RESUMO
Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-ß (phospho-IR-ß), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.
Assuntos
Receptor Nuclear Órfão DAX-1/metabolismo , Hormônios Esteroides Gonadais/biossíntese , Resistência à Insulina , Insulina/metabolismo , Células Intersticiais do Testículo/metabolismo , Obesidade/metabolismo , Animais , Linhagem Celular , Receptor Nuclear Órfão DAX-1/genética , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/genética , Hormônios Esteroides Gonadais/genética , Insulina/genética , Células Intersticiais do Testículo/patologia , Masculino , Camundongos , Obesidade/induzido quimicamente , Obesidade/patologia , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais/genéticaRESUMO
Growth hormone (GH) is a key metabolic regulator mediating glucose and lipid metabolism. Ataxia telangiectasia mutated (ATM) is a member of the phosphatidylinositol 3-kinase superfamily and regulates cell cycle progression. The orphan nuclear receptor small heterodimer partner (SHP: NR0B2) plays a pivotal role in regulating metabolic processes. Here, we studied the role of ATM on GH-dependent regulation of hepatic gluconeogenesis in the liver. GH induced phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase gene expression in primary hepatocytes. GH treatment and adenovirus-mediated STAT5 overexpression in hepatocytes increased glucose production, which was blocked by a JAK2 inhibitor, AG490, dominant negative STAT5, and STAT5 knockdown. We identified a STAT5 binding site on the PEPCK gene promoter using reporter assays and point mutation analysis. Up-regulation of SHP by metformin-mediated activation of the ATM-AMP-activated protein kinase pathway led to inhibition of GH-mediated induction of hepatic gluconeogenesis, which was abolished by an ATM inhibitor, KU-55933. Immunoprecipitation studies showed that SHP physically interacted with STAT5 and inhibited STAT5 recruitment on the PEPCK gene promoter. GH-induced hepatic gluconeogenesis was decreased by either metformin or Ad-SHP, whereas the inhibition by metformin was abolished by SHP knockdown. Finally, the increase of hepatic gluconeogenesis following GH treatment was significantly higher in the liver of SHP null mice compared with that of wild-type mice. Overall, our results suggest that the ATM-AMP-activated protein kinase-SHP network, as a novel mechanism for regulating hepatic glucose homeostasis via a GH-dependent pathway, may be a potential therapeutic target for insulin resistance.
Assuntos
Gluconeogênese/genética , Hepatócitos/metabolismo , Hormônio do Crescimento Humano/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fator de Transcrição STAT5/metabolismo , Ativação Transcricional , Adenilato Quinase/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Ativadores de Enzimas/farmacologia , Genes Reporter , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Células Hep G2 , Humanos , Fígado/citologia , Fígado/metabolismo , Metformina/farmacologia , Camundongos , Fosfoenolpiruvato Carboxiquinase (GTP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Fator de Transcrição STAT5/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.
Assuntos
Glucose-6-Fosfatase/biossíntese , Células Intersticiais do Testículo/enzimologia , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , AMP Cíclico/genética , AMP Cíclico/metabolismo , Receptor Nuclear Órfão DAX-1/genética , Receptor Nuclear Órfão DAX-1/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose-6-Fosfatase/genética , Células HeLa , Humanos , Células Intersticiais do Testículo/citologia , Masculino , Camundongos , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Regiões Promotoras Genéticas/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Botulinum toxin A (BoNT-A) is used clinically for various muscle disorders and acts by preventing the release of the neurotransmitter acetylcholine into the synapse space. Here, we compared the efficacy of prabotulinumtoxinA (PRA) and onabotulinumtoxinA (ONA) for the reduction in hypertrophy in myostatin-deficient (Mstn-/- ) mice. Two different BoNT-A products (2.5, 10 and 25 U/kg) were injected to paralyse the hindlimb for 2 months, after which sciatic nerve conduction study, 3D micro-CT, haematoxylin and eosin (H&E) and dystrophin staining were conducted. Administration of BoNT-A products induced denervation-mediated atrophy and alleviated muscle hypertrophy generated in Mstn-/- mice. The present study revealed that each BoNT-A regulates skeletal muscle size, myofibre number and myofibre diameter in Mstn-/- mice. The potential applicability of BoNT-A for the treatment of rare muscle hypertrophic diseases was demonstrated. Compared with ONA, PRA had a comparable ability to act in the local area.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Toxinas Botulínicas/farmacologia , Hipertrofia/tratamento farmacológico , Doenças Musculares/tratamento farmacológico , Miostatina/genética , Animais , Toxinas Botulínicas/administração & dosagem , Toxinas Botulínicas Tipo A/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Membro Posterior , Hipertrofia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/patologia , Doenças Musculares/fisiopatologia , Fármacos Neuromusculares/administração & dosagem , Fármacos Neuromusculares/farmacologiaRESUMO
Bisphenol A bis (2,3-dihydroxypropyl) ether (BADGE.2H(2)O) is a component of commercial liquid epoxy resins commonly used in the food-packing industry and in dental sealants. There is evidence that it has significant estrogenic activity. Nur77 plays a crucial role in the regulation of certain genes involved in LH-mediated steroidogenesis in testicular Leydig cells. It was previously demonstrated that Bisphenol A (BPA) stimulates Nur77 gene induction and steroidogenesis. In this study, we investigated the effects of BADGE.2H(2)O on Nur77 gene expression and steroidogenesis. Northern blot analysis showed that it increased the expression of Nur77 mRNA and protein, and transient transfection assays demonstrated that it increased the promoter activity and transactivation of Nur77. It also increased the expression of certain steroidogenic genes, such as StAR and 3 beta-HSD. Finally, over-expression of a dominant negative Nur77 cDNA via adenoviral infection reduced BADGE.2H(2)O-mediated progesterone biosynthesis. These results indicate that BADGE.2H(2)O disrupts testicular steroidogenesis by increasing Nur77 gene expression.
Assuntos
Proteínas de Ligação a DNA/genética , Compostos de Epóxi/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Progesterona/metabolismo , Receptores de Esteroides/genética , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Adenoviridae/genética , Animais , Compostos Benzidrílicos , Northern Blotting , Western Blotting , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Radioimunoensaio , Receptores de Esteroides/metabolismo , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
Atopic dermatitis (AD) is a common inflammatory skin disease characterized by a complex, heterogeneous pathogenesis including skin barrier dysfunction, immunology, and pruritus. Although epidermal growth factor (EGF) is essential for epithelial homeostasis and wound healing, the effect of EGF on AD remains to be explored. To develop a new therapy for AD, the anti-AD potential of EGF was investigated by inducing AD-like skin lesions in NC/Nga mice using 2,4-dinitrochlorobenzene (DNCB). EGF was administrated to NC/Nga mice to evaluate its therapeutic effect on DNCB-induced AD. EGF treatment improved dermatitis score, ear thickness, epidermal hyperplasia, serum total immunoglobulin E level, and transepidermal water loss in NC/Nga mice with DNCB-induced AD. In addition, levels of skin barrier-related proteins such as filaggrin, involucrin, loricrin, occludin, and zonula occludens-1 (ZO-1) were increased by EGF treatment. These beneficial effects of EGF on AD may be mediated by EGF regulation of Th1/Th2-mediated cytokines, mast cell hyperplasia, and protease activated receptor-2 (PAR-2) and thymic stromal lymphopoietin (TSLP), which are triggers of AD. Taken together, our findings suggest that EGF may potentially protect against AD lesional skin via regulation of skin barrier function and immune response.
Assuntos
Dermatite Atópica/prevenção & controle , Fator de Crescimento Epidérmico/farmacologia , Mastócitos/efeitos dos fármacos , Pele/efeitos dos fármacos , Administração Tópica , Animais , Citocinas/imunologia , Citocinas/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/imunologia , Dinitroclorobenzeno , Fator de Crescimento Epidérmico/administração & dosagem , Proteínas Filagrinas , Proteínas de Filamentos Intermediários/imunologia , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Receptor PAR-2/imunologia , Receptor PAR-2/metabolismo , Pele/imunologia , Pele/metabolismo , Proteína da Zônula de Oclusão-1/imunologia , Proteína da Zônula de Oclusão-1/metabolismo , Linfopoietina do Estroma do TimoRESUMO
The estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the nuclear receptor superfamily that activates transcription in the absence of ligand. However, the detailed mechanism of gene regulation by ERRgamma is not fully understood. In this study we have found that the orphan nuclear receptor ERRgamma activates the DAX-1 promoter, which, in turn, represses transactivation by ERRgamma. Serial deletions of mouse DAX-1 (mDAX-1) gene promoter have revealed that the region responding to ERRgamma is located between -129 and -121 bp and -334 and -326 bp. Gel shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated that ERRgamma binds directly to the mDAX-1 promoter. Site-directed mutagenesis results demonstrated that ERRE1 (-129 to -121 bp) is more important than ERRE2 (-334 to -326 bp) which is not conserved in the human DAX-1 promoter. In addition, adenovirus-mediated overexpression of ERRgamma induced DAX-1 gene expression in MCF-7 breast cancer cells that co-expressed ERRgamma and DAX-1. Moreover, yeast two-hybrid and glutathione S-transferase (GST)-pull down assays demonstrated that DAX-1 physically interacted with ERRgamma and inhibited ERRgamma transactivation, and that this interaction was dependent on the AF-2 domain of ERRgamma. In addition, in vitro competition assays showed that DAX-1 inhibited PGC-1alpha mediated ERRgamma transactivation, via competition between these two factors for the AF-2 binding domain. We thus propose a novel autoregulatory loop that controls DAX-1 gene expression by ERRgamma.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Receptores de Estrogênio/fisiologia , Transativadores/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular , Receptor Nuclear Órfão DAX-1 , Proteínas de Ligação a DNA/metabolismo , Homeostase , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta , Ativação TranscricionalRESUMO
Adrenal insufficiency is an uncommon cause of hypercalcemia and not easily considered as an etiology of adrenal insufficiency in clinical practice, as not all cases of adrenal insufficiency manifest as hypercalcemia. We report a case of secondary adrenal insufficiency presenting as hypercalcemia and acute kidney injury in a 66-year-old female. The patient was admitted to the emergency department with general weakness and poor oral intake. Hypercalcemia (11.5 mg/dL) and moderate renal dysfunction (serum creatinine 4.9 mg/dL) were shown in her initial laboratory findings. Studies for malignancy and hyperparathyroidism showed negative results. Basal cortisol and adrenocorticotropic hormone levels and adrenocorticotropic hormone stimulation test confirmed the diagnosis of adrenal insufficiency. With the administration of oral hydrocortisone, hypercalcemia was dramatically resolved within 3 days. This case shows that adrenal insufficiency may manifest as hypercalcemia and acute kidney injury, which implicates that adrenal insufficiency should be considered a cause of hypercalcemia in clinical practice.
RESUMO
In this study, we prospectively investigated the impact of kidney transplantation (KT) on the status of hypertension, including circadian rhythm in end-stage renal disease (ESRD) patients. We performed 24-hour ambulatory blood pressure (BP) monitoring and office BP measurement in 48 patients before and 1 year after KT. According to the nocturnal reduction in systolic BP (ΔSBP), the patients were divided into dippers, non-dippers, and reverse dippers. After KT, the mean BP value in office BP and 24-hour ambulatory BP monitoring did not change, but the proportion of patients taking anti-hypertensive drugs and the pill number significantly decreased. In contrast, the mean ΔSBP significantly decreased, and the proportion of non-dippers and reverse dippers did not decrease. Decrease in ΔSBP after KT was associated with inferior allograft function during follow-up. Our study suggests that KT improved the overall BP level, but it did not affect abnormal circadian rhythm in ESRD patients.
Assuntos
Monitorização Ambulatorial da Pressão Arterial , Hipertensão/diagnóstico , Falência Renal Crônica/cirurgia , Transplante de Rim/métodos , Adulto , Análise de Variância , Anti-Hipertensivos/uso terapêutico , Ritmo Circadiano , Estudos de Coortes , Feminino , Seguimentos , Humanos , Hipertensão/tratamento farmacológico , Falência Renal Crônica/diagnóstico , Transplante de Rim/efeitos adversos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Prospectivos , Medição de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Kruppel-associated box (KRAB) domain-containing proteins consist of potential transcriptional repression modules. Previously, gonadotropin-inducible ovarian transcription factor-1 (GIOT-1) was identified as a novel KRAB-containing zinc finger protein and shown to have transcriptional repression activity. Here, we demonstrate that orphan nuclear receptor Nur77 regulates GIOT-1 gene expression in testicular Leydig cell lines and that GIOT-1 acts as a novel corepressor of the orphan nuclear receptor steroidogenic factor 1 (SF-1). Mutation analysis of the GIOT-1 promoter and overexpression analysis of dominant-negative Nur77 revealed that luteinizing hormone activates GIOT-1 gene expression through Nur77. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that Nur77 directly binds to the GIOT-1 promoter. GIOT-1 represses the SF-1 transactivation, and specific interaction between GIOT-1 and SF-1 was observed. We also demonstrate an interaction between GIOT-1 and histone deacetylase 2 (HDAC2). GIOT-1-mediated transrepression was recovered by down-regulation of HDAC2 expression with small interfering RNA of HDAC2. Knock down of the endogenous GIOT-1 results in significant enhancement of CYP17 expression in Leydig cells. In conclusion, this study of cross-talk between GIOT-1 and orphan nuclear receptors will provide new insights into the role of KRAB-containing zinc finger proteins in nuclear receptor action.