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Steroid 5ß-reductase (AKR1D1) plays important role in hepatic bile acid synthesis and glucocorticoid clearance. Bile acids and glucocorticoids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown. Akr1d1-/- mice were generated on a C57BL/6 background. Liquid chromatography/mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin tolerance were evaluated. Molecular changes were assessed by RNA-Seq and Western blotting. Male Akr1d1-/- mice were challenged with a high fat diet (60% kcal from fat) for 20 weeks. Akr1d1-/- mice had a sex-specific metabolic phenotype. At 30 weeks of age, male, but not female, Akr1d1-/- mice were more insulin tolerant and had reduced lipid accumulation in the liver and adipose tissue yet had hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was associated with sexually dimorphic changes in bile acid metabolism and composition but without overt effects on circulating glucocorticoid levels or glucocorticoid-regulated gene expression in the liver. Male Akr1d1-/- mice were not protected against diet-induced obesity and insulin resistance. In conclusion, this study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin tolerance and lipid homeostasis in a sex-dependent manner.
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Glucocorticoides , Oxirredutases , Animais , Ácidos e Sais Biliares , Dieta Hiperlipídica , Feminino , Glucocorticoides/metabolismo , Insulina/metabolismo , Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases/genética , FenótipoRESUMO
Adverse effects of calcineurin inhibitors (CNI), such as hypertension, hyperkalemia, acidosis, hypomagnesemia and hypercalciuria, have been linked to dysfunction of the distal convoluted tubule (DCT). To test this, we generated a mouse model with an inducible DCT-specific deletion of the calcineurin regulatory subunit B alpha (CnB1-KO). Three weeks after CnB1 deletion, these mice exhibited hypomagnesemia and acidosis, but no hypertension, hyperkalemia or hypercalciuria. Consistent with the hypomagnesemia, CnB1-KO mice showed a downregulation of proteins implicated in DCT magnesium transport, including TRPM6, CNNM2, SLC41A3 and parvalbumin but expression of calcium channel TRPV5 in the kidney was unchanged. The abundance of the chloride/bicarbonate exchanger pendrin was increased, likely explaining the acidosis. Plasma aldosterone levels, kidney renin expression, abundance of phosphorylated sodium chloride-cotransporter and abundance of the epithelial sodium channel were similar in control and CnB1-KO mice, consistent with a normal sodium balance. Long-term potassium homeostasis was maintained in CnB1-KO mice, but in-vivo and ex-vivo experiments indicated that CnB1 contributes to acute regulation of potassium balance and sodium chloride-cotransporter. Tacrolimus treatment of control and CnB1-KO mice demonstrated that CNI-related hypomagnesemia is linked to impaired calcineurin-signaling in DCT, while hypocalciuria and hyponatremia occur independently of CnB1 in DCT. Transcriptome and proteome analyses of isolated DCTs demonstrated that CnB1 deletion impacts the expression of several DCT-specific proteins and signaling pathways. Thus, our data support a critical role of calcineurin for DCT function and provide novel insights into the pathophysiology of CNI side effects and involved molecular players in the DCT.
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Acidose , Magnésio , Animais , Calcineurina/genética , Túbulos Renais Distais , Camundongos , Proteoma/genética , TranscriptomaRESUMO
BACKGROUND AND PURPOSE: 11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) regulates tissue-specific glucocorticoid metabolism and its impaired expression and activity are associated with major diseases. Pharmacological inhibition of 11ß-HSD1 is considered a promising therapeutic strategy. This study investigated whether alternative 7-oxo bile acid substrates of 11ß-HSD1 or the ratios to their 7-hydroxy products can serve as biomarkers for decreased enzymatic activity. EXPERIMENTAL APPROACH: Bile acid profiles were measured by ultra-HPLC tandem-MS in plasma and liver tissue samples of four different mouse models with decreased 11ß-HSD1 activity: global (11KO) and liver-specific 11ß-HSD1 knockout mice (11LKO), mice lacking hexose-6-phosphate dehydrogenase (H6pdKO) that provides cofactor NADPH for 11ß-HSD1 and mice treated with the pharmacological inhibitor carbenoxolone. Additionally, 11ß-HSD1 expression and activity were assessed in H6pdKO- and carbenoxolone-treated mice. KEY RESULTS: The enzyme product to substrate ratios were more reliable markers of 11ß-HSD1 activity than absolute levels due to large inter-individual variations in bile acid concentrations. The ratio of the 7ß-hydroxylated ursodeoxycholyltaurine (UDC-Tau) to 7-oxolithocholyltaurine (7oxoLC-Tau) was diminished in plasma and liver tissue of all four mouse models and decreased in H6pdKO- and carbenoxolone-treated mice with moderately reduced 11ß-HSD1 activity. The persistence of 11ß-HSD1 oxoreduction activity in the face of H6PD loss indicates the existence of an alternative NADPH source in the endoplasmic reticulum. CONCLUSIONS AND IMPLICATIONS: The plasma UDC-Tau/7oxo-LC-Tau ratio detects decreased 11ß-HSD1 oxoreduction activity in different mouse models. This ratio may be a useful biomarker of decreased 11ß-HSD1 activity in pathophysiological situations or upon pharmacological inhibition. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.
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11-beta-Hidroxiesteroide Desidrogenase Tipo 1 , Glucocorticoides , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Animais , Ácidos e Sais Biliares , Biomarcadores , Camundongos , Camundongos KnockoutRESUMO
The renal distal convoluted tubule (DCT) is critical for the fine-tuning of urinary ion excretion and the control of blood pressure. Ion transport along the DCT is tightly controlled by posttranscriptional mechanisms including a complex interplay of kinases, phosphatases, and ubiquitin ligases. Previous work identified the transcription factor Prox-1 as a gene significantly enriched in the DCT of adult mice. To test if Prox-1 contributes to the transcriptional regulation of DCT function and structure, we developed a novel mouse model (NCCcre:Prox-1flox/flox) for an inducible deletion of Prox-1 specifically in the DCT. The deletion of Prox-1 had no obvious impact on DCT structure and growth independent whether the deletion was achieved in newborn or adult mice. Furthermore, DCT-specific Prox-1 deficiency did not alter DCT-proliferation in response to loop diuretic treatment. Likewise, the DCT-specific deletion of Prox-1 did not cause other gross phenotypic abnormalities. Body weight, urinary volume, Na+ and K+ excretion as well as plasma Na+, K+, and aldosterone levels were similar in Prox-1DCTKO and Prox-1DCTCtrl mice. However, Prox-1DCTKO mice exhibited a significant hypomagnesemia with a profound downregulation of the DCT-specific apical Mg2+ channel TRPM6 and the NaCl cotransporter (NCC) at both mRNA and protein levels. The expression of other proteins involved in distal tubule Mg2+ and Na+ handling was not affected. Thus, Prox-1 is a DCT-enriched transcription factor that does not control DCT growth but contributes to the molecular control of DCT-dependent Mg2+ homeostasis in the adult kidney.
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Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Túbulos Renais Distais/efeitos dos fármacos , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Canais de Cátion TRPM/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Deleção de Genes , Proteínas de Homeodomínio/genética , Túbulos Renais Distais/citologia , Magnésio/metabolismo , Camundongos , Potássio/metabolismo , Sódio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Canais de Cátion TRPM/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Bile acids control lipid homeostasis by regulating uptake from food and excretion. Additionally, bile acids are bioactive molecules acting through receptors and modulating various physiological processes. Impaired bile acid homeostasis is associated with several diseases and drug-induced liver injury. Individual bile acids may serve as disease and drug toxicity biomarkers, with a great demand for improved bile acid quantification methods. We developed, optimized, and validated an LC-MS/MS method for quantification of 36 bile acids in serum, plasma, and liver tissue samples. The simultaneous quantification of important free and taurine- and glycine-conjugated bile acids of human and rodent species has been achieved using a simple workflow. The method was applied to a mouse model of statin-induced myotoxicity to assess a possible role of bile acids. Treatment of mice for three weeks with 5, 10, and 25 mg/kg/d simvastatin, causing adverse skeletal muscle effects, did not alter plasma and liver tissue bile acid profiles, indicating that bile acids are not involved in statin-induced myotoxicity. In conclusion, the established LC-MS/MS method enables uncomplicated sample preparation and quantification of key bile acids in serum, plasma, and liver tissue of human and rodent species to facilitate future studies of disease mechanisms and drug-induced liver injury.
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BACKGROUND: The lumen of the endoplasmic reticulum (ER) acts as a cellular Ca2+ store and a site for oxidative protein folding, which is controlled by the reduced glutathione (GSH) and glutathione-disulfide (GSSG) redox pair. Although depletion of luminal Ca2+ from the ER provokes a rapid and reversible shift towards a more reducing poise in the ER, the underlying molecular basis remains unclear. RESULTS: We found that Ca2+ mobilization-dependent ER luminal reduction was sensitive to inhibition of GSH synthesis or dilution of cytosolic GSH by selective permeabilization of the plasma membrane. A glutathione-centered mechanism was further indicated by increased ER luminal glutathione levels in response to Ca2+ efflux. Inducible reduction of the ER lumen by GSH flux was independent of the Ca2+-binding chaperone calreticulin, which has previously been implicated in this process. However, opening the translocon channel by puromycin or addition of cyclosporine A mimicked the GSH-related effect of Ca2+ mobilization. While the action of puromycin was ascribable to Ca2+ leakage from the ER, the mechanism of cyclosporine A-induced GSH flux was independent of calcineurin and cyclophilins A and B and remained unclear. CONCLUSIONS: Our data strongly suggest that ER influx of cytosolic GSH, rather than inhibition of local oxidoreductases, is responsible for the reductive shift upon Ca2+ mobilization. We postulate the existence of a Ca2+- and cyclosporine A-sensitive GSH transporter in the ER membrane. These findings have important implications for ER redox homeostasis under normal physiology and ER stress.
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Cálcio/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glutationa/metabolismo , Calreticulina/metabolismo , Humanos , Ligação ProteicaRESUMO
Gestational diabetes mellitus (GDM) is one of the most common diseases associated with pregnancy, however, the underlying mechanisms remain unclear. Based on the well documented role of inflammation in type 2 diabetes, the aim was to investigate the role of inflammation in GDM. We established a mouse model for GDM on the basis of its two major risk factors, obesity and aging. In these GDM mice, we observed increased Interleukin-1ß (IL-1ß) expression in the uterus and the placenta along with elevated circulating IL-1ß concentrations compared to normoglycemic pregnant mice. Treatment with an anti-IL-1ß antibody improved glucose-tolerance of GDM mice without apparent deleterious effects for the fetus. Finally, IL-1ß antagonism showed a tendency for reduced plasma corticosterone concentrations, possibly explaining the metabolic improvement. We conclude that IL-1ß is a causal driver of impaired glucose tolerance in GDM.
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Diabetes Gestacional/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Interleucina-1beta/antagonistas & inibidores , Animais , Diabetes Gestacional/sangue , Modelos Animais de Doenças , Feminino , Hormônios/sangue , Hiperglicemia/sangue , Interleucina-1beta/metabolismo , Camundongos Endogâmicos C57BL , Gravidez , Esteroides/sangueRESUMO
The kidney needs to adapt daily to variable dietary K+ contents via various mechanisms including diuretic, acid-base and hormonal changes that are still not fully understood. In this study, we demonstrate that following a K+-deficient diet in wildtype mice, the serine protease CAP2/Tmprss4 is upregulated in connecting tubule and cortical collecting duct and also localizes to the medulla and transitional epithelium of the papilla and minor calyx. Male CAP2/Tmprss4 knockout mice display altered water handling and urine osmolality, enhanced vasopressin response leading to upregulated adenylate cyclase 6 expression and cAMP overproduction, and subsequently greater aquaporin 2 (AQP2) and Na+-K+-2Cl- cotransporter 2 (NKCC2) expression following K+-deficient diet. Urinary acidification coincides with significantly increased H+,K+-ATPase type 2 (HKA2) mRNA and protein expression, and decreased calcium and phosphate excretion. This is accompanied by increased glucocorticoid receptor (GR) protein levels and reduced 11ß-hydroxysteroid dehydrogenase 2 activity in knockout mice. Strikingly, genetic nephron-specific deletion of GR leads to the mirrored phenotype of CAP2/Tmprss4 knockouts, including increased water intake and urine output, urinary alkalinisation, downregulation of HKA2, AQP2 and NKCC2. Collectively, our data unveil a novel role of the serine protease CAP2/Tmprss4 and GR on renal water handling upon dietary K+ depletion.
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11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aquaporina 2/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Rim/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Potássio na Dieta/metabolismo , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismoRESUMO
Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways. However, recent research has emphasized the roles of oxysterols in essential physiologic processes and in various diseases. Despite these discoveries, the metabolic pathways leading to the different oxysterols are still largely unknown and the biosynthetic origin of several oxysterols remains unidentified. Earlier studies demonstrated that the glucocorticoid metabolizing enzymes, 11ß-hydroxysteroid dehydrogenase (11ß-HSD) types 1 and 2, interconvert 7-ketocholesterol (7kC) and 7ß-hydroxycholesterol (7ßOHC). We examined the role of 11ß-HSDs in the enzymatic control of the intracellular availability of 7ß,27-dihydroxycholesterol (7ß27OHC), a retinoid-related orphan receptor γ (RORγ) ligand. We used microsomal preparations of cells expressing recombinant 11ß-HSD1 and 11ß-HSD2 to assess whether 7ß27OHC and 7-keto,27-hydroxycholesterol (7k27OHC) are substrates of these enzymes. Binding of 7ß27OHC and 7k27OHC to 11ß-HSDs was studied by molecular modeling. To our knowledge, the stereospecific oxoreduction of 7k27OHC to 7ß27OHC by human 11ß-HSD1 and the reverse oxidation reaction of 7ß27OHC to 7k27OHC by human 11ß-HSD2 were demonstrated for the first time. Apparent enzyme affinities of 11ß-HSDs for these novel substrates were equal to or higher than those of the glucocorticoids. This is supported by the fact that 7k27OHC and 7ß27OHC are potent inhibitors of the 11ß-HSD1-dependent oxoreduction of cortisone and the 11ß-HSD2-dependent oxidation of cortisol, respectively. Furthermore, molecular docking calculations explained stereospecific enzyme activities. Finally, using an inducible RORγ reporter system, we showed that 11ß-HSD1 and 11ß-HSD2 controlled RORγ activity. These findings revealed a novel glucocorticoid-independent prereceptor regulation mechanism by 11ß-HSDs that warrants further investigation.
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11-beta-Hidroxiesteroide Desidrogenases/metabolismo , Receptores Nucleares Órfãos/metabolismo , Receptores de Mineralocorticoides/metabolismo , 11-beta-Hidroxiesteroide Desidrogenases/genética , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Glucocorticoides/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Oxisteróis/metabolismo , Espectrometria de Massas em TandemRESUMO
We describe two cases of hypertension and hypokalemia due to mineralocorticoid excess caused by posaconazole treatment of coccidioidomycosis and rhinocerebral mucormycosis infections, respectively. Clinical laboratory evaluations, including a comprehensive analysis of blood and urine steroid profiles, revealed low renin and aldosterone and indicated as the underlying mechanism primarily a block of 11ß-hydroxylase activity in patient 1, whereas patient 2 displayed weaker 11ß-hydroxylase but more pronounced 11ß-hydroxysteroid dehydrogenase 2 inhibition. The results show that both previously suggested mechanisms must be considered and emphasize significant interindividual differences in the contribution of each enzyme to the observed mineralocorticoid excess phenotype. The mineralocorticoid symptoms of patient 1 resolved after replacement of posaconazole therapy by isavoconazole, and posaconazole dosage de-escalation ameliorated the effects in patient 2. By providing a thorough analysis of the patients' blood and urine steroid metabolites, this report adds further evidence for two individually pronounced mechanisms of posaconazole-induced hypertension and hypokalemia. The elucidation of the factors responsible for the individual phenotype warrants further research.
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CONTEXT: Mammalian target of rapamycin complex 1 (mTORC1) activity is often increased in the adrenal cortex of patients with primary aldosteronism (PA), and mTORC1 inhibition decreases aldosterone production in adrenocortical cells, suggesting the mTORC1 pathway as a target for treatment of PA. OBJECTIVE: To investigate the effect of mTORC1 inhibition on adrenal steroid hormones and hemodynamic parameters in mice and in patients with PA. DESIGN: (i) Plasma aldosterone, corticosterone, and angiotensin II (Ang II) were measured in mice treated for 24 hours with vehicle or rapamycin. (ii) Plasma aldosterone levels after a saline infusion test, plasma renin, and 24-hour urine steroid hormone metabolome and hemodynamic parameters were measured during an open-label study in 12 patients with PA, before and after 2 weeks of treatment with everolimus and after a 2-week washout. MAIN OUTCOME MEASURES: (i) Change in plasma aldosterone levels. (ii) Change in other steroid hormones, renin, Ang II, and hemodynamic parameters. RESULTS: Treatment of mice with rapamycin significantly decreased plasma aldosterone levels (P = 0.007). Overall, treatment of PA patients with everolimus significantly decreased blood pressure (P < 0.05) and increased renin levels (P = 0.001) but did not decrease aldosterone levels significantly. However, prominent reduction of aldosterone levels upon everolimus treatment was observed in four patients. CONCLUSION: In mice, mTORC1 inhibition was associated with reduced plasma aldosterone levels. In patients with PA, mTORC1 inhibition was associated with improved blood pressure and renin suppression. In addition, mTORC1 inhibition appeared to reduce plasma aldosterone in a subset of patients.
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Everolimo/uso terapêutico , Hiperaldosteronismo/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Sirolimo/uso terapêutico , Adulto , Aldosterona/sangue , Angiotensina II/sangue , Animais , Pressão Sanguínea/efeitos dos fármacos , Everolimo/farmacologia , Feminino , Hemodinâmica/efeitos dos fármacos , Humanos , Hiperaldosteronismo/sangue , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Projetos Piloto , Estudo de Prova de Conceito , Renina/sangue , Sirolimo/farmacologiaRESUMO
Humans are constantly exposed to a multitude of environmental chemicals that may disturb endocrine functions. It is crucial to identify such chemicals and uncover their mode-of-action to avoid adverse health effects. 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) catalyze the formation of cortisol and aldosterone, respectively, in the adrenal cortex. Disruption of their synthesis by exogenous chemicals can contribute to cardio-metabolic diseases, chronic kidney disease, osteoporosis, and immune-related disorders. This study applied in silico screening and in vitro evaluation for the discovery of xenobiotics inhibiting CYP11B1 and CYP11B2. Several databases comprising environmentally relevant pollutants, chemicals in body care products, food additives and drugs were virtually screened using CYP11B1 and CYP11B2 pharmacophore models. A first round of biological testing used hamster cells overexpressing human CYP11B1 or CYP11B2 to analyze 25 selected virtual hits. Three compounds inhibited CYP11B1 and CYP11B2 with IC50 values below 3 µM. The most potent inhibitor was epoxiconazole (IC50 value of 623 nM for CYP11B1 and 113 nM for CYP11B2, respectively); flurprimidol and ancymidol were moderate inhibitors. In a second round, these three compounds were tested in human adrenal H295R cells endogenously expressing CYP11B1 and CYP11B2, confirming the potent inhibition by epoxiconazole and the more moderate effects by flurprimidol and ancymidol. Thus, the in silico screening, prioritization of chemicals for initial biological tests and use of H295R cells to provide initial mechanistic information is a promising strategy to identify potential endocrine disruptors inhibiting corticosteroid synthesis. A critical assessment of human exposure levels and in vivo evaluation of potential corticosteroid disrupting effects by epoxiconazole is required.
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Citocromo P-450 CYP11B2/antagonistas & inibidores , Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Fungicidas Industriais/farmacologia , Esteroide 11-beta-Hidroxilase/antagonistas & inibidores , Triazóis/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Neoplasias das Glândulas Suprarrenais/enzimologia , Neoplasias das Glândulas Suprarrenais/patologia , Sobrevivência Celular , Inibidores Enzimáticos/química , Compostos de Epóxi/química , Fungicidas Industriais/química , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Triazóis/química , Células Tumorais CultivadasRESUMO
Brain susceptibility to a neurotoxic insult may be increased in a compromised health status, such as metabolic syndrome. Both metabolic syndrome and exposure to trimethyltin (TMT) are known to promote neurodegeneration. In combination the two factors may elicit additive or compensatory/regulatory mechanisms. Combined effects of TMT exposure (0.5-1 µM) and mimicked metabolic syndrome-through modulation of insulin and glucocorticoid (GC) levels-were investigated in three models: tridimensional rat brain cell cultures for neuron-glia effects; murine microglial cell line BV-2 for a mechanistic analysis of microglial reactivity; and db/db mice as an in vivo model of metabolic syndrome. In 3D cultures, low insulin condition significantly exacerbated TMT's effect on GABAergic neurons and promoted TMT-induced neuroinflammation, with increased expression of cytokines and of the regulator of intracellular GC activity, 11ß-hydroxysteroid dehydrogenase 1 (11ß-Hsd1). Microglial reactivity increased upon TMT exposure in medium combining low insulin and high GC. These results were corroborated in BV-2 microglial cells where lack of insulin exacerbated the TMT-induced increase in 11ß-Hsd1 expression. Furthermore, TMT-induced microglial reactivity seems to depend on mineralocorticoid receptor activation. In diabetic BKS db mice, a discrete exacerbation of TMT neurotoxic effects on GABAergic neurons was observed, together with an increase of interleukin-6 (IL-6) and of basal 11ß-Hsd1 expression as compared to controls. These results suggest only minor additive effects of the two brain insults, neurotoxicant TMT exposure and metabolic syndrome conditions, where 11ß-Hsd1 appears to play a key role in the regulation of neuroinflammation and of its protective or neurodegenerative consequences.
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Glucocorticoides/metabolismo , Inflamação/metabolismo , Secreção de Insulina/efeitos dos fármacos , Degeneração Neural/metabolismo , Compostos de Trimetilestanho/toxicidade , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/biossíntese , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/efeitos dos fármacos , Animais , Linhagem Celular , Células Cultivadas , Citocinas/biossíntese , Técnicas In Vitro , Inflamação/induzido quimicamente , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos , Degeneração Neural/induzido quimicamente , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/efeitos dos fármacos , Reprodutibilidade dos TestesRESUMO
Oxysterols are cholesterol metabolites derived through either autoxidation or enzymatic processes. They consist of a large family of bioactive lipids that have been associated with the progression of multiple pathologies. In order to unravel (patho-)physiological mechanisms involving oxysterols, it is crucial to elucidate the underlying formation and degradation of oxysterols. A role of 11ß-hydroxysteroid dehydrogenases (11ß-HSDs) in oxysterol metabolism by catalyzing the interconversion of 7-ketocholesterol (7kC) and 7ß-hydroxycholesterol (7ßOHC) has already been reported. The present study addresses a function of 11ß-HSD1 in the enzymatic generation of 7ß,25-dihydroxycholesterol (7ß25OHC) from 7-keto,25-hydroxycholesterol (7k25OHC) and tested whether 11ß-HSD2 is able to catalyze the reverse reaction. For the first time, using recombinant enzymes, the formation of 7k25OHC from 7kC by cholesterol 25-hydroxylase (CH25H) and further stereospecific oxoreduction to 7ß25OHC by human and mouse 11ß-HSD1 could be demonstrated. Additionally, experiments using human 11ß-HSD2 showed the oxidation of 7ß25OHC to 7k25OHC. Molecular modeling provided an explanation for the stereospecific interconversion of 7ß25OHC and 7k25OHC. Production of the Epstein-Barr virus-induced gene 2 (EBI2) ligand 7ß25OHC from 7k25OHC in challenged tissue by 11ß-HSD1 may be important in inflammation. In conclusion, these results demonstrate a novel glucocorticoid-independent pre-receptor regulation mediated by 11ß-HSDs.
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11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Hidroxicolesteróis/metabolismo , Cetocolesteróis/metabolismo , Animais , Células HEK293 , Humanos , Hidroxilação , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Oxirredução , Células RAW 264.7RESUMO
The novel d-amphetamine prodrug lisdexamfetamine is applied to treat attention-deficit/hyperactivity disorder (ADHD). d-Amphetamine releases dopamine and norepinephrine and stimulates the hypothalamic-pituitary-adrenal (HPA) axis, which may contribute to its reinforcing effects and risk of abuse. However, no data is currently available on the effects of lisdexamfetamine on circulating steroids. This randomized, double-blind, placebo-controlled, cross-over study evaluated the effects of equimolar doses of d-amphetamine (40 mg) and lisdexamfetamine (100 mg) and placebo on circulating steroids in 24 healthy subjects. Plasma steroid and d-amphetamine levels were determined up to 24 h. Delayed increase and peak levels of plasma d-amphetamine concentrations were observed following lisdexamfetamine treatment compared with d-amphetamine administration, however the maximal concentrations and total exposure (area under the curve [AUC]) were similar. Lisdexamfetamine and d-amphetamine significantly enhanced plasma levels of adrenocorticotropic hormone, glucocorticoids (cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, and 11-deoxycortisol), androgens (dehydroepiandrosterone, dehydroepiandrosterone sulfate, and Δ4-androstene-3,17-dione [androstenedione]), and progesterone (only in men) compared with placebo. Steroid concentration-time curves were shifted to later time points due to a non-significantly later onset following lisdexamfetamine administration than after d-amphetamine, however maximal plasma steroid concentrations and AUCs did not differ between the active treatments. None of the active treatments altered plasma levels of the mineralocorticoids aldosterone and 11-deoxycorticosterone or the androgen testosterone compared with placebo. The effects of the amphetamines on glucocorticoid production were similar to those that were previously reported for methylphenidate (60 mg) but weaker than those for the serotonin releaser 3,4-methylenedioxymethamphetamine (MDMA; 125 mg) or direct serotonin receptor agonist lysergic acid diethylamide (LSD; 0.2 mg). Lisdexamfetamine produced comparable HPA axis activation and had similar pharmacokinetics than d-amphetamine, except for a delayed time of onset. Thus, serotonin (MDMA, LSD) may more effectively stimulate the HPA axis than dopamine and norepinephrine (D-amphetamine).
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Estimulantes do Sistema Nervoso Central/farmacologia , Dextroanfetamina/farmacologia , Dimesilato de Lisdexanfetamina/farmacologia , Esteroides/sangue , Hormônio Adrenocorticotrópico/sangue , Adulto , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Masculino , Efeito Placebo , Adulto JovemRESUMO
Hexose-6-phosphate dehydrogenase (H6PD) is thought to be the major source of NADPH within the endoplasmic reticulum (ER), determining 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) reaction direction to convert inert 11-oxo- to potent 11ß-hydroxyglucocorticoids. Here, we tested the hypothesis whether H6pd knock-out (KO) in primary murine bone marrow-derived macrophages results in a switch from 11ß-HSD1 oxoreduction to dehydrogenation, thereby inactivating glucocorticoids (GC) and affecting macrophage phenotypic activation as well as causing a more aggressive M1 macrophage phenotype. H6pdKO did not lead to major disturbances of macrophage activation state, although a slightly more pronounced M1 phenotype was observed with enhanced proinflammatory cytokine release, an effect explained by the decreased 11ß-HSD1-dependent GC activation. Unexpectedly, ablation of H6pd did not switch 11ß-HSD1 reaction direction. A moderately decreased 11ß-HSD1 oxoreduction activity by 40-50% was observed in H6pdKO M1 macrophages but dehydrogenation activity was undetectable, providing strong evidence for the existence of an alternative source of NADPH in the ER. H6pdKO M1 activated macrophages showed decreased phagocytic activity, most likely a result of the reduced 11ß-HSD1-dependent GC activation. Other general macrophage functions reported to be influenced by GC, such as nitrite production and cholesterol efflux, were altered negligibly or not at all. Importantly, assessment of energy metabolism using an extracellular flux analyzer and lactate measurements revealed reduced overall glucose consumption in H6pdKO M1 activated macrophages, an effect that was GC independent. The GC-independent influence of H6PD on energy metabolism and the characterization of the alternative source of NADPH in the ER warrant further investigations. ENZYMES: 11ß-HSD1, EC 1.1.1.146; H6PD, EC 1.1.1.47.
Assuntos
11-beta-Hidroxiesteroide Desidrogenases/metabolismo , Desidrogenases de Carboidrato/fisiologia , Glucocorticoides/metabolismo , Glucose/metabolismo , Ativação de Macrófagos , 11-beta-Hidroxiesteroide Desidrogenases/genética , Animais , Células Cultivadas , Retículo Endoplasmático/metabolismo , Metabolismo Energético , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADP/metabolismo , OxirreduçãoRESUMO
Androgen biosynthesis in males occurs to a large extent in testicular Leydig cells. This study focused on the evaluation of three murine Leydig cell lines as potential screening tool to test xenobiotics interfering with gonadal androgen synthesis. The final step of testosterone (T) production in Leydig cells is catalyzed by the enzyme 17ß-hydroxysteroid dehydrogenase 3 (17ß-hsd3). The endogenous 17ß-hsd3 mRNA expression and Δ4-androstene-3,17-dione (AD) to T conversion were determined in the murine cell lines MA-10, BLTK1 and TM3. Additionally, effects of 8-Br-cAMP and forskolin stimulation on steroidogenesis and T production were analyzed. Steroids were quantified in supernatants of cells using liquid chromatography-tandem mass spectrometry. Unstimulated cells incubated with AD produced only very low T but substantial amounts of the inactive androsterone. Stimulated cells produced low amounts of T, moderate amounts of AD, but high amounts of progesterone. Gene expression analyses revealed barely detectable 17ß-hsd3 levels, absence of 17ß-hsd5 (Akr1c6), but substantial 17ß-hsd1 expression in all three cell lines. Thus, MA-10, BLTK1 and TM3 cells are not suitable to study the expression and activity of the gonadal T synthesizing enzyme 17ß-hsd3. The low T production reported in stimulated MA-10 cells are likely a result of the expression of 17ß-hsd1. This study substantiates that the investigated Leydig cell lines MA-10, BLTK1, and TM3 are not suitable to study gonadal androgen biosynthesis due to altered steroidogenic pathways. Furthermore, this study emphasizes the necessity of mass spectrometry-based steroid quantification in experiments using steroidogenic cells such as Leydig cells.
RESUMO
NaPi-IIb/Slc34a2 is a Na+-dependent phosphate transporter that accounts for the majority of active phosphate transport into intestinal epithelial cells. Its abundance is regulated by dietary phosphate, being high during dietary phosphate restriction. Intestinal ablation of NaPi-IIb in mice leads to increased fecal excretion of phosphate, which is compensated by enhanced renal reabsorption. Here we compared the adaptation to dietary phosphate of wild type (WT) and NaPi-IIb-/- mice. High phosphate diet (HPD) increased fecal and urinary excretion of phosphate in both groups, though NaPi-IIb-/- mice still showed lower urinary excretion than WT. In both genotypes low dietary phosphate (LDP) resulted in reduced fecal excretion and almost undetectable urinary excretion of phosphate. Consistently, the expression of renal cotransporters after prolonged LDP was similar in both groups. Plasma phosphate declined more rapidly in NaPi-IIb-/- mice upon LDP, though both genotypes had comparable levels of 1,25(OH)2vitamin D3, parathyroid hormone and fibroblast growth factor 23. Instead, NaPi-IIb-/- mice fed LDP had exacerbated hypercalciuria, higher urinary excretion of corticosterone and deoxypyridinoline, lower bone mineral density and higher number of osteoclasts. These data suggest that during dietary phosphate restriction NaPi-IIb-mediated intestinal absorption prevents excessive demineralization of bone as an alternative source of phosphate.
Assuntos
Densidade Óssea , Osso e Ossos/fisiologia , Dieta/métodos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Fosfatos/administração & dosagem , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo , Animais , Camundongos , Camundongos Knockout , Fosfatos/sangue , Plasma/química , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/deficiênciaRESUMO
DHRS7 (SDR34C1) has been associated with potential tumor suppressor effects in prostate cancer; however, its function remains largely unknown. Recent experiments using purified recombinant human DHRS7 suggested several potential substrates, including the steroids cortisone and Δ4-androstene-3,17-dione (androstenedione). However, the substrate and cofactor concentrations used in these experiments were very high and the physiological relevance of these observations needed to be further investigated. In the present study, recombinant human DHRS7 was expressed in intact HEK-293 cells in order to investigate whether glucocorticoids and androgens serve as substrates at sub-micromolar concentrations and at physiological cofactor concentrations. Furthermore, the membrane topology of DHRS7 was revisited using redox-sensitive green-fluorescent protein fusions in living cells. The results revealed that (1) cortisone is a substrate of DHRS7; however, it is not reduced to cortisol but to 20ß-dihydrocortisone, (2) androstenedione is not a relevant substrate of DHRS7, (3) DHRS7 catalyzes the oxoreduction of 5α-dihydrotestosterone (5αDHT) to 3α-androstanediol (3αAdiol), with a suppressive effect on androgen receptor (AR) transcriptional activity, and (4) DHRS7 is anchored in the endoplasmic reticulum membrane with a cytoplasmic orientation. Together, the results show that DHRS7 is a cytoplasmic oriented enzyme exhibiting 3α/20ß-hydroxysteroid dehydrogenase activity, with a possible role in the modulation of AR function. Further research needs to address the physiological relevance of DHRS7 in the inactivation of 5αDHT and AR regulation.
Assuntos
Androgênios/metabolismo , Di-Hidrotestosterona/metabolismo , Regulação para Baixo , Retículo Endoplasmático/enzimologia , Oxirredutases/metabolismo , Receptores Androgênicos/metabolismo , Androgênios/química , Androstano-3,17-diol/química , Androstano-3,17-diol/metabolismo , Cortisona/análogos & derivados , Cortisona/química , Cortisona/metabolismo , Di-Hidrotestosterona/química , Glucocorticoides/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Ligantes , Conformação Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Concentração Osmolar , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Receptores Androgênicos/química , Receptores Androgênicos/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Vascular calcification resulting from hyperphosphatemia is a major determinant of mortality in chronic kidney disease (CKD). Vascular calcification is driven by aldosterone-sensitive osteogenic transformation of vascular smooth muscle cells (VSMCs). We show that even in absence of exogenous aldosterone, silencing and pharmacological inhibition (spironolactone, eplerenone) of the mineralocorticoid receptor (MR) ameliorated phosphate-induced osteo-/chondrogenic transformation of primary human aortic smooth muscle cells (HAoSMCs). High phosphate concentrations up-regulated aldosterone synthase (CYP11B2) expression in HAoSMCs. Silencing and deficiency of CYP11B2 in VSMCs ameliorated phosphate-induced osteogenic reprogramming and calcification. Phosphate treatment was followed by nuclear export of APEX1, a CYP11B2 transcriptional repressor. APEX1 silencing up-regulated CYP11B2 expression and stimulated osteo-/chondrogenic transformation. APEX1 overexpression blunted the phosphate-induced osteo-/chondrogenic transformation and calcification of HAoSMCs. Cyp11b2 expression was higher in aortic tissue of hyperphosphatemic klotho-hypomorphic (kl/kl) mice than in wild-type mice. In adrenalectomized kl/kl mice, spironolactone treatment still significantly ameliorated aortic osteoinductive reprogramming. Our findings suggest that VSMCs express aldosterone synthase, which is up-regulated by phosphate-induced disruption of APEX1-dependent gene suppression. Vascular CYP11B2 may contribute to stimulation of VSMCs osteo-/chondrogenic transformation during hyperphosphatemia.