RESUMO
BACKGROUND: Oral cholic acid therapy is an effective therapy in children with primary bile acid synthesis deficiencies. Most reported patients with this treatment have 3ß-hydroxy-Δ5-C27-steroid oxidoreductase deficiency. The aim of the study was the evaluation of cholic acid therapy in a cohort of patients with the rarer Δ4-3-oxosteroid 5ß-reductase (Δ4-3-oxo-R) deficiency. METHODS: Sixteen patients with Δ4-3-oxo-R deficiency confirmed by AKR1D1 gene sequencing who received oral cholic acid were retrospectively analyzed. RESULTS: First symptoms were reported early in life (median 2 months of age), with 14 and 3 patients having cholestatic jaundice and severe bleeding respectively. Fifteen patients received ursodeoxycholic acid before diagnosis, with partial improvement in 8 patients. Four patients had liver failure at the time of cholic acid initiation. All 16 patients received cholic acid from a median age of 8.1 months (range 3.1-159) and serum liver tests normalized in all within 6-12 months of treatment. After a median cholic acid therapy of 4.5 years (range 1.1-24), all patients were alive with their native liver. Median daily cholic acid dose at last follow-up was 8.3 mg/kg of body weight. All patients, but one, had normal physical examination and all had normal serum liver tests. Fibrosis, evaluated using liver biopsy (n = 4) or liver elastography (n = 9), had stabilized or improved. Cholic acid therapy enabled a 12-fold decrease of 3-oxo-∆4 derivatives in urine. Patients had normal growth and quality of life. The treatment was well tolerated without serious adverse events and signs of hepatotoxicity. CONCLUSIONS: Oral cholic acid therapy is a safe and effective treatment for patients with Δ4-3-oxo-R deficiency.
Assuntos
Ácidos e Sais Biliares , Doenças Metabólicas , Criança , Humanos , Ácido Cólico/uso terapêutico , Estudos Retrospectivos , Qualidade de Vida , Doenças Metabólicas/tratamento farmacológico , Oxirredutases/genéticaRESUMO
Exposure to xenobiotics can adversely affect biochemical reactions, including hepatic bile acid synthesis. Bile acids are essential for dissolving lipophilic compounds in the hydrophilic environment of the gastrointestinal tract. The critical micellar concentration of bile acids depends on the Δ4-reduction stereochemistry, with the 3-oxo-5ß-steroid-Δ4-dehydrogenase (AKR1D1) introducing the cis ring A/B conformation. Loss-of-function mutations in AKR1D1 cause hepatic cholestasis, which, if left untreated can progress into steatosis and liver cirrhosis. Furthermore, AKR1D1 is involved in clearing steroids with an A-ring Δ4-double bond. Here, we tested whether anabolic-androgenic steroids (AAS), often taken off-label at high doses, might inhibit AKR1D1, thereby potentially causing hepatotoxicity. A computational molecular model was established and used for virtual screening of the DrugBank database consisting of 2740 molecules, yielding mainly steroidal hits. Fourteen AAS were selected for in vitro evaluation, as such compounds can reach high hepatic concentrations in an abuse situation. Nandrolone, clostebol, methasterone, drostanolone, and methenolone inhibited to various extent the AKR1D1-mediated reduction of testosterone. Molecular modeling suggests that 9 out of 14 investigated AAS are competitive inhibitors. Moreover quantum mechanical calculations show that nadrolone and clostebol are substrates of AKR1D1 with different activation energy barriers for the hydrogen transfer from cofactor to the C5 position affecting their turnover. In this multidisciplinary approach, we established a molecular model of AKR1D1, identified several AAS as inhibitors, and described their binding mode. This approach may be applied to study other classes of inhibitors including non-steroidal compounds.
Assuntos
Anabolizantes , Esteróides Androgênicos Anabolizantes , Humanos , Ácidos e Sais Biliares , Esteroides , Mutação , Fígado/metabolismo , Anabolizantes/toxicidadeRESUMO
In mammals there are two 3-oxo-4-ene steroid reductases that generate either A/B-trans or A/B cis-ring junctions in the steroid nucleus known as steroid 5α- and 5ß- reductases, respectively. There is only one steroid 5ß- reductase in each species and these are members of the aldo-keto-reductase (AKR) protein superfamily. The corresponding human enzyme is AKR1D1, and it plays an essential role in bile-acid biosynthesis. Germline mutations in AKR1D1 give rise to bile-acid deficiency. Because of its central role in steroid metabolism and need for detailed structure-function studies there is a need to purify the enzyme to homogeneity and in high yield. We report the purification of milligram amounts of crystallographic quality homogeneous recombinant protein for structure-function studies and its characterization.
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Oxirredutases , Esteroides , Animais , Humanos , Oxirredutases/química , Esteroides/química , Esteroides/metabolismo , Ácidos e Sais Biliares , Mamíferos/metabolismoRESUMO
Δ4-3-Oxosteroid 5ß-reductase (AKR1D1) deficiency typically causes severe cholestasis occurs in newborns, leading to death unless patients are treated with primary bile acids. However, we encountered an AKR1D1 deficiency patient treated with only ursodeoxycholic acid who had cholestasis until about 1 year of age but then grew up healthy without further treatment. We also have been following other healthy patients with AKR1D1 mutation who have never developed cholestasis and have not been treated. However, reports are few, involving 3 patients. To better understand and clinically manage a diverse group of patients with AKR1D1 mutation who do not develop potentially fatal cholestasis in the neonatal period, ongoing accumulation and study of informative cases is needed.
RESUMO
Hepatocellular carcinoma (HCC) is the most common histological type of primary liver cancer and the majority of patients are diagnosed at an advanced stage and have a poor prognosis. AKR1C3 (Aldo-keto reductase family 1 member C3) and AKR1D1 (Aldo-keto reductase family 1 member D1) catalyze the conversion of aldehydes and ketones to alcohols and play crucial roles in multiple cancers. However, the functions of AKR1C3 and AKR1D1 in HCC remain unclear. In our study, data from the public databases were selected as training and validation sets, then 76 HCC patients in our center were chosen as a test set. Bioinformatics methods suggested AKR1C3 was overexpressed in HCC and AKR1D1 was down-regulated. The receiver operating characteristic curve (ROC) analysis was performed and the area under curve (AUC) values of AKR1C3 and AKR1D1 were above 0.7 (0.948, 0.836, respectively). Also, the high expression of AKR1C3 and low expression of AKR1D1 predicted poor prognosis and short median survival time. Then, the knockdown of AKR1C3 and overexpression of AKR1D1 in HCC cells were achieved with lentivirus. And both decreased cell proliferation, restrained cell viability, and inhibited tumorigenesis. Moreover, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted and the results showed that AKR1C3 and AKR1D1 might participate in the MAPK/ERK and androgen receptor (AR) signaling pathway. Furthermore, the AR and phosphorylated ERK1/2 were significantly reduced after the suppression of AKR1C3 or overexpression of AKR1D1. Collectively, AKR1C3 and AKR1D1 might serve as candidate diagnostic and prognostic biomarkers for HCC and provide potential targets for HCC treatment.
Assuntos
Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Oxirredutases/genética , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Área Sob a Curva , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Ontologia Genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Oxirredutases/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Mapas de Interação de Proteínas , RNA Mensageiro/metabolismo , Curva ROC , Taxa de Sobrevida , Regulação para CimaRESUMO
The 5-reductases (5α-reductase types 1, 2 and 3 [5αR1-3], 5ß-reductase [5ßR]) are steroid hormone metabolising enzymes that hold fundamental roles in human physiology and pathology. They possess broad substrate specificity converting many steroid hormones to their 5α- and 5ß-reduced metabolites, as well as catalysing crucial steps in bile acid synthesis. 5αRs are fundamentally important in urogenital development by converting testosterone to the more potent androgen 5α-dihydrotestosterone (5αDHT); inactivating mutations in 5αR2 lead to disorders of sexual development. Due to the ability of the 5αRs to generate 5αDHT, they are an established drug target, and 5αR inhibitors are widely used for the treatment of androgen-dependent benign or malignant prostatic diseases. There is an emerging body of evidence to suggest that the 5-reductases can impact upon aspects of health and disease (other than urogenital development); alterations in their expression and activity have been associated with metabolic disease, polycystic ovarian syndrome, inflammation and bone metabolism. This review will outline the evidence base for the extra-urogenital role of 5-reductases from in vitro cell systems, pre-clinical models and human studies, and highlight the potential adverse effects of 5αR inhibition in human health and disease.
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3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Inibidores de 5-alfa Redutase/uso terapêutico , Doenças Metabólicas/genética , Esteroides/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Androgênios/metabolismo , Animais , Humanos , Doenças Metabólicas/enzimologia , Doenças Metabólicas/metabolismo , Especificidade por Substrato , Testosterona/metabolismoRESUMO
AIMS: The present observational cohort study evaluated the association between the AKR1D1*36 (rs1872930) allele and the risk of major adverse cardiovascular and cerebrovascular events (MACCE) in clopidogrel treated patients. METHODS: We screened 198 consecutive cardiovascular patients on clopidogrel therapy admitted in October to November 2010 with cardiovascular or cerebrovascular symptoms; of these 118 met the study protocol entry criteria; the median age of the cohort was 62.5 years (IQR 57-66 years), and 55% were females. RESULTS: The median follow up time was 38.5 (IQR 24-48) months; Kaplan-Meier/Log-rank analysis showed that patients carrying the AKR1D1*36 allelic variant have a shorter event-free-survival compared to wild type patients, hazard ratio = 2.193 (95% CI, 1.091 to 4.406); p = 0.0155. Multivariable Cox regression analysis confirmed the AKR1D1*36 allele as an independent risk factor (HR = 2.36; 95% CI, 1.34 to 4.18) and identified 3 other risk factors for MACCE; previous percutaneous interventions (PCI), HR = 2.78; (95% CI, 1.34 to 5.78), and a history of myocardial infarction, HR = 2.62; (95% CI, 1.48 to 4.64) at baseline and the previously reported CYP2C19*2 polymorphism (HR = 2.33; 95% CI, 1.33 to 4.06). CONCLUSION: The AKR1D1*36 (rs1872930) variant is independently associated with a higher risk for MACCE and shorter event-free survival time.
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Steroid hormones, including glucocorticoids and androgens, have potent actions to regulate many cellular processes within the liver. The steroid A-ring reductase, 5ß-reductase (AKR1D1), is predominantly expressed in the liver, where it inactivates steroid hormones and, in addition, plays a crucial role in bile acid synthesis. However, the precise functional role of AKR1D1 to regulate steroid hormone action in vitro has not been demonstrated. We have therefore hypothesised that genetic manipulation of AKR1D1 has the potential to regulate glucocorticoid availability and action in human hepatocytes. In both liver (HepG2) and non-liver cell (HEK293) lines, AKR1D1 over-expression increased glucocorticoid clearance with a concomitant decrease in the activation of the glucocorticoid receptor and the down-stream expression of glucocorticoid target genes. Conversely, knockdown of AKR1D1 using siRNA decreased glucocorticoid clearance and reduced the generation of 5ß-reduced metabolites. In addition, the two 5α-reductase inhibitors finasteride and dutasteride failed to effectively inhibit AKR1D1 activity in either cell-free or hepatocellular systems. Through manipulation of AKR1D1 expression and activity, we have demonstrated its potent ability to regulate glucocorticoid availability and receptor activation within human hepatoma cells. These data suggest that AKR1D1 may have an important role in regulating endogenous (and potentially exogenous) glucocorticoid action that may be of particular relevance to physiological and pathophysiological processes affecting the liver.
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Carcinoma Hepatocelular/metabolismo , Glucocorticoides/metabolismo , Neoplasias Hepáticas/metabolismo , Oxirredutases/metabolismo , Receptores de Glucocorticoides/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Fígado/metabolismoRESUMO
The relative contribution of CYP2C9 allelic variants to the pharmacokinetics (PK) of ibuprofen (IBP) enantiomers has been studied extensively, but the potential clinical benefit of pharmacogenetically guided IBP treatment is not evident yet. The role of AKR1D1*36C>T (rs 1872930) allelic variant in interindividual variability of CYP450 mediated drug metabolism was recently elucidated. A total of 27 healthy male subjects, volunteers in IBP single-dose two-way cross-over bioequivalence studies were genotyped for CYP2C9*2, CYP2C9*3 and AKR1D1*36 polymorphisms. The correlation between CYP2C9 and AKR1D1 genetic profile and the PK parameters for S-(+) and R-(-)-IBP was evaluated. Remarkable changes in the PK values pointing to reduced CYP2C9 enzyme activity were detected only in the CYP2C9*2 allelic variant carriers. Statistically significant association between the AKR1D1*36 allele and the increased IBP metabolism (low AUC0-t and 0-∞, high Cltot and short tmax values for both enantiomers) was observed in subjects carrying the CYP2C9 *1/*3 or CYP2C9*1/*1 genotype. The clinical value of concomitant CYP2C9 and AKR1D1 genotyping has to be further verified.
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Citocromo P-450 CYP2C9/genética , Ibuprofeno/farmacocinética , Oxirredutases/genética , Polimorfismo Genético/genética , Adolescente , Adulto , Alelos , Estudos Cross-Over , Genótipo , Voluntários Saudáveis , Humanos , Masculino , Taxa de Depuração Metabólica/genética , Pessoa de Meia-Idade , Projetos Piloto , Estereoisomerismo , Adulto JovemRESUMO
BACKGROUND: Oral cholic acid (CA) replacement has been shown to be an effective therapy in children with primary bile acid synthesis defects, which are rare and severe genetic liver diseases. To date there has been no report of the effects of this therapy in children reaching adulthood. The aim of the study was to evaluate the long-term effectiveness and safety of CA therapy. METHODS: Fifteen patients with either 3ß-hydroxy-Δ5-C27-steroid oxidoreductase (3ß-HSD) (n = 13) or Δ4-3-oxosteroid 5ß-reductase (Δ4-3-oxo-R) (n = 2) deficiency confirmed by mass spectrometry and gene sequencing received oral CA and were followed prospectively. RESULTS: The median age at last follow-up and the median time of follow-up with treatment were 24.3 years (range: 15.3-37.2) and 21.4 years (range: 14.6-24.1), respectively. At last evaluation, physical examination findings and blood laboratory test results were normal in all patients. Liver sonograms were normal in most patients. Mean daily CA dose was 6.9 mg/kg of body weight. Mass spectrometry analysis of urine showed that excretion of the atypical metabolites remained low or traces in amount with CA therapy. Liver fibrosis scored in liver biopsies or assessed by elastography in 14 patients, after 10 to 24 years with CA therapy, showed a marked improvement with disappearance of cirrhosis (median score < F1; range: F0-F2). CA was well tolerated in all patients, including five women having 10 uneventful pregnancies during treatment. CONCLUSIONS: Oral CA therapy is a safe and effective long-term treatment of 3ß-HSD and Δ4-3-oxo-R deficiencies and allows affected children to reach adulthood in good health condition without the need for a liver transplantation.
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3-Oxo-5-alfa-Esteroide 4-Desidrogenase/deficiência , Hiperplasia Suprarrenal Congênita/tratamento farmacológico , Ácidos e Sais Biliares/biossíntese , Ácido Cólico/uso terapêutico , Adolescente , Adulto , Ácido Cólico/administração & dosagem , Esquema de Medicação , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Aldo-keto reductase family 1 member D1 (AKR1D1) is a Δ4-3-oxosteroid 5ß-reductase required for bile acid synthesis and steroid hormone metabolism. Both bile acids and steroid hormones, especially glucocorticoids, play important roles in regulating body metabolism and energy expenditure. Currently, our understanding on AKR1D1 regulation and its roles in metabolic diseases is limited. We found that AKR1D1 expression was markedly repressed in diabetic patients. Consistent with repressed AKR1D1 expression, hepatic bile acids were significantly reduced in diabetic patients. Mechanistic studies showed that activation of peroxisome proliferator-activated receptor-α (PPARα) transcriptionally down-regulated AKR1D1 expression in vitro in HepG2 cells and in vivo in mice. Consistently, PPARα signaling was enhanced in diabetic patients. In summary, dysregulation of AKR1D1 disrupted bile acid and steroid hormone homeostasis, which may contribute to the pathogenesis of diabetes. Restoring bile acid and steroid hormone homeostasis by modulating AKR1D1 expression may represent a new approach to develop therapies for diabetes.
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Diabetes Mellitus/enzimologia , Oxirredutases/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Animais , Ácidos e Sais Biliares/metabolismo , Estudos de Casos e Controles , Ácido Quenodesoxicólico/metabolismo , Colesterol 7-alfa-Hidroxilase/metabolismo , Diabetes Mellitus/patologia , Feminino , Células Hep G2 , Homeostase , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Oxirredutases/genética , PPAR alfa/metabolismo , Regiões Promotoras Genéticas/genética , Transdução de SinaisRESUMO
OBJECTIVE: Gut microbiota may promote positive energy balance; however, germfree mice can be either resistant or susceptible to diet-induced obesity (DIO) depending on the type of dietary intervention. We here sought to identify the dietary constituents that determine the susceptibility to body fat accretion in germfree (GF) mice. METHODS: GF and specific pathogen free (SPF) male C57BL/6N mice were fed high-fat diets either based on lard or palm oil for 4 wks. Mice were metabolically characterized at the end of the feeding trial. FT-ICR-MS and UPLC-TOF-MS were used for cecal as well as hepatic metabolite profiling and cecal bile acids quantification, respectively. Hepatic gene expression was examined by qRT-PCR and cecal gut microbiota of SPF mice was analyzed by high-throughput 16S rRNA gene sequencing. RESULTS: GF mice, but not SPF mice, were completely DIO resistant when fed a cholesterol-rich lard-based high-fat diet, whereas on a cholesterol-free palm oil-based high-fat diet, DIO was independent of gut microbiota. In GF lard-fed mice, DIO resistance was conveyed by increased energy expenditure, preferential carbohydrate oxidation, and increased fecal fat and energy excretion. Cecal metabolite profiling revealed a shift in bile acid and steroid metabolites in these lean mice, with a significant rise in 17ß-estradiol, which is known to stimulate energy expenditure and interfere with bile acid metabolism. Decreased cecal bile acid levels were associated with decreased hepatic expression of genes involved in bile acid synthesis. These metabolic adaptations were largely attenuated in GF mice fed the palm-oil based high-fat diet. We propose that an interaction of gut microbiota and cholesterol metabolism is essential for fat accretion in normal SPF mice fed cholesterol-rich lard as the main dietary fat source. This is supported by a positive correlation between bile acid levels and specific bacteria of the order Clostridiales (phylum Firmicutes) as a characteristic feature of normal SPF mice fed lard. CONCLUSIONS: In conclusion, our study identified dietary cholesterol as a candidate ingredient affecting the crosstalk between gut microbiota and host metabolism.