this enzyme.


Assuntos
Peixes-Gato/genética , Cortisona Redutase/genética , Oncorhynchus mykiss/genética , Regiões Promotoras Genéticas , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Sequência de Bases , Peixes-Gato/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , DNA/genética , DNA/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Dados de Sequência Molecular , Oncorhynchus mykiss/metabolismo , Ovário/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes/genética , Especificidade da Espécie , Xenobióticos/metabolismo , beta-Naftoflavona/farmacologia
3.
Mol Cell Endocrinol ; 349(2): 202-13, 2012 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-22061621

RESUMO

Hydroxysteroid dehydrogenases (HSDs) are involved in metabolism and pre-receptor regulation of steroid hormones. While 17beta-HSDs and 11beta-HSDs are extensively studied in mammals, only few orthologs are characterized in fish. We discovered a novel zebrafish HSD candidate closely related to 17beta-HSD types 3 and 12, which has orthologs in other species. The enzyme catalyzes the conversion of cortisone to 20beta-hydroxycortisone identified by LC-MS/MS. We named the new enzyme 20beta-HSD type 2. All 20beta-HSD type 2 orthologs localize in the endoplasmic reticulum. Zebrafish 20beta-HSD type 2 is expressed during embryonic development showing the same expression pattern as 11beta-HSD type 2 known to oxidize cortisol to cortisone. In adult tissues 20beta-HSD type 2 shows a ubiquitous expression pattern with some minor sex-specific differences. In contrast to other enzymes metabolizing C21-steroids and being mostly involved in reproduction we propose that novel type 2 20beta-HSDs in teleost fish are important enzymes in cortisol catabolism.


Assuntos
Cortisona Redutase/metabolismo , Proteínas de Peixes/metabolismo , Hidrocortisona/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cortisona/metabolismo , Cortisona Redutase/genética , Retículo Endoplasmático/metabolismo , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Estudos de Associação Genética , Células HEK293 , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
4.
Gen Comp Endocrinol ; 175(1): 48-54, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21945117

RESUMO

The maturation inducing hormone, 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DP) is required for the meiotic maturation and is produced from the precursor 17α-hydroxyprogesterone by the enzyme 20ß-hydroxysteroid dehydrogenase (20ß-HSD) in several teleosts. Central role of 20ß-HSD in ovarian cycle and final oocyte maturation is well studied when compared to spermatogenesis. In the present study, we investigated the localization and expression of 20ß-HSD in testicular cycle and gonadotropin induced sperm maturation. During testicular ontogeny, 20ß-HSD expression was detectable at 50 and 100 days post-hatch (dph), while the expression was high at 150 dph. In testicular cycle, highest levels of mRNA and protein of 20ß-HSD were observed during spawning phase. Intraperitoneal injection of human chorionic gonadotropin (hCG) to prespawning catfish elevated both 20ß-HSD transcripts and protein levels when compared to saline treated controls in a time-dependent manner. Serum 17α,20ß-DP levels, measured during different phases of testicular cycle as well as following the treatment of hCG, showed a positive correlation with the expression of 20ß-HSD. Immunolocalization revealed the presence of 20ß-HSD protein predominantly in interstitial cells and spermatogonia/spermatocytes while 20ß-HSD was undetectable in haploid cells (spermatids/sperm). These results together with high expression during spawning phase of testicular cycle and after hCG treatment in the prespawning catfish suggests a pivotal role for 20ß-HSD during testicular recrudescence leading to sperm maturation. Further studies using various fish models on testicular 20ß-HSD may provide interesting details to understand its importance in teleostean spermatogenesis.


Assuntos
Peixes-Gato/metabolismo , Gonadotropina Coriônica/farmacologia , Cortisona Redutase/metabolismo , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Testículo/enzimologia , Animais , Gonadotropina Coriônica/administração & dosagem , Cortisona Redutase/genética , DNA Complementar/genética , Humanos , Hidroxiprogesteronas/sangue , Injeções Intraperitoneais , Células Intersticiais do Testículo/enzimologia , Masculino , Estações do Ano , Espermatócitos/enzimologia , Testículo/citologia
5.
Front Biosci (Landmark Ed) ; 16: 1898-914, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21196272

RESUMO

Meiotic maturation is a complex process that involves resumption of meiosis in response to preovulatory luteinizing hormone (LH) surge just before ovulation. High levels of cAMP in oocytes maintain meiotic arrest at diplotene of prophase I in mammals and pisces. In mammals, the process by which LH induces recommencement of meiosis involves breakdown of oocyte-somatic cells communication, which is followed by a drop in intracellular cAMP levels that in turn causes exit from meiotic arrest. Maturation promoting factor (MPF) then accomplishes progression of oocytes to reach first metaphase followed by second metaphase after reinitiating meiosis. Pisces require precise completion of oocyte growth involving vitellogenesis before the entry of meiotic maturation. Then, both mammalian and fish oocytes enters resumption of meiosis involving germinal vesicle breakdown, chromosome condensation, assembly of meiotic spindle, and formation of first polar body. However, this process in pisces is regulated by three major mediators, LH, 17alpha,20beta-dihydroxy progesterone and MPF which are unique. The molecular mechanisms of meiotic maturation and ovulation by comparing mammalian and piscine research have been dealt in this review.


Assuntos
Peixes/fisiologia , Mamíferos/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Ovulação/fisiologia , 3-Hidroxiesteroide Desidrogenases/metabolismo , Proteínas de Ancoragem à Quinase A/fisiologia , Animais , Cortisona Redutase/metabolismo , AMP Cíclico/metabolismo , Feminino , Humanos , Fator Promotor de Maturação/fisiologia , Folículo Ovariano/fisiologia , Fosfoproteínas/fisiologia , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroides/fisiologia , Fatores de Transcrição/fisiologia
6.
Biol Chem ; 391(1): 1-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19804362

RESUMO

Hexose-6-phosphate dehydrogenase (H6PD) is a luminal enzyme of the endoplasmic reticulum that is distinguished from cytosolic glucose-6-phosphate dehydrogenase by several features. H6PD converts glucose-6-phosphate and NADP(+) to 6-phosphogluconate and NADPH, thereby catalyzing the first two reactions of the pentose-phosphate pathway. Because the endoplasmic reticulum has a separate pyridine nucleotide pool, H6PD provides NADPH for luminal reductases. One of these enzymes, 11beta-hydroxysteroid dehydrogenase type 1 responsible for prereceptorial activation of glucocorticoids, has been the focus of much attention as a probable factor in the pathomechanism of several human diseases including insulin resistance and the metabolic syndrome. This review summarizes recent advances related to the functions of H6PD.


Assuntos
Retículo Endoplasmático/enzimologia , Glucosefosfato Desidrogenase/metabolismo , NADP/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Animais , Cortisona Redutase/deficiência , Glucosefosfato Desidrogenase/genética , Humanos , Camundongos , Camundongos Knockout , Via de Pentose Fosfato
7.
Artigo em Inglês | MEDLINE | ID: mdl-19036348

RESUMO

We have studied in vivo, the effects of physiological androgen (11-ketotestosterone: 11-KT and testosterone: T) concentrations on the growth of cod previtellogenic oocytes and steroidogenic gene expression patterns. Immature female Atlantic cod were injected three times (days 0, 7 and 14) with 0.05, 0.5 and 5 mg/kg of 11-KT and T. The control group was injected with the carrier solvent (ethanol diluted 1:10 in sunflower oil). Quantitative histological analyses demonstrated growth and development of previtellogenic oocytes after exposure to androgens. The oocyte developmental effect of androgens was more pronounced in fish receiving 11-KT. Quantitative PCR analysis demonstrated dose- and androgen-specific modulation of mRNA expression for genes involved in steroidogenesis (StAR (steroidogenic acute regulatory) protein, P450scc (P450-mediated cholesterol side-chain cleavage), 20beta-HSD (20beta-hydroxysteroid dehydrogenase)) and cell growth control, namely--opioid growth factor receptor (OGF-R), progesterone receptor protein p23 (PR23P) and apoptosis-inducing TAF9-like domain 1 (TAF9). Messenger RNA species associated with the zona pelucida, namely--the zona pellucida protein A domain (ZPA) and egg envelope glycoprotein (EeG) were modulated based on dose and androgen type. Cyclin-B mRNA expression was not affected by androgen exposure. Interestingly, we showed recently that these transcripts were responsive to in vitro androgen exposure in previtellogenic cod ovary. In conclusion, the present study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, suggesting androgen control of early oocyte growth in cod. The enhanced effects of 11-KT on oocyte growth support our hypothesis that non-aromatizable androgens play significant roles in the regulation of early previtellogenic oocyte growth and development.


Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Cortisona Redutase/genética , Gadus morhua/genética , Oócitos/efeitos dos fármacos , Fosfoproteínas/genética , Testosterona/farmacologia , Transcrição Genética/efeitos dos fármacos , Androgênios/química , Animais , Ciclina B/efeitos dos fármacos , Ciclina B/genética , Relação Dose-Resposta a Droga , Proteínas do Ovo/efeitos dos fármacos , Proteínas do Ovo/genética , Feminino , Gadus morhua/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Oócitos/crescimento & desenvolvimento , RNA Mensageiro/genética , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Receptores Opioides/efeitos dos fármacos , Receptores Opioides/genética , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Associados à Proteína de Ligação a TATA/efeitos dos fármacos , Fatores Associados à Proteína de Ligação a TATA/genética , Testosterona/análogos & derivados , Testosterona/sangue , Fatores de Tempo , Transcrição Genética/genética , Glicoproteínas da Zona Pelúcida
8.
Curr Opin Pediatr ; 20(4): 453-7, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18622204

RESUMO

PURPOSE OF REVIEW: Inactive cortisone is converted to active cortisol by the reductase activity of 11 beta-hydroxysteroid dehydrogenase type 1, which can thus increase glucocorticoid effects in target tissues. This paper reviews the functional role(s) of 11 beta-hydroxysteroid dehydrogenase type 1 and examines factors influencing its activity. RECENT FINDINGS: In obese humans, 11 beta-hydroxysteroid dehydrogenase type 1 is relatively highly expressed in adipose tissue. In mice, overexpression of 11 beta-hydroxysteroid dehydrogenase type 1 in adipose or liver causes obesity or insulin resistance, respectively, whereas mice lacking 11 beta-hydroxysteroid dehydrogenase type 1 resist diet-induced obesity and are insulin-sensitive. Thus, 11 beta-hydroxysteroid dehydrogenase type 1 is a promising drug target for treating the metabolic syndrome and type 2 diabetes. Studies in vitro and in mutant mice demonstrate that the reductase activity of 11 beta-hydroxysteroid dehydrogenase type 1 depends on reduced nicotinamide adenine dinucleotide phosphate synthesized within the endoplasmic reticulum by hexose-6-phosphate dehydrogenase. Apparent cortisone reductase deficiency is characterized by androgen excess in women or children and decreased urinary excretion of cortisol metabolites. Although polymorphisms in the genes encoding 11 beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase were initially implicated in this condition, subsequent reports have not confirmed this. SUMMARY: Hexose-6-phosphate dehydrogenase and 11 beta-hydroxysteroid dehydrogenase type 1 may play important roles in the pathogenesis of obesity and metabolic syndrome. Although the importance of polymorphisms in the corresponding genes remains uncertain, rare mutations have not been ruled out.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/fisiologia , Desidrogenases de Carboidrato/fisiologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Animais , Desidrogenases de Carboidrato/genética , Cortisona Redutase/deficiência , Cortisona Redutase/genética , Retículo Endoplasmático/metabolismo , Glucocorticoides/metabolismo , Humanos , Obesidade/fisiopatologia , Polimorfismo Genético
9.
J Clin Endocrinol Metab ; 93(10): 3827-32, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18628520

RESUMO

CONTEXT: Cortisone reductase deficiency (CRD) is characterized by a failure to regenerate cortisol from cortisone via 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), resulting in increased cortisol clearance, activation of the hypothalamic-pituitary-axis (HPA) and ACTH-mediated adrenal androgen excess. 11beta-HSD1 oxoreductase activity requires the reduced nicotinamide adenine dinucleotide phosphate-generating enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the endoplasmic reticulum. CRD manifests with hyperandrogenism resulting in hirsutism, oligo-amenorrhea, and infertility in females and premature pseudopuberty in males. Recent association studies have failed to corroborate findings that polymorphisms in the genes encoding H6PDH (R453Q) and 11beta-HSD1 (Intron 3 inserted adenine) interact to cause CRD. OBJECTIVE: Our objective was to reevaluate the genetics and steroid biochemistry of patients with CRD. DESIGN: We analyzed 24-h urine collection for steroid biomarkers by gas chromatography/mass spectrometry and sequenced the HSD11B1 and H6PD genes in our CRD cohort. PATIENTS: Patients included four cases presenting with hyperandrogenism and biochemical features clearly indicative of CRD. RESULTS: Gas chromatography/mass spectrometry identified steroid biomarkers that correlated with CRD in each case. Three cases were identified as homozygous (R109AfsX3, Y316X, and G359D) and one case identified as compound heterozygous (c.960G-->A and D620fsX3) for mutations in H6PD. No mutations affecting enzyme activity were identified in the HSD11B1 gene. Expression and activity assays demonstrate loss of function for all reported H6PDH mutations. CONCLUSIONS: CRD is caused by inactivating mutations in the H6PD gene, rendering the 11beta-HSD1 enzyme unable to operate as an oxoreductase, preventing local glucocorticoid regeneration. These data highlight the importance of the redox control of cortisol metabolism and the 11beta-HSD1-H6PDH pathway in regulating hypothalamic-pituitary-adrenal axis activity.


Assuntos
Biomarcadores/análise , Desidrogenases de Carboidrato/genética , Cortisona Redutase/deficiência , Análise Mutacional de DNA , Doenças Metabólicas/genética , Adulto , Alopecia/complicações , Alopecia/genética , Alopecia/metabolismo , Biomarcadores/metabolismo , Criança , Cortisona Redutase/genética , Feminino , Hirsutismo/complicações , Hirsutismo/genética , Hirsutismo/metabolismo , Humanos , Masculino , Doenças Metabólicas/complicações , Doenças Metabólicas/enzimologia , Doenças Metabólicas/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Mutação/fisiologia , Linhagem , Puberdade Precoce/complicações , Puberdade Precoce/genética , Puberdade Precoce/metabolismo , Esteroides/metabolismo
10.
J Mol Endocrinol ; 41(3): 125-33, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18586838

RESUMO

The reductase activity of 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) plays an important role in the growth and differentiation of adipose tissue via the prereceptorial activation of glucocorticoids. This enzyme colocalizes with hexose-6-phosphate dehydrogenase (H6PD) at the luminal surface of the endoplasmic reticulum membrane, and the latter enzyme provides NADPH to the former, which can thus act as an 11beta-reductase. It was suggested that, during adipogenesis, the increased expression of H6PD causes a dehydrogenase-to-reductase switch in the activity of HSD11B1. However, only the expression of the HSD11B1 has been extensively studied, and little is known about the expression of H6PD. Here, we investigated the expression and the activity of H6PD in the course of the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) and murine 3T3-L1 cells. It was found that H6PD is already present in adipose-derived stem cells and in 3T3-L1 fibroblasts even before the induction of adipogenesis. Moreover, mRNA and protein levels, as well as the microsomal H6PD activities remained unchanged during the differentiation. At the same time a great induction of HSD11B1 was observed in both cell types. The observed constant expression of H6PD suggests that HSD11B1 acts as a reductase throughout the adipogenesis process in human ADMSCs and murine 3T3-L1 cells.


Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/enzimologia , Desidrogenases de Carboidrato/genética , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/enzimologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/biossíntese , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Células 3T3-L1 , Adipogenia , Animais , Desidrogenases de Carboidrato/biossíntese , Linhagem da Célula , Cortisona/metabolismo , Cortisona Redutase/metabolismo , Indução Enzimática , Humanos , Hidrocortisona/metabolismo , Camundongos , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
J Mol Endocrinol ; 39(4): 319-28, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17909270

RESUMO

In order to better quantify the molecular mechanisms regulating final oocyte maturation and spawning, complete coding sequences with partially or fully untranslated regions for the steroidogenic enzymes, cytochrome P450 aromatase and 20 beta-hydroxysteroid dehydrogenase, were cloned from ovaries of Atlantic cod (Gadus morhua). The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species, and conserved features important for functionality were identified in both predicted proteins. The sequences of the corresponding genomic loci were also determined, allowing the design of mRNA-specific quantitative PCR assays. As a reference gene for the real-time RT-PCR assays, eukaryotic elongation factor 1 alpha was chosen, and the mRNA as well as the genomic sequence was determined. In addition, a real-time quantitative PCR assay for the 18S rRNA was adapted to be used in cod. Analysis of immature and maturing female cod from July to January respectively showed that the enzyme genes showed the expected quantitative changes associated with physiological regulation. However, mRNA for eukaryotic elongation factor 1 alpha, and to a lesser extent even 18S rRNA, showed variable expression in these samples as well. To find accurate standards for real-time PCR in such a dynamic organ as the cod ovary is not an easy task, and several possible solutions are discussed.


Assuntos
Aromatase/genética , Cortisona Redutase/genética , Gadus morhua/genética , Regulação Enzimológica da Expressão Gênica , Maturidade Sexual/genética , Sequência de Aminoácidos , Animais , Aromatase/metabolismo , Sequência de Bases , Cortisona Redutase/metabolismo , Fator de Iniciação 1 em Eucariotos/genética , Fator de Iniciação 1 em Eucariotos/metabolismo , Feminino , Masculino , Dados de Sequência Molecular , Estações do Ano , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
12.
Ecotoxicol Environ Saf ; 68(1): 33-9, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17543385

RESUMO

Proteomic analyses were performed to identify regulated liver proteins in rainbow trout (Oncorhynchus mykiss) caged upstream and downstream from a sewage treatment works (STW). Two-dimensional gel electrophoresis, image analysis and FT-ICR mass-spectrometry revealed four regulated protein spots. The three down-regulated spots contained betaine aldehyde dehydrogenase, lactate dehydrogenase and an unidentified protein respectively. The only up-regulated spot consisted of both mitochondrial ATP synthase alpha-subunit and carbonyl reductase/20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD). Further studies using quantitative PCR revealed a 13.5-fold induction of CR/20beta-HSD B mRNA following STW effluent exposure. The CR/20beta-HSD B gene was not regulated by 17alpha-ethinylestradiol, suggesting that its induction downstream from the STW is due to other factors than exposure to estrogens. Image analysis was initially performed on four gels from each group. These analyses suggested 15 regulated spots. However, validation of the 15 spots by increasing the number of replicates confirmed only four regulated spots. Hence, the present study also demonstrates the need for sufficient biological/technical replication in the interpretation of proteomic data.


Assuntos
Cortisona Redutase/biossíntese , Fígado/efeitos dos fármacos , Fígado/enzimologia , Oncorhynchus mykiss/metabolismo , Proteômica , Esgotos , Poluentes Químicos da Água/toxicidade , Animais , Cortisona Redutase/genética , Eletroforese em Gel Bidimensional , Indução Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , ATPases Mitocondriais Próton-Translocadoras/biossíntese , ATPases Mitocondriais Próton-Translocadoras/genética , RNA Mensageiro/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Espectrometria de Massas em Tandem
13.
J Clin Endocrinol Metab ; 92(1): 359-62, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17062770

RESUMO

CONTEXT: Recently, it was proposed that a combination of the 83,557insA polymorphism in the 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) gene and the R453Q polymorphism in the hexose-6-phosphate dehydrogenase (H6PD) gene interacts to cause cortisone reductase deficiency (CRD) when at least three alleles are affected. OBJECTIVE: The aim was to study the separate and combined effects of these polymorphisms on body composition, adrenal androgen production, blood pressure, glucose metabolism, and the incidence of dementia in the healthy elderly population. DESIGN/SETTING/PARTICIPANTS: The Rotterdam study (n = 6105) and the Frail Old Men study (n = 347) are population-based cohort studies in the elderly. MAIN OUTCOME MEASURES: Genotype distributions and influences of (combined) genotypes on body mass index, adrenal androgen production, waist to hip ratio, systolic and diastolic blood pressure, fasting glucose levels, glucose tolerance test, and incidence of dementia were measured. RESULTS: No influence of the HSD11B1 83,557insA (allele frequencies 22.0 and 21.5%) and H6PD R453Q (allele frequencies 22.9 and 20.2%) variants was found for the different outcome measures that were investigated, either separately or when at least three alleles were affected. CONCLUSIONS: Two population-based studies among Caucasian elderly showed no evidence for (combined) effects of two polymorphisms in the HSD11B1 and H6PD genes on body composition, adrenal androgen production, blood pressure, glucose metabolism, and incidence of dementia. Moreover, the high frequencies observed for these two polymorphisms do not correspond to the low incidence of CRD observed in the general population. Altogether, it is unlikely that these polymorphisms cause CRD.


Assuntos
11-beta-Hidroxiesteroide Desidrogenases/genética , Androgênios/biossíntese , Pressão Sanguínea , Composição Corporal , Desidrogenases de Carboidrato/genética , Cortisona Redutase/deficiência , Demência/genética , Glucose/metabolismo , Polimorfismo de Nucleotídeo Único , Glândulas Suprarrenais/metabolismo , Idoso , Índice de Massa Corporal , Demência/enzimologia , Humanos , Masculino , Relação Cintura-Quadril
14.
Clin Endocrinol (Oxf) ; 65(1): 64-70, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16817821

RESUMO

OBJECTIVE: There are close phenotypic similarities between cortisone reductase deficiency (CRD), a rare abnormality of cortisone metabolism, and polycystic ovary syndrome (PCOS). As there is evidence that CRD results from digenic mutations involving the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) and hexose-6-phosphate dehydrogenase (H6PD), we sought to establish whether CRD-associated variants in these genes, individually or in combination, influence susceptibility to PCOS. DESIGN: Case-control, family-based association and quantitative-trait analyses. PATIENTS: A UK case sample comprising 256 nuclear families ascertained from a PCOS offspring and 213 singleton PCOS cases plus 549 control subjects. MEASUREMENTS: All subjects were genotyped for CRD-related variants in HSD11B1 (rs12086634) and H6PD (rs6688832). Testosterone was measured with an in-house radioimmunoassay using ether extraction and dextran-coated charcoal separation. RESULTS: Case-control analyses revealed no differences in genotype distribution between PCOS and controls for rs12086634 or rs6688832 (both P = 0.84). Three per cent of cases and 2.4% of controls had genotype combinations (three or more variant alleles at the two sites) considered characteristic of CRD (P = 0.73). There were no departures from expectation in the family-based association studies, and no significant associations between genotypes (individually or in combination) and BMI, WHR or testosterone. CONCLUSIONS: The variants in HSD11B1 and H6PD typed, though implicated in causation of CRD, do not influence susceptibility to PCOS. It seems likely that additional variants within these genes are required for the development of CRD.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Desidrogenases de Carboidrato/genética , Cortisona Redutase/genética , Síndrome do Ovário Policístico/enzimologia , Síndrome do Ovário Policístico/genética , Adulto , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Cortisona Redutase/deficiência , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Característica Quantitativa Herdável
15.
J Biol Chem ; 281(8): 4671-7, 2006 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-16373343

RESUMO

The redox state of the intraluminal pyridine nucleotide pool was investigated in rat liver microsomal vesicles. The vesicles showed cortisone reductase activity in the absence of added reductants, which was dependent on the integrity of the membrane. The intraluminal pyridine nucleotide pool could be oxidized by the addition of cortisone or metyrapone but not of glutathione. On the other hand, intraluminal pyridine nucleotides were slightly reduced by cortisol or glucose 6-phosphate, although glutathione was completely ineffective. Redox state of microsomal protein thiols/disulfides was not altered either by manipulations affecting the redox state of pyridine nucleotides or by the addition of NAD(P)+ or NAD(P)H. The uncoupling of the thiol/disulfide and NAD(P)+/NAD(P)H redox couples was not because of their subcompartmentation, because enzymes responsible for the intraluminal oxidoreduction of pyridine nucleotides were distributed equally in smooth and rough microsomal subfractions. Instead, the phenomenon can be explained by the negligible representation of glutathione reductase in the endoplasmic reticulum lumen. The results demonstrated the separate existence of two redox systems in the endoplasmic reticulum lumen, which explains the contemporary functioning of oxidative folding and of powerful reductive reactions.


Assuntos
Retículo Endoplasmático/metabolismo , Microssomos Hepáticos/metabolismo , Oxirredução , Oxigênio/metabolismo , Piridinas/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Animais , Transporte Biológico , Western Blotting , Desidrogenases de Carboidrato/química , Cortisona Redutase/metabolismo , Citosol/metabolismo , Glucose-6-Fosfato/química , Glutationa/metabolismo , Glutationa Redutase/química , Hidrocortisona/química , Luz , Masculino , NADP/química , NADPH Oxidases/metabolismo , Ratos , Ratos Sprague-Dawley , Espalhamento de Radiação , Espectrometria de Fluorescência , Frações Subcelulares , Temperatura , Fatores de Tempo
16.
Reprod Biol Endocrinol ; 3: 53, 2005 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-16197550

RESUMO

BACKGROUND: TGF-beta is a multifunctional growth factor involved in regulating a variety of cellular activities. Unlike mammals, the function of TGF-beta in the reproduction of lower vertebrates, such as fish, is not clear. Recently, we showed that TGF-beta1 inhibits gonadotropin- and 17alpha, 20beta-dihydroxyprogesterone (DHP)-induced maturation in zebrafish. The aim of the present study was to investigate the mechanisms underlying this action. METHOD: To determine if the effect of TGF-beta1 on oocyte maturation involves transcription and/or translation, ovarian follicles were pre-treated with actinomycin D, a blocker of transcription, and cyclohexamide, an inhibitor of translation, and incubated with hCG or DHP, either alone or in combination with TGF-beta1 and oocyte maturation scored. To determine the effect of TGF-beta1 on mRNA levels of several key effectors of oocyte maturation, three sets of experiments were performed. First, follicles were treated with control medium or TGF-beta1 for 2, 6, 12, and 24 h. Second, follicles were treated with different concentrations of TGF-beta1 (0 to 10 ng/ml) for 18 h. Third, follicles were incubated with hCG in the absence or presence of TGF-beta1 for 18 h. At the end of each experiment, total RNA was extracted and reverse transcribed. PCR using primers specific for 20beta-hydroxysteroid dehydrogenase (20beta-HSD) which is involved in DHP production, follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), the two forms of membrane progestin receptor: mPR-alpha and mPR-beta, as well as GAPDH (control), were performed. RESULTS: Treatment with actinomycin D, a blocker of transcription, reduced the inhibitory effect of TGF-beta1 on DHP-induced oocyte maturation, indicating that the inhibitory action of TGF-beta1 is in part due to regulation of gene transcription. Treatment with TGF-beta1 caused a dose and time-dependent decrease in mRNA levels of 20beta-HSD, LHR and mPR-beta in follicles. On the other hand, TGF-beta1 had no effect on mPR-alpha mRNA expression and increased FSHR mRNA levels. Furthermore, hCG upregulated 20beta-HSD, LHR and mPR-beta mRNA levels, but this stimulatory effect was blocked by TGF-beta1. CONCLUSION: These findings suggest that TGF-beta1 acts at multiple sites, including LHR, 20beta-HSD and mPR-beta, to inhibit zebrafish oocyte maturation.


Assuntos
Oócitos/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Gonadotropina Coriônica/farmacologia , Cortisona Redutase/biossíntese , Cortodoxona/análogos & derivados , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação para Baixo , Feminino , Expressão Gênica/efeitos dos fármacos , Hidroxiprogesteronas/antagonistas & inibidores , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Receptores do FSH/biossíntese , Receptores do LH/biossíntese , Receptores do LH/metabolismo , Receptores de Progesterona/biossíntese , Fator de Crescimento Transformador beta1 , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese
17.
Gen Comp Endocrinol ; 144(3): 224-31, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16102755

RESUMO

In salmonid fishes, estradiol-17beta (E2) and 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) are the major steroid hormones controlling oocyte growth (vitellogenesis) and final maturation (resumption of meiosis). The aim of this study was to determine changes in mRNAs encoding ovarian steroidogenic enzymes and steroidogenic acute regulatory protein (StAR) during ovarian development in female rainbow trout. We analyzed the levels of mRNAs encoding the enzymes P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17alpha-hydroxylase/C17-C20 lyase (P450c17), aromatase (P450arom), and carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase (20beta-HSD), and StAR in developing ovarian follicles of rainbow trout by Northern blot, in relation to the pattern of serum E2 and 17,20beta-P levels. Serum E2 levels were elevated during vitellogenesis and decreased prior to an ovulatory increase in 17,20beta-P. Transcripts for P450scc and P450c17 increased in late vitellogenic follicles, then decreased in post-ovulatory follicles. In contrast, P450arom transcripts were abundant during vitellogenesis and then declined as vitellogenesis was completed and were barely detectable in post-ovulatory follicles. 3beta-HSD mRNA levels increased in late vitellogenic follicles and were maintained at high levels in post-ovulatory follicles. 20beta-HSD and StAR mRNA levels were very low during vitellogenesis, and then strongly increased during late vitellogenesis to a peak in post-ovulatory follicles. These results indicate that the expression of genes encoding steroidogenic enzymes and StAR change dynamically, dependent on the developmental stages of rainbow trout follicles. The acquisition of the ability of later stage follicles to rapidly produce large quantities of 17,20beta-P appears to be supported by a preparatory increase in mRNAs encoding StAR and other steroidogenic enzymes.


Assuntos
Oncorhynchus mykiss/crescimento & desenvolvimento , Ovário/enzimologia , Ovário/crescimento & desenvolvimento , Fosfoproteínas/genética , RNA Mensageiro/análise , Esteroides/biossíntese , 3-Hidroxiesteroide Desidrogenases/genética , Animais , Aromatase/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Cortisona Redutase/genética , Feminino , Oncorhynchus mykiss/metabolismo , Folículo Ovariano/química , Folículo Ovariano/enzimologia , Ovário/química , Esteroide 17-alfa-Hidroxilase/genética
18.
J Clin Endocrinol Metab ; 90(10): 5880-3, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16091483

RESUMO

CONTEXT: Apparent cortisone reductase deficiency (ACRD) is a rarely ascertained condition characterized by signs of androgen excess in women or children and decreased urinary excretion of cortisol metabolites compared with cortisone metabolites. These findings suggest a deficiency of 11beta-hydroxysteroid dehydrogenase type 1 (11-HSD1; encoded by the HSD11B1 gene), which normally converts cortisone to cortisol. Common polymorphisms in both HSD11B1 and the hexose-6-phosphate dehydrogenase (H6PD) gene encoding hexose-6-phosphate dehydrogenase have been found together in ACRD patients, who carry three of a possible four minor alleles at the two loci. OBJECTIVE: The objective of this study was to confirm the postulated digenic inheritance mechanism for ACRD. DESIGN: This was a population-based association study (Dallas Heart Study). Subjects were genotyped for the 1971T>G polymorphism in intron 3 of HSD11B1 and the R453Q polymorphism in H6PD. SUBJECTS: The study comprised 3551 individuals in a population-based sample (50% black, 35% white, and 15% Hispanic). MAIN OUTCOME MEASURE: The main outcome measure was association between genotypes and risk for polycystic ovarian syndrome. RESULTS: Both polymorphisms occurred more frequently than previously reported. Thus, ACRD genotypes (at least three of four minor alleles) occurred in 7.0% of subjects. There were no associations between genotype and body mass index; waist/hip ratio; visceral adiposity; measures of insulin sensitivity; levels of testosterone, FSH, or LH (in females); or risk of polycystic ovarian syndrome. There was no genotype effect on urinary free cortisol/cortisone or corticosteroid metabolite ratios, which were measured in 10 subjects, each carrying zero, three, or four minor alleles. CONCLUSIONS: Previously reported associations of ACRD with HSD11B1 and H6PD alleles represent ascertainment bias. However, rare severe mutations in these genes cannot be ruled out.


Assuntos
11-beta-Hidroxiesteroide Desidrogenases/genética , Desidrogenases de Carboidrato/genética , Cortisona Redutase/deficiência , Adolescente , Adulto , Idoso , Alelos , Feminino , Frequência do Gene , Testes Genéticos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/urina , População , Fatores de Risco , Esteroides/urina , Texas/epidemiologia
19.
J Clin Endocrinol Metab ; 90(7): 4157-62, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15827106

RESUMO

CONTEXT: The R453Q variant in the hexose-6-phosphate dehydrogenase gene (H6PD) and 83557insA mutations in 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 gene (HSD11B1) interact, resulting in cortisone reductase deficiency (CRD), a rare disorder characterized by a polycystic ovary syndrome (PCOS)-like phenotype. OBJECTIVE: The objective was to study these mutations in PCOS. DESIGN: The design was a case-control study. SETTING: The study was conducted in an academic hospital. PARTICIPANTS: A total of 116 PCOS patients and 76 nonhyperandrogenic controls participated. MAIN OUTCOME MEASURES: Genotype distributions and influence of genotypes on clinical and biochemical variables and, in 28 patients and 12 controls, estimates of 11betaHSD oxoreductase activity were the main outcome measures. RESULTS: Four controls and five patients presented three of four mutant alleles in H6PD R453Q and HSD11B1 83557insA, which is the genotype observed in some subjects with CRD. Estimates of 11betaHSD oxoreductase activity were measured in six of these nine women, ruling out CRD. Moreover, H6PD R453Q and HSD11B1 83557insA genotypes, either separately or in combination, did not influence 11betaHSD oxoreductase activity. The distribution of H6PD R453Q genotypes (R/R, R/Q, and Q/Q) was different in patients and controls (42% of controls and 63% of PCOS patients were R/R; 53% of controls and 31% of PCOS patients were R/Q; and 5% of controls and 6% of PCOS patients were Q/Q; chi(2) = 9.1; P = 0.011). Patients homozygous for R453 alleles presented with increased cortisol and 17-hydroxyprogesterone levels, compared with carriers of Q453 alleles, but these differences were not observed in controls. On the contrary, HSD11B1 83557insA genotypes were not associated with PCOS and did not influence any phenotypic variable. CONCLUSIONS: Digenic triallelic genotypes of the H6PD R453Q variant and HSD11B1 83557insA mutation do not always cause CRD. On the contrary, the H6PD R453Q variant is associated with PCOS and might influence its phenotype by influencing adrenal activity.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Desidrogenases de Carboidrato/genética , Síndrome do Ovário Policístico/genética , Polimorfismo Genético , Adulto , Estudos de Casos e Controles , Cortisona Redutase/deficiência , Feminino , Genótipo , Humanos , Síndrome do Ovário Policístico/enzimologia , Síndrome do Ovário Policístico/etiologia
20.
Endocr Rev ; 25(5): 831-66, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15466942

RESUMO

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) interconverts inactive cortisone and active cortisol. Although bidirectional, in vivo it is believed to function as a reductase generating active glucocorticoid at a prereceptor level, enhancing glucocorticoid receptor activation. In this review, we discuss both the genetic and enzymatic characterization of 11beta-HSD1, as well as describing its role in physiology and pathology in a tissue-specific manner. The molecular basis of cortisone reductase deficiency, the putative "11beta-HSD1 knockout state" in humans, has been defined and is caused by intronic mutations in HSD11B1 that decrease gene transcription together with mutations in hexose-6-phosphate dehydrogenase, an endoluminal enzyme that provides reduced nicotinamide-adenine dinucleotide phosphate as cofactor to 11beta-HSD1 to permit reductase activity. We speculate that hexose-6-phosphate dehydrogenase activity and therefore reduced nicotinamide-adenine dinucleotide phosphate supply may be crucial in determining the directionality of 11beta-HSD1 activity. Therapeutic inhibition of 11beta-HSD1 reductase activity in patients with obesity and the metabolic syndrome, as well as in glaucoma and osteoporosis, remains an exciting prospect.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/fisiologia , Glucocorticoides/farmacologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Sequência de Aminoácidos , Animais , Desidrogenases de Carboidrato/genética , Clonagem Molecular , Cortisona Redutase/deficiência , Cortisona Redutase/genética , Inibidores Enzimáticos , Regulação Enzimológica da Expressão Gênica , Glaucoma/enzimologia , Humanos , Hidrocortisona/metabolismo , Dados de Sequência Molecular , Mutação , NADP/metabolismo , Obesidade/enzimologia , Especificidade de Órgãos , Osteoporose/enzimologia , Proteínas Recombinantes , Alinhamento de Sequência , Especificidade por Substrato , Transcrição Genética
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