RESUMEN
Paraquat, a widely used nonselective herbicide, is a serious hazard to human health. However, the effects of paraquat on the male reproductive system remain unclear. In this study, adult male Sprague Dawley rats were intraperitoneally injected ethane dimethane sulfonate (EDS, 75â¯mg/kg) to initiate a regeneration of Leydig cells. EDS-treated rats were orally exposed to paraquat (0.5, 2, 8â¯mg/kg/day) from post-EDS day 17 to day 28 and effects of paraquat on Leydig and Sertoli cell functions on post-EDS day 35 and day 56 were investigated. Paraquat significantly decreased serum testosterone levels at 2 and 8â¯mg/kg. Paraquat lowered Leydig cell Hsd17b3, Srd5a1, and Hsd11b1 mRNA levels but increased Hsd3b1 on post-EDS day 35. Paraquat lowered Cyp11a1, Cyp17a1, and Hsd11b1 but increased Srd5a1 on post-EDS day 56. However, paraquat did not alter Leydig cell number and PCNA labeling index. Epididymal staining showed that few sperms were observed in paraquat-treated rats. Primary culture of adult Leydig cells showed that paraquat diminished testosterone output and induced reactive oxygen species generation at 1 and 10⯵M and apoptosis rate at 10⯵M. In conclusion, a short-term exposure to paraquat delays Leydig cell regeneration from stem/progenitor Leydig cells, causing low production of testosterone and an arrest of spermatogenesis.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Herbicidas/toxicidad , Células Intersticiales del Testículo/citología , Paraquat/toxicidad , Regeneración/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 17-Hidroxiesteroide Deshidrogenasas/análisis , 17-Hidroxiesteroide Deshidrogenasas/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/análisis , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Animales , Apoptosis/efectos de los fármacos , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Mesilatos/farmacología , Progesterona Reductasa/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Esteroide 17-alfa-Hidroxilasa/análisis , Testosterona/sangreRESUMEN
Glucocorticoid action in target organs is regulated by relative activities of 11ß-HSD type 1 (HSD11B1) that mainly converts cortisone to active cortisol and type 2 (HSD11B2) that inactivates cortisol to cortisone. HSD11Bs have been shown to be expressed in the ovary of various species. However, little is known about the expression and activity of HSD11Bs in the bovine cumulus-oocyte complex (COC). In the present study, we investigated the expression and activities of HSD11Bs in in vitro-matured (IVM) bovine COCs. Bovine COCs were matured in M199 supplemented with or without FSH and FCS. The expression of HSD11B1 and HSD11B2 was measured by using quantitative RT-PCR in denuded oocytes (DO) and cumulus cells (CC). Reductive and oxidative activities of HSD11Bs were determined by radiometric conversion assay using labeled cortisol, cortisone or dexamethasone in intact COCs, DO or CC in the presence or absence of 11-keto-progesterone (11kP), a selective inhibitor of HSD11B2. The presence of HSD11Bs in the oocyte was examined by immunofluorescence microscopy. Oocytes exclusively expressed HSD11B2 and its expression and activity were largely unchanged during IVM. CC, on the other hand, exclusively expressed HSD11B1 and its expression and activity were upregulated as IVM progressed. As a result, the net glucocorticoid metabolism shifted from inactivation to activation towards the end of IVM. These results indicate that the bovine COC is capable of modulating local glucocorticoid concentration and, by doing so, may create an environment that is favorable to ovulating oocyte for maturation, fertilization and subsequent development.
Asunto(s)
Bovinos/metabolismo , Células del Cúmulo/metabolismo , Glucocorticoides/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Animales , Cortisona/metabolismo , Células del Cúmulo/enzimología , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Hidrocortisona/metabolismo , Oocitos/enzimología , Oxidación-Reducción , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinariaRESUMEN
Diminished glucocorticoid signaling is associated with an age-related decline in hippocampal functioning. In this study we demonstrate the effect of intermittent, every other day (EOD) feeding on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the hippocampus of middle-aged (18-month-old) and aged (24-month-old) Wistar rats. In aged ad libitum-fed rats, a decrease in the level of total GR and GR phosphorylated at Ser(232) (pGR) was detected. Conversely, aged rats subjected to EOD feeding, starting from 6 months of age, showed an increase in GR and pGR levels and a higher content of hippocampal corticosterone. Furthermore, prominent nuclear staining of pGR was observed in CA1 pyramidal and DG granule neurons of aged EOD-fed rats. These changes were accompanied by increased Sgk-1 and decreased GFAP transcription, pointing to upregulated transcriptional activity of GR. EOD feeding also induced an increase in the expression of the mineralocorticoid receptor. Our results reveal that intermittent feeding restores impaired GR signaling in the hippocampus of aged animals by inducing rather than by stabilizing GR signaling during aging.
Asunto(s)
Envejecimiento , Privación de Alimentos/fisiología , Hipocampo/fisiología , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Animales , Corticosterona/análisis , Corticosterona/metabolismo , Proteínas HSP90 de Choque Térmico/análisis , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Inmediatas-Precoces/genética , Masculino , Fosfotransferasas/análisis , Fosfotransferasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptores de Glucocorticoides/análisis , Proteínas de Unión a Tacrolimus/análisis , Proteínas de Unión a Tacrolimus/metabolismo , Regulación hacia ArribaRESUMEN
Although the effects of glucocorticoids on proliferation, differentiation and apoptosis are well known, and steroid hormones have been identified to play a role in pathogenesis and the development of various cancers, limited data are available regarding the relationship between the local metabolism of glucocorticoids and colorectal adenocarcinoma (CRC) formation. Glucocorticoid metabolism is determined by 11ß-hydroxysteroid dehydrogenases type 1 and 2 (11HSD1, 11HSD2), which increase the local concentration of cortisol due to the reduction of cortisone, or decrease this concentration due to the oxidation of cortisol. The objective of this study was to evaluate the extent of 11HSD1 and 11HSD2 mRNA in pre-malignant colorectal polyps and in CRC. The specimens were retrieved from patients by endoscopic or surgical resection and the expression of 11HSD1 and 11HSD2 was measured by real-time PCR. The polyps were of the following histological types: hyperplastic polyps and adenomas with low- or high-grade dysplasia. The neoplastic tissue of CRC obtained during tumor surgery was also studied. It was found that 11HSD2 was not only downregulated in CRC but already in the early stages of neoplastic transformation (adenoma with low-grade dysplasia). In contrast, the level of 11HSD1 was significantly increased in CRC but not in pre-malignant polyps. The results demonstrate that the downregulation of 11HSD2 gene expression is a typical feature of the development of colorectal polypous lesions and their transformation into CRC.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/biosíntesis , Adenocarcinoma/enzimología , Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/análisis , Pólipos Adenomatosos/enzimología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Regulación hacia Abajo , Femenino , Humanos , Captura por Microdisección con Láser , Masculino , Persona de Mediana Edad , Lesiones Precancerosas/enzimología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma , Adulto JovenRESUMEN
The objective of the present study is to determine whether di(2-ethylhexyl) phthalate (DEHP) exposure at adulthood increases rat Leydig cell number and to investigate the possible mechanism. 90-day-old Long-Evans rats were randomly divided into 3 groups, and were gavaged with the corn oil (control) or 10 or 750 mg/kg DEHP daily for 7 days, and then received an intraperitoneal injection of 75 mg/kg ethane dimethanesulfonate (EDS) to eliminate Leydig cells. Serum testosterone concentrations were assessed by RIA, and the mRNA levels of Leydig cell genes were measured by qPCR. EDS eliminated all Leydig cells in the control testis on day 4 post-EDS, as judged by undetectable serum testosterone level and no 3ß-hydroxysteroid dehydrogenase positive (3ß-HSD(pos)) cells in the interstitium. However, in DEHP-treated groups, there were detectable serum testosterone concentrations and some oval-shaped 3ß-HSD(pos) cells in the interstitium. These 3ß-HSD(pos) cells were not stained by the antibody against 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), a marker for Leydig cells at a more advanced stage. The disappearance of mRNAs of Leydig cell biomarkers including Lhcgr, Cyp11a1, Cyp17a1, Insl3 and Hsd11b1 in the control testis was observed on day 4 post-EDS. However, there were detectable concentrations of Lhcgr, Cyp11a1 and Cyp17a1 mRNAs but undetectable concentrations of Insl3, Hsd17b3 and Hsd11b1 in the DEHP-treated testes, indicating that these 3ß-HSD(pos) cells were newly formed progenitor Leydig cells. The mRNA level for nestin (Nes, biomarker for stem Leydig cells) was significantly increased in the control testis on day 4 post-EDS, but not in the DEHP treated testes, suggesting that these nestin positive stem cells were differentiated into progenitor Leydig cells in the DEHP-treated testes. The present study suggests that DEHP increases the differentiation of stem cells into progenitor Leydig cells.
Asunto(s)
Dietilhexil Ftalato/toxicidad , Células Intersticiales del Testículo/efectos de los fármacos , Plastificantes/toxicidad , Testículo/efectos de los fármacos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/ultraestructura , Hormona Luteinizante/sangre , Masculino , Mesilatos/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , ARN/química , ARN/genética , Distribución Aleatoria , Ratas , Ratas Long-Evans , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/citología , Testículo/metabolismo , Testosterona/sangreRESUMEN
BACKGROUND: Visceral fat deposition and its associated atherogenic complications are mediated by glucocorticoids. Cardiac visceral fat comprises mediastinal adipose tissue (MAT) and epicardial adipose tissue (EAT), and MAT is a potential biomarker of risk for obese patients. AIM: Our objective was to evaluate the role of EAT and MAT 11beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1) and glucocorticoid receptor (GCR) expression in comparison with subcutaneous adipose tissue (SAT) in the development of coronary atherosclerosis in obese patients with coronary artery disease (CAD), and to assess their correlations with CD68 and fatty acids from these tissues. METHODS AND RESULTS: Expression of 11ß-HSD-1 and GCR was measured by qRT-PCR in EAT, MAT and SAT of thirty-one obese patients undergoing coronary artery bypass grafting due to CAD (obese CAD group) and sixteen obese patients without CAD undergoing heart valve surgery (controls). 11ß-HSD-1 and GCR expression in MAT were found to be significantly increased in the obese CAD group compared with controls (p < 0.05). In the obese CAD group, 11ß-HSD-1 and GCR mRNA levels were strongly correlated in MAT. Stearidonic acid was significantly increased in EAT and MAT of the obese CAD group and arachidonic acid was significantly expressed in MAT of the obese male CAD group (p < 0.05). CONCLUSIONS: We report for the first time the increased expression of 11ß-HSD-1 and GCR in MAT compared with EAT and SAT, and also describe the interrelated effects of stearidonic acid, HOMA-IR, plasma cortisol and GCR mRNA levels, explaining 40.2% of the variance in 11ß-HSD-1 mRNA levels in MAT of obese CAD patients. These findings support the hypothesis that MAT contributes locally to the development of coronary atherosclerosis via glucocorticoid action.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , Enfermedad de la Arteria Coronaria/enzimología , Hidrocortisona/análisis , Grasa Intraabdominal/enzimología , Isquemia Miocárdica/enzimología , Obesidad/enzimología , Receptores de Glucocorticoides/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Anciano , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Ácido Araquidónico/análisis , Estudios de Casos y Controles , Puente de Arteria Coronaria , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/cirugía , Ácidos Grasos Omega-3/análisis , Femenino , Humanos , Modelos Lineales , Masculino , Mediastino , Persona de Mediana Edad , Análisis Multivariante , Isquemia Miocárdica/genética , ARN Mensajero/análisis , Receptores de Glucocorticoides/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Grasa Subcutánea/enzimologíaRESUMEN
Gingerols and their corresponding dehydration products shogaols were considered as the active principles of ginger, the rhizome of the plant Zingiber officinale, for its antioxidant, anti-inflammatory, and antitumor activities. Ginger (Z. officinale) has been cultivated for thousands of years as a spice and for medicinal purposes in China. Tongling (Anhui province, China) has traditionally been regarded as an ideal cultivation place. "Tongling White Ginger" enjoys a reputation for being one of the top gingers in China for its thin white peel, tender flesh, rich juice, and flavor. In this study, we have isolated and identified two novel gingerdione dimers, bisgingerdiones A (1) and B (2); two new gingerol derivatives, (5R)-5-acetoxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptan-3-one (3) and methyl (Z)-neral acetal-[6]-gingerdiol (4); and 38 known compounds (5-42) from rhizomes of Zingiber officinale collected from Tongling, China. Their structures were elucidated by means of spectroscopic methods. Compounds 1-4 showed weak cytotoxic and anti-HIV-1 activities. Compounds 6, 8, and 26 showed inhibitory activities against human and mouse 11ß-HSD1 (11ß-hydroxysteroid dehydrogenases) with IC(50) values between 1.09 and 1.30 µM.
Asunto(s)
Catecoles/química , Inhibidores Enzimáticos/química , Alcoholes Grasos/química , Extractos Vegetales/química , Zingiber officinale/química , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Animales , Catecoles/farmacología , Línea Celular Tumoral , China , Inhibidores Enzimáticos/farmacología , Alcoholes Grasos/farmacología , Humanos , Ratones , Estructura Molecular , Extractos Vegetales/farmacología , Rizoma/químicaRESUMEN
In healthy individuals measures of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) enzyme activity predict the change in bone formation markers in response to therapeutic glucocorticoids. It is unclear whether these measures remain predictive in inflammatory disease. We therefore examined whether 11ß-HSD1 activity predicts changes in bone markers and bone mineral density (BMD) in patients with inflammatory bowel disease (IBD) treated with therapeutic glucocorticoids. Prospective and cross-sectional studies were carried out in patients attending a gastroenterology clinic with active (n = 39) or clinically inactive (n = 34) IBD and healthy controls (n = 51). Urinary corticosteroid metabolite profiles were obtained on a spot urine sample and total corticosteroid metabolite excretion and 11ß-HSD1 activity (measured as the ratio of tetrahydrocortisol to tetrahydrocortisone metabolites, [THF+alloTHF]/THE) determined. Patients with active disease were treated with an 8-week reducing course of oral prednisolone. The (THF+alloTHF)/THE ratio was significantly increased in patients with IBD, even those in clinical remission. The baseline (THF+alloTHF)/THE ratio failed to predict the decrease in bone formation markers or hip BMD. Measures of 11ß-HSD activity do not predict bone loss during glucocorticoid treatment of active IBD, probably due to disease-related increases in 11ß-HSD1 activity. Our observation of elevated 11ß-HSD1 activity in clinically inactive IBD implicates gastrointestinal glucocorticoid activation in the maintenance of disease remission.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Resorción Ósea/diagnóstico , Huesos/efectos de los fármacos , Glucocorticoides/efectos adversos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/fisiología , Adulto , Antiinflamatorios/efectos adversos , Antiinflamatorios/uso terapéutico , Resorción Ósea/inducido químicamente , Resorción Ósea/epidemiología , Huesos/metabolismo , Resistencia a Medicamentos/fisiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Femenino , Glucocorticoides/uso terapéutico , Humanos , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/epidemiología , Enfermedades Inflamatorias del Intestino/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
BACKGROUND: Numerous studies have explored the etiologic or permissive role of 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) in obesity and Type 2 diabetes, biochemical conditions often with concurrent hyperinsulinism. In contrast, there are limited data on the effect of insulin deficiency (i.e. Type 1 diabetes) on 11ß-HSD1 or endoplasmic reticulum enzymes that generate the reduced pyridine cofactor NADPH. Thus, the aim of the present study was to examine the effect of insulin-deficient, streptozotozin diabetes on key microsomal enzymes involved in rat hepatic corticosterone production. METHODS: After rats had been rendered diabetic with streptozotocin and some had been treated with insulin (2-6 units, s.c., long-acting insulin once daily) for 7 days, hepatic microsomes were isolated. Serum corticosterone and fructosamine were obtained premortem. Intact microsomes were incubated in vitro and 11ß-HSD1, hexose-6-phosphate dehydrogenase (H6PDH), and isocitrate dehydrogenase (IDH) measured. RESULTS: Although diabetes markedly altered body weight gain and serum protein glycosylation (assessed by fructosamine), there was no significant change in hepatic 11ß-HSD1 reductase activity, with or without insulin treatment. However, serum corticosterone levels were significantly correlated with 11ß-HSD1 reductase activity when all groups were analyzed together (P < 0.05). Untreated diabetes modified (P < 0.01) two hepatic microsomal NADPH-generating enzymes, namely H6PDH and IDH, resulting in a 37% decrease and 14% increase in enzyme levels, respectively. CONCLUSIONS: Consistent with most in vivo studies, chronic insulin deficiency with attendant hyperglycemia does not significantly modify hepatic 11ß-HSD1 reductase activity, but does alter the activity of two microsomal enzymes coupled with pyridine cofactors.
Asunto(s)
Corticosterona/biosíntesis , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 2/enzimología , Hígado/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , Animales , Deshidrogenasas de Carbohidratos/análisis , Corticosterona/sangre , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Fructosamina/sangre , Hiperglucemia/enzimología , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Isocitrato Deshidrogenasa/análisis , Masculino , Microsomas Hepáticos/enzimología , Ratas , Ratas Sprague-Dawley , Aumento de PesoRESUMEN
Recent investigations have demonstrated that activation of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in liver and adipose tissue is closely related to the pathogenesis of obesity and diabetes. However, the relationship between alteration of 11beta-HSD1 and the pathogenesis of type 2 diabetes in skeletal muscle is still unclear. A rat model of Type 2 diabetes was developed by high fat diet feeding combined with multiple low dose streptozotocin injection (30 mg/kg, i.p. twice). Intraperitoneal glucose tolerance test, insulin tolerance test were performed. Fasting blood glucose, fasting insulin, total cholesterol, triglyceride were measured. The protein and mRNA level of 11beta-HSD1 and glucocorticoid receptor in gastrocnemius muscle were determined. The alteration of insulin signaling pathway related protein was investigated. We found that the protein levels of 11beta-HSD1 and glucocorticoid receptor were significantly increased (P < 0.05); the mRNA level of 11beta-HSD1 was also elevated (P < 0.05); the mRNA level of glucocorticoid receptor was decreased (P < 0.05). After insulin stimulation, diabetic rats had no significant changes in the level of the insulin receptor beta-subunit (IR-beta), AKT, as in phosphorylated AKT in the gastrocnemius muscle compared to its basal state. Similar results were observed in the protein expression level of glucose transporter 4 (GLUT4). Our data indicate that the alteration of 11beta-HSD1 at protein and mRNA level may be related to the abnormality of insulin signal pathway in skeletal muscle, this effect may be mediated by glucocorticoid receptor.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Músculo Esquelético/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Animales , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/patología , Insulina/farmacología , ARN Mensajero/análisis , Ratas , Receptor de Insulina/metabolismo , Receptores de Glucocorticoides/genética , Transducción de SeñalRESUMEN
The liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an increasingly common tool in the clinical laboratory. Established applications include routine assays for detecting inborn errors of metabolism and for monitoring therapeutic drugs and steroids. Steroid profiling is a very effective method for distinguishing almost all steroid related disorders. It allows accurate diagnosis and is very useful in many clinical situations. Most methods for the determination of steroid hormones are based on immunoassays, which are rapid and easy to perform. However, the reliability of steroid immunoassays has been shown to be doubtful because of the lack of specificity and of matrix effects. Immunological methods, especially direct assays, often overestimate true steroid values. This is of particular importance in the newborn period and in early infancy. Problems with steroid immunoassays have further been reported for female patients or when analysing different media, e.g. saliva. Patient follow-up over time or between laboratories, as well as longitudinal studies are extremely difficult. In contrast to immunoassays, which allow the measurement of only a single steroid at a time, LC-MS/MS has the advantage that a wide spectrum of steroid hormones can be measured simultaneously. The applicability for clinical samples and problems in pediatric endocrinology will be discussed.
Asunto(s)
Endocrinología/métodos , Esteroides/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 17-alfa-Hidroxiprogesterona/análisis , Animales , Calibración , Niño , Cromatografía Liquida , Humanos , Hidrocortisona/análisis , Espectrometría de Masas , Pregnanotriol/orina , Ratas , Saliva/química , Esteroides/biosíntesis , Esteroides/sangre , Esteroides/químicaRESUMEN
CONTEXT: Glucocorticoids are crucial in fetal lung function. The amount of cortisol available to its receptors is increased by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Glucocorticoids and IL-1beta are known to induce 11beta-HSD1 expression in a number of tissues, but controversial results were obtained with regard to 11beta-HSD1 expression in human fetal lung. OBJECTIVE: We examined the expression of 11beta-HSD1 and its regulation by cortisol and IL-1beta in human fetal lung. RESULTS: Immunohistochemistry revealed 11beta-HSD1 expression in the epithelium and mesenchymal layer of the small bronchus and bronchiole of human fetal lung at 8 months but not at 4 months gestation, which was confirmed by PCR revealing 11beta-HSD1 mRNA expression in the fetal lung tissue. By using a cell line derived from human fetal lung fibroblasts, we demonstrated that cortisol (10(-5) to 10(-3) mmol/liter) or IL-1beta (0.1 to 10 ng/ml) induced 11beta-HSD1 mRNA expression in a concentration-dependent manner. The induction of 11beta-HSD1 by IL-1beta was further increased by cortisol, whereas the induction of cyclooxygenase 2 by IL-1beta was inhibited by cortisol. Nuclear factor kappaB activation inhibitor could only block the induction of cyclooxygenase 2 but not 11beta-HSD1 by IL-1beta, suggesting that different mechanisms were utilized by IL-1beta in the regulation of 11beta-HSD1 versus proinflammatory mediators. Global inhibition of CCAAT-enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression plasmid could attenuate the induction of 11beta-HSD1 by IL-1beta, suggesting that C/EBPs may mediate the induction of 11beta-HSD1 by IL-1beta. CONCLUSIONS: 11beta-HSD1 is expressed in human fetal lung; cortisol and IL-1beta could synergistically induce its expression.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Feto/enzimología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hidrocortisona/farmacología , Interleucina-1beta/farmacología , Pulmón/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Ciclooxigenasa 2/genética , Humanos , InmunohistoquímicaRESUMEN
Glucocorticoid excess promotes visceral obesity and cardiovascular disease. Similar features are found in the highly prevalent metabolic syndrome in the absence of high levels of systemic cortisol. Although elevated activity of the glucocorticoid-amplifying enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) within adipocytes might explain this paradox, the potential role of 11beta-HSD1 in preadipocytes is less clear; human omental adipose stromal vascular (ASV) cells exhibit 11beta-dehydrogenase activity (inactivation of glucocorticoids) probably due to the absence of cofactor provision by hexose-6-phosphate dehydrogenase. To clarify the depot-specific impact of 11beta-HSD1, we assessed whether preadipocytes in ASV from mesenteric (as a representative of visceral adipose tissue) and sc tissue displayed 11beta-HSD1 activity in mice. 11beta-HSD1 was highly expressed in freshly isolated ASV cells, predominantly in preadipocytes. 11beta-HSD1 mRNA and protein levels were comparable between ASV and adipocyte fractions in both depots. 11beta-HSD1 was an 11beta-reductase, thus reactivating glucocorticoids in ASV cells, consistent with hexose-6-phosphate dehydrogenase mRNA expression. Unexpectedly, glucocorticoid reactivation was higher in intact mesenteric ASV cells despite a lower expression of 11beta-HSD1 mRNA and protein (homogenate activity) levels than sc ASV cells. This suggests a novel depot-specific control over 11beta-HSD1 enzyme activity. In vivo, high-fat diet-induced obesity was accompanied by increased visceral fat preadipocyte differentiation in wild-type but not 11beta-HSD1(-/-) mice. The results suggest that 11beta-HSD1 reductase activity is augmented in mouse mesenteric preadipocytes where it promotes preadipocyte differentiation and contributes to visceral fat accumulation in obesity.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/fisiología , Adipocitos/enzimología , Grasa Intraabdominal/metabolismo , Obesidad/etiología , Células Madre/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Animales , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Grasas de la Dieta/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisisRESUMEN
OBJECTIVE: We examined whether chronic leptin treatment of diet-induced obese rats promotes or alleviates the susceptibility to continued high-fat feeding. Second, we examined if voluntary wheel running is beneficial in reducing the trajectory of weight gain in high-fat-raised leptin-resistant rats. RESEARCH DESIGN AND METHODS: Sprague-Dawley rats were fed a standard diet or a high-fat diet for 5 months, and then hypothalamic leptin overexpression was induced through central administration of adeno-associated virus-encoding leptin while continuing either the standard or high-fat diet. Two weeks later, half of the rats in each group were provided access to running wheels for 38 days while being maintained on either a standard or high-fat diet. RESULTS; In standard diet-raised rats, either wheel running or leptin reduced the trajectory of weight gain, and the combined effect of both treatments was additive. In high-fat-raised leptin-resistant rats, leptin overexpression first transiently reduced weight gain but then accelerated the weight gain twofold over controls. Wheel running in high-fat-raised rats was sixfold less than in standard diet-raised rats and did not affect weight gain. Surprisingly, wheel running plus leptin completely prevented weight gain. This synergy was associated with enhanced hypothalamic signal transducer and activator of transcription (STAT) 3 phosphorylation and suppressor of cytokine signaling 3 expression in wheel running plus leptin compared with leptin-treated sedentary high-fat counterparts. This enhanced STAT3 signaling associated with the combination treatment occurred only in high-fat-raised, leptin-resistant rats and not in standard diet-raised, leptin-responsive rats. CONCLUSIONS: Chronic leptin treatment in diet-induced obese rats accelerates dietary obesity. However, leptin combined with wheel running prevents further dietary weight gain. Thus, this combination therapy may be a viable antiobesity treatment.
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Dieta , Leptina/genética , Leptina/metabolismo , Actividad Motora/fisiología , Obesidad/terapia , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , Tejido Adiposo/química , Animales , Corticosterona/sangre , Ingestión de Alimentos , Regulación de la Expresión Génica , Terapia Genética , Masculino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) exists in two isoforms, 11beta-HSD1 and 11beta-HSD2. 11beta-HSD1 generates active cortisol from cortisone and appears to be involved in insulin resistant states. 11beta-HSD2 protects the mineralocorticoid receptor from inappropriate activation by glucocorticoids and is important to prevent sodium retention and hypertension. The purposes of the present study were to develop two real-time PCR assays to assess 11beta-HSD1 and 11beta-HSD2 mRNA expression and to evaluate the tissue distribution of the two isoforms in dogs. Thirteen different tissues of 10 healthy dogs were evaluated. Both real-time PCR assays were highly specific, sensitive and reproducible. Highest 11beta-HSD1 mRNA expression was seen in liver, lung, and renal medulla; highest 11beta-HSD2 mRNA expression in renal cortex, adrenal gland, and renal medulla. Higher 11beta-HSD1 than 11beta-HSD2 mRNA levels were found in all tissues except adrenal gland, colon, and rectum. Our results demonstrate that the basic tissue distribution of 11beta-HSD1 and 11beta-HSD2 in dogs corresponds to that in humans and rodents. In a next step 11beta-HSD1 and 11beta-HSD2 expression should be assessed in diseases like obesity, hypercortisolism, and hypertension to improve our knowledge about 11beta-HSD activity, to evaluate the dog as a model for humans and to potentially find new therapeutic options.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/análisis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Animales , Cartilla de ADN/análisis , Perros , Eficiencia , Femenino , Salud , Masculino , Caracteres Sexuales , Especificidad por SustratoRESUMEN
11Beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes modulate the target cell actions of corticosteroids by catalysing metabolism of the physiological glucocorticoid (GC), cortisol, to inert cortisone. Recent studies have implicated GCs in boar sperm apoptosis. Hence, the objective of this study was to characterise 11betaHSD enzyme expression and activities in the boar testis and reproductive tract. Although 11betaHSD1 and 11betaHSD2 mRNA transcripts and proteins were co-expressed in all tissues, cortisol-cortisone interconversion was undetectable in the corpus and cauda epididymides, vas deferens, vesicular and prostate glands, irrespective of nucleotide cofactors. In contrast, homogenates of boar testis, caput epididymidis and bulbourethral gland all displayed pronounced 11betaHSD activities in the presence of NADPH/NADP(+) and NAD(+), and the penile urethra exhibited NAD(+)-dependent 11beta-dehydrogenase activity. In kinetic studies, homogenates of boar testis, caput epididymidis and bulbourethral gland oxidised cortisol with K(m) values of 237-443 and 154-226 nmol/l in the presence of NADP(+) and NAD(+) respectively. Maximal rates of NADP(+)-dependent cortisol oxidation were 7.4- to 28.5-fold greater than the V(max) for NADPH- dependent reduction of cortisone, but were comparable with the rates of NAD(+)-dependent cortisol metabolism. The relatively low K(m) estimates for NADP(+) -dependent cortisol oxidation suggest that either the affinity of 11betaHSD1 has been increased or the cortisol inactivation is catalysed by a novel NADP(+)-dependent 11betaHSD enzyme in these tissues. We conclude that in the boar testis, caput epididymidis and bulbourethral gland, NADP(+)- and NAD(+)-dependent 11betaHSD enzymes catalyse net inactivation of cortisol, consistent with a physiological role in limiting any local actions of GCs within these reproductive tissues.
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11-beta-Hidroxiesteroide Deshidrogenasas/análisis , Genitales Masculinos/enzimología , Glucocorticoides/fisiología , Sus scrofa/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasas/genética , Animales , Secuencia de Bases , Western Blotting/métodos , Glándulas Bulbouretrales/enzimología , Cortisona/metabolismo , Epidídimo/enzimología , Hidrocortisona/metabolismo , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , NAD/metabolismo , NADP/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Testículo/enzimologíaRESUMEN
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/química , Electroquímica/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Hidrocortisona/análisis , Mediciones Luminiscentes/métodos , Microquímica/métodos , Humanos , Técnica de Dilución de Radioisótopos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Glucocorticoids play a critical role in fetal development, but inappropriate exposure is associated with reduced fetal growth. We investigated cortisol exposure and supply in a porcine model of differential fetal growth. This model compares the smallest fetus of a litter with an average-sized sibling at three stages of gestation. At day 45, small fetuses had reduced plasma cortisol (16.8 +/- 3.4 ng/ml) relative to average fetuses (34.4 +/- 3.4 ng/ml, P < 0.001). At day 65 levels had reduced in small and average fetuses to similar concentrations (5.7 +/- 1.0 vs 4.8 +/- 0.5 ng/ml, P = 0.128). By day 100, elevated levels were found in small fetuses (10.7 +/- 1.5 vs 7.6 +/- 0.7 ng/ml, P < 0.001). Maternal plasma cortisol was unchanged over gestation (day 45, 56.7 +/- 21.6 ng/ml; day 65, 57.8 +/- 14.4 ng/ml; day 100, 55.7 +/- 6.5 ng/ml). We examined the cause of altered cortisol by investigating the fetal hypothalamic-pituitary-adrenal axis through the measurement of adrenocorticotropic hormone and assessing exposure to maternal cortisol by quantifying placental 11beta-hydroxysteroid dehydrogenase-isoform 2 (11beta HSD-2) gene expression. These data suggest that altered cortisol supply was of fetal origin. We examined organ glucocorticoid (GC) metabolism by the measurement of GC receptor (GR) and 11beta-hydroxysteroid dehydrogenase-isoform 1 (11beta HSD-1) gene expression. We found that fetal organs have different temporal patterns of 11beta HSD-1 and GR expression, with the liver particularly sensitive to cortisol in late gestation. This study examines GC exposure in naturally occurring differential growth and simultaneously explores tissue GC sensitivity and handling, at three key stages of gestation.
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11-beta-Hidroxiesteroide Deshidrogenasas/análisis , Hidrocortisona/sangre , Intercambio Materno-Fetal , Sistema Hipófiso-Suprarrenal/embriología , Receptores de Glucocorticoides/análisis , Porcinos/embriología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasas/genética , Hormona Adrenocorticotrópica/sangre , Animales , Biomarcadores/análisis , Northern Blotting/métodos , Peso Corporal , Femenino , Sangre Fetal/química , Desarrollo Fetal/fisiología , Expresión Génica , Edad Gestacional , Hígado/química , Modelos Biológicos , Embarazo , ARN Mensajero/análisis , Receptores de Glucocorticoides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Glucocorticoids (GCs) have a profound effect on adipose biology increasing tissue mass causing central obesity. The pre-receptor regulation of GCs by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) that activates cortisol from cortisone has been postulated as a fundamental mechanism underlying the metabolic syndrome mediating adipocyte hyperplasia and hypertrophy in the omental (OM) depot. Orbital adipose tissue (OF) is the site of intense inflammation and tissue remodelling in several orbital inflammatory disease states. In this study, we describe features of the GC metabolic pathways in normal human OF depot and compare it with subcutaneous (SC) and OM depots. Using an automated histological characterisation technique, OF adipocytes were found to be significantly smaller (parameters: area, maximum diameter and perimeter) than OM and SC adipocytes (P<0 x 001). Although immunohistochemical analyses demonstrated resident CD68+ cells in all three whole tissue adipose depots, OF CD68 mRNA and protein expression exceeded that of OM and SC (mRNA, P<0 x 05; protein, P<0 x 001). In addition, there was higher expression of glucocorticoid receptor (GR)alpha mRNA in the OF whole tissue depot (P<0 x 05). Conversely, 11beta-HSD1 mRNA together with the markers of late adipocyte differentiation (FABP4 and G3PDH) were significantly lower in OF. Primary cultures of OF preadipocytes demonstrated predominant 11beta-HSD1 oxo-reductase activity with minimal dehydrogenase activity. Orbital adipocytes are smaller, less differentiated, and express low levels of 11beta-HSD1 but abundant GRalpha compared with SC and OM. OF harbours a large CD68+ population. These characteristics define an orbital microenvironment that has the potential to respond to sight-threatening orbital inflammatory disease.