RESUMEN

The adipose organ comprises two main fat depots termed white and brown adipose tissues. Adipogenesis is a process leading to newly differentiated adipocytes starting from precursor cells, which requires the contribution of many cellular activities at the genome, transcriptome, proteome, and metabolome levels. The adipogenic program is accomplished through two sequential phases; the first includes events favoring the commitment of adipose tissue stem cells/precursors to preadipocytes, while the second involves mechanisms that allow the achievement of full adipocyte differentiation. While there is a very large literature about the mechanisms involved in terminal adipogenesis, little is known about the first stage of this process. Growing interest in this field is due to the recent identification of adipose tissue precursors, which include a heterogenous cell population within different types of adipose tissue as well as within the same fat depot. In addition, the alteration of the heterogeneity of adipose tissue stem cells and of the mechanisms involved in their commitment have been linked to adipose tissue development defects and hence to the onset/progression of metabolic diseases, such as obesity. For this reason, the characterization of early adipogenic events is crucial to understand the etiology and the evolution of adipogenesis-related pathologies, and to explore the adipose tissue precursors' potential as future tools for precision medicine.

Asunto(s)

Adipocitos Blancos/citología , Adipogénesis , Diferenciación Celular , Obesidad/fisiopatología , Termogénesis , Animales , Humanos

RESUMEN

BACKGROUND/OBJECTIVES: In the last decade, a strict link between epigenetics and metabolism has been demonstrated. Histone deacetylases (HDACs) have emerged as key epigenetic regulators involved in metabolic homeostasis in normal and pathologic conditions. Here we investigated the effect of the class I HDAC inhibitor MS-275 in a model of obesity induced by a high-fat diet (HFD). METHODS: C57BL6/J male mice were fed HFD for 17 weeks and then randomized in two groups, treated intraperitoneally with vehicle dimethylsulfoxide (DMSO) or with the class I selective HDAC inhibitor MS-275 every other day for 22 days. Glucose tolerance test and measurement of body temperature during cold exposure were performed. Adipose tissues and liver were phenotypically characterized through histological analysis. Gene and protein expression analysis of brown and white adipose tissues (WATs) were performed. RESULTS: MS-275 treated mice showed 10% reduction of body weight, lower adipocyte size and improved glucose tolerance. Inhibition of class I HDAC determined reduction of adipocyte size and of fat mass, paralleled by higher expression of adipose functionality markers and by increased rate of lipolysis and fatty acid ß-oxidation. MS-275 also promoted thermogenic capacity, related to 'browning' of visceral and subcutaneous WAT, showing increased expression of uncoupling protein 1. In brown adipose tissue, we observed limited effects on gene expression and only reduction of brown adipocyte size. CONCLUSIONS: This study provides evidence that class I HDAC inhibition stimulated functionality and oxidative potential of adipose tissue, improving glucose tolerance and ameliorating the metabolic profile in diet-induced obese mice.

Asunto(s)

Adipocitos Marrones/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Blanco/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Inhibidores de Histona Desacetilasas/farmacología , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Benzamidas/farmacología , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Histona Desacetilasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/enzimología , Piridinas/farmacología , Termogénesis/efectos de los fármacos , Termogénesis/genética

RESUMEN

Cholesterol is required in the brain for synaptogenesis and its turnover is critical for cerebral functions. Several proteins involved in cholesterol handling and metabolism are transcriptionally regulated by the nuclear liver X receptor (LXR) alpha and beta. Sterol 27-hydroxylase (CYP27) is a ubiquitously expressed enzyme involved in cholesterol metabolism. Notably, its deficiency causes a disease characterized by progressive neurologic impairment. With the final goal to understand the pathophysiological role of CYP27A1 in the CNS, we studied the expression pattern of Cyp27a1 and other related genes in primary cultures of rat glia and neurons. Secondly, given the pivotal role of LXR in the regulation of cholesterol homeostasis, we investigated the effects of its activation on the expression of Cyp27a1.We found that primary astrocytes express different sterol hydroxylases and are able to uptake exogenous 27-hydroxycholesterol. We found that both microglia and astrocytes express preferentially Lxrbeta. However, despite this similarity, we observed cell-specific responsiveness of known and novel (including Cyp27a1) target genes to LXR activation. The increase of mRNA and protein levels in treated astrocytes is paralleled by transactivation of the proximal Cyp27a1 promoter in transfected astrocytes. We suggest that the astrocyte-restricted up-regulation of Cyp27a1 may be ascribable to differential expression of transcriptional co-activators. Given the role of astrocytes in maintaining brain homeostasis, we hypothesize that impairment of CYP27 activity in these cells may alter critical features of the astrocytes, from the handling and delivery of cholesterol to neurons to the release of signaling molecules.

Asunto(s)

Encéfalo/metabolismo , Colestanotriol 26-Monooxigenasa/metabolismo , Microglía/metabolismo , Neuroglía/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Animales , Encéfalo/enzimología , Células Cultivadas , Colestanotriol 26-Monooxigenasa/genética , Colesterol/metabolismo , Regulación de la Expresión Génica , Homeostasis/genética , Hidroxicolesteroles/metabolismo , Receptores X del Hígado , Microglía/enzimología , Neuroglía/enzimología , Neuronas/enzimología , Neuronas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Esteroide Hidroxilasas/metabolismo , Transcripción Genética

RESUMEN

The field of bile acids has witnessed an impulse in the last two decades. This has been the result of cloning the genes encoding enzymes of bile acid synthesis and their transporters. There is no doubt that the identification of Farnesoid X Receptor (FXR, NR1H4) as the bile acid receptor has contributed substantially to attract the interest of scientists in this area. When FXR was cloned by Forman et al. [1], farnesol metabolites were initially considered the physiological ligands. After identifying FXR and other nuclear receptors as bile acid sensors [2-4], it has become clear that bile acids are involved in the regulation of lipid and glucose metabolism and that these molecules are eclectic regulators of diverse cellular functions. In this review, we will summarize the current knowledge of the functions regulated by bile acids and how their physiological receptors mediate the signaling underlying numerous cellular responses.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Ácidos y Sales Biliares , Glucosa/metabolismo , Humanos , Metabolismo de los Lípidos , Transducción de Señal

RESUMEN

BACKGROUND: Recent data highlighted the role of nuclear receptors in the transcriptional regulation of the limiting enzyme of bile acid synthesis, cholesterol 7alpha-hydroxylase, in cellular and animal models. This study was designed to analyze the effects of age on cholesterol 7alpha-hydroxylase and related nuclear receptor expression in human livers. DESIGN: Surgical liver biopsies were obtained in 23 patients requiring operation on the gastrointestinal tract. mRNA levels of cholesterol 7alpha-hydroxylase and related nuclear receptors and co-activators were assayed by quantitative real-time RT-PCR. Serum levels of 7alpha-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis, were assayed by gas-liquid chromatography:mass spectrometry. RESULTS: Ageing was inversely correlated with serum 7alpha-hydroxy-4-cholesten-3-one and with cholesterol 7alpha-hydroxylase mRNA levels (r = -0.44 and r = -0.45 on a semi-log scale, respectively, P < 0.05). Among different nuclear factors, cholesterol 7alpha-hydroxylase mRNA best correlated with hepatocyte nuclear factor-4 (r = 0.55 on a log scale, P < 0.05); hepatocyte nuclear factor-4 levels were also inversely correlated with age (r = -0.64 on a semi-log scale, P < 0.05). Age was inversely correlated with serum insulin-like growth factor-I levels, which were directly correlated with hepatocyte nuclear factor-4 and cholesterol 7alpha-hydroxylase expression. No suppressive effect of short heterodimer partner expression on cholesterol 7alpha-hydroxylase was observed. CONCLUSIONS: Ageing associates with reduced bile acid synthesis, possibly related to decreased hepatic expression of hepatocyte nuclear factor-4 and consequently of cholesterol 7alpha-hydroxylase. Age-related modifications of the growth hormone/insulin-like growth factor axis might play a role. These findings may help to elucidate the pathophysiology of age-related modifications of cholesterol metabolism.

Asunto(s)

Envejecimiento/metabolismo , Ácidos y Sales Biliares/biosíntesis , Colesterol 7-alfa-Hidroxilasa/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Hígado/enzimología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Factor Nuclear 4 del Hepatocito/análisis , Humanos , Lipogénesis , Masculino , Persona de Mediana Edad , Receptores Citoplasmáticos y Nucleares

RESUMEN

BACKGROUND: Cholesterol cholelithiasis (gallstone disease) is a common disease in the Western world. The aim of the present study was to analyze the hepatic expression of a number of nuclear receptors involved in bile acid metabolism in human cholesterol gallstone disease. MATERIALS AND METHODS: Surgical liver biopsies were obtained from 11 patients with untreated cholesterol cholelithiasis and nine gallstone-free subjects; mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) and related nuclear receptors and coactivators were assayed by quantitative real-time RT-PCR. RESULTS: No differences between the two groups were detected in mRNA levels of CYP7A1 and related nuclear receptors, with the exception of peroxysome proliferator-activated receptor-gamma coactivator 1 (PGC-1), which was significantly (P < 0.01) less expressed in gallstone subjects. Expression of PGC-1 was linearly correlated with farnesoid X receptor (FXR) in gallstone patients (r = 0.87 on a log scale, P < 0.01), but not in control subjects; in gallstone patients PGC-1 expression was also correlated with hepatocyte nuclear factor 4 (HNF-4) (r = 0.78, P < 0.01). CONCLUSION: These findings suggest that PGC-1 can play a role in the prevention of cholesterol gallstone disease in humans; this might take place via interaction with the bile acid receptor FXR, whose protective role in cholelithiasis has been suggested by recent evidence in animal models and other coactivators. The present data might help to understand the pathophysiology and possibly focus on new therapeutical targets in cholesterol gallstone disease.

Asunto(s)

Colelitiasis/metabolismo , Hígado/química , Factores de Transcripción/análisis , Ácidos y Sales Biliares/biosíntesis , Biomarcadores/sangre , Colestenonas/sangre , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilasa/análisis , Colesterol 7-alfa-Hidroxilasa/genética , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Femenino , Regulación de la Expresión Génica/genética , Factor Nuclear 4 del Hepatocito/análisis , Factor Nuclear 4 del Hepatocito/genética , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares , Factores de Transcripción/genética

RESUMEN

Cholesterol homoeostasis is the result of the fine tuning between intake and disposal of this molecule. High levels of cholesterol in the blood are detrimental as they may lead to excessive accumulation in vessel walls, a condition predisposing to the development of atherosclerotic lesions. Cholesterol is removed from the vessel wall and transported to the liver through a process called reverse cholesterol transport. Nuclear receptors are among the most important transcription factors regulating genes involved in different steps of reverse cholesterol transport. Here, we discuss the role of the nuclear receptors LXR (liver X receptor) and HNF-4alpha (hepatocyte nuclear factor-4alpha) in different steps of reverse cholesterol transport. LXR controls the transcription of crucial genes in cholesterol efflux from macrophages and its transport to the liver, such as ABCA1 (ATP binding cassette A1), CYP27A1 (sterol 27-hydroxylase), CLA-1 (scavenger receptor type B1) and apolipoprotein E. Some oxysterols present in oxidized low-density lipoproteins and proinflammatory cytokines modulate the activity of LXR by antagonizing the effect of activators of this receptor, thus contributing to cholesterol accumulation in macrophages. Bile acid synthesis, which represents the final step of reverse cholesterol transport, is transcriptionally regulated by several nuclear receptors at the level of the liver-specific cytochrome P450 cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme of this metabolic pathway. Bile acids returning to the liver through the enterohepatic circulation down-regulate CYP7A1 transcription via the bile acid sensors farnesoid X receptor and HNF-4alpha. Based on this evidence, these nuclear receptors are candidate targets of new drugs for the treatment and prevention of atherosclerotic disease.

Asunto(s)

Colesterol/metabolismo , Proteínas de Unión al ADN/metabolismo , Hígado/metabolismo , Fosfoproteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Transporte Biológico , Factor Nuclear 4 del Hepatocito , Humanos

RESUMEN

Regulation of bile acid synthesis, a key determinant of cholesterol homeostasis, is still incompletely understood. To elucidate the feedback control exerted on bile acid biosynthesis in humans with obstructive cholestasis, 16 patients with bile duct obstruction were studied. In vivo 7alpha-hydroxylation, reflecting bile acid synthesis, was assayed in 13 of them by tritium release analysis. Serum 27-hydroxycholesterol was determined by gas chromatography-mass spectrometry. In a subgroup, hepatic cholesterol 7alpha-hydroxylase mRNA was assayed by real-time polymerase chain reaction (PCR), enzyme activity was determined by isotope incorporation, and microsomal cholesterol content was assayed by gas chromatography-mass spectrometry. Age-matched control subjects were studied in parallel. Hydroxylation rates were lower in cholestatic patients (108 +/- 33 mg of cholesterol per day, mean +/- SEM; controls: 297 +/- 40 mg/d; P <.01). The reduction was proportional to the severity of cholestasis, and synthetic rates were normalized in 4 subjects restudied after resolution of biliary obstruction. Consistent findings were obtained by analysis of serum 7alpha-hydroxycholesterol levels. On the other hand, hepatic cholesterol 7alpha-hydroxylase mRNA, microsomal enzyme activity, and cholesterol content tended to be increased in cholestasis. Finally, serum 27-hydroxycholesterol levels were slightly reduced in cholestatic subjects and were not related with the severity of the disease. Suppression of in vivo bile acid synthesis with no corresponding reduction in tissue 7alpha-hydroxylase expression and activity is consistent with nontranscriptional, posttranslational levels of regulation; these may play a role in the feedback control of bile acid synthesis in particular conditions. Alteration of the alternate biosynthetic pathway seems unlikely according to the present data.

Asunto(s)

Ácidos y Sales Biliares/antagonistas & inhibidores , Colestasis/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Hígado/enzimología , Anciano , Anciano de 80 o más Años , Ácidos y Sales Biliares/biosíntesis , Colestasis/fisiopatología , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilasa/genética , Femenino , Humanos , Hidroxicolesteroles/sangre , Hidroxilación , Hígado/metabolismo , Masculino , Microsomas Hepáticos/enzimología , Persona de Mediana Edad , ARN Mensajero/metabolismo , Valores de Referencia , Índice de Severidad de la Enfermedad

RESUMEN

Bile acids regulate the cholesterol 7alpha-hydroxylase gene (CYP7A1), which encodes the rate-limiting enzyme in the classical pathway of bile acid synthesis. Here we report a novel mechanism whereby bile acid feedback regulates CYP7A1 transcription through the nuclear receptor hepatocyte nuclear factor-4 (HNF-4), which binds to the bile acid response element (BARE) at nt -149/-118 relative to the transcription start site. Using transient transfection assays of HepG2 cells with Gal4-HNF-4 fusion proteins, we show that chenodeoxycholic acid (CDCA) dampened the transactivation potential of HNF-4. Overexpression of a constitutive active form of MEKK1, an upstream mitogen-activated protein kinase (MAPK) module triggered by stress signals, strongly repressed the promoter activity of CYP7A1 via the consensus sequence for HNF-4 embedded in the BARE. Similarly, MEKK1 inhibited the activity of HNF-4 in the Gal4-based assay. The involvement of the MEKK1-dependent pathway in the bile acid-mediated repression of CYP7A1 was confirmed by co-transfecting a dominant negative form of the stress-activated protein kinase kinase, SEK, which abolished the effect of CDCA upon CYP7A1 transcription. Treatment of transfected HepG2 cells with tumor necrosis factor alpha (TNF-alpha), an activator of the MEKK1 pathway, led to the repression of CYP7A1 via the HNF-4 site in the BARE. TNF-alpha also inhibited the transactivation potential of HNF-4. Collectively, our results demonstrate for the first time that HNF-4, in combination with a MAPK signaling pathway, acts as a bile acid sensor in the liver. Furthermore, the effects of CDCA and TNF-alpha converge to HNF-4, which binds to the BARE of CYP7A1, suggesting a link between the cascades elicited by bile acids and pro-inflammatory stimuli in the liver.

Asunto(s)

Ácidos y Sales Biliares/farmacología , Colesterol 7-alfa-Hidroxilasa/genética , Proteínas de Unión al ADN , Quinasa 1 de Quinasa de Quinasa MAP , Fosfoproteínas/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Ácidos y Sales Biliares/biosíntesis , Células Cultivadas , Retroalimentación , Factor Nuclear 4 del Hepatocito , Humanos , Proteínas Serina-Treonina Quinasas/fisiología , Elementos de Respuesta , Activación Transcripcional

RESUMEN

Data currently available are insufficient to demonstrate a real utility for CA 15-3 in the diagnosis, staging or surveillance of breast cancer patients following primary treatment. The aim of this study was to determine if there was a correlation between supranormal CA 15-3 serum levels and clinical and biological variables in breast cancer patients at first disease relapse. From October 1988 to March 1998, 430 consecutive patients entered the study. Overall CA 15-3 sensitivity was 60.7%. Elevated CA 15-3 levels were found more frequently in patients with liver metastases (74.6%) and in those with pleural effusion (75.7%). CA 15-3 sensitivity was 70.4% in patients with estrogen-receptor-positive (ER+) primary tumors and 45.9% in those with estrogen-receptor-negative (ER-) tumors (p < 0.0001). In patients with a limited extent of disease, marker sensitivity was 57.7% in ER+ tumors and 25.7% in ER- tumors (p < 0.0001). Logistic regression analysis showed ER status, disease extent and pleural effusion as independent variables associated with CA 15-3 positivity. The multivariate Cox analysis showed ER and disease extent as independent variables predicting overall survival, whereas CA 15-3 failed to be statistically significant. CA 15-3 was an independent variable only when the disease extent variable was removed. This study suggests that CA 15-3 in advanced breast cancer patients is a marker of both disease extent and ER status. The direct relationship with ER status indicates that CA 15-3 diagnostic sensitivity in the early detection of disease recurrence could be greater in ER+ patients than in ER- ones. Furthermore, this suggests that patients with elevated CA 15-3 levels could have disease that is more sensitive to hormone manipulation than those with normal CA 15-3 values.

Asunto(s)

Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Mucina-1/sangre , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Femenino , Humanos , Persona de Mediana Edad , Análisis Multivariante , Valor Predictivo de las Pruebas , Pronóstico , Recurrencia

RESUMEN

Bile acid biosynthesis occurs primarily through a pathway initiated by the 7alpha-hydroxylation of cholesterol, catalysed by cholesterol 7alpha-hydroxylase (encoded by CYP7A1). Insulin down-regulates CYP7A1 transcription. The aim of our study was to characterize the sequences of hamster CYP7A1 promoter, mediating the response to insulin. We therefore performed transient transfection assays with CYP7A1 promoter/luciferase chimaeras mutated at putative response elements and studied protein-DNA interactions by means of gel electrophoresis mobility-shift assay. Here we show that two sequences confer insulin responsiveness on hamster CYP7A1 promoter: a canonical insulin response sequence TGTTTTG overlapping a binding site for hepatocyte nuclear factor 3 (HNF-3) (at nt -235 to -224) and a binding site for HNF-4 at nt -203 to -191. In particular we show that the hamster CYP7A1 insulin response sequence is part of a complex unit involved in specific interactions with multiple transcription factors such as members of the HNF-3 family; this region does not bind very strongly to HNF-3 and as a consequence partly contributes to the transactivation of the gene. Another sequence located at nt -138 to -128 binds to HNF-3 and is involved in the tissue-specific regulation of hamster CYP7A1. The sequence at nt -203 to -191 is not only essential for insulin effect but also has a major role in the liver-specific expression of CYP7A1; it is the target of HNF-4. Therefore the binding sites for liver-enriched factors, present in the hamster CYP7A1 proximal promoter in close vicinity and conserved between species, constitute a regulatory unit important for basal hepatic expression and tissue restriction of the action of hormones such as insulin.

Asunto(s)

Colesterol 7-alfa-Hidroxilasa/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Regiones Promotoras Genéticas/genética , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Cricetinae , Proteínas de Unión al ADN/metabolismo , Factor Nuclear 3-alfa del Hepatocito , Factor Nuclear 4 del Hepatocito , Humanos , Luciferasas/genética , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , TATA Box , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas

RESUMEN

The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by accumulation of cholesta-5,7-dien-3 beta-0l caused by a deficiency of the enzyme desaturating this sterol to cholesterol. In addition to other unusual sterols recently found in plasma of patients with SLOS, namely cholesta-5,8-dien-3 beta-ol and 19-nor-cholesta-5,7,9 (10)-trien-3 beta-ol we have detected a trienol and we describe here its identification as cholesta-5,7,9 (11)-trien-3 beta-ol by GC-MS and by comparison with a synthetic standard. We tested the possibility that the trienol may be formed by radical oxidation of cholesta-5,7-dien-3 beta-ol accumulated in plasma of patients with SLOS because it is known to be formed by decomposition of 7-hydroperoxy-cholesta-5,8-dien-3 beta-ol, which is a product of cholesta-5,7-dien-3 beta-ol photooxidation. Incubation of cholesta-5,7-dien-3 beta-ol with rat liver microsomes in the presence of ADP/Fe2+ and NADPH gave rise to a number of oxygenated sterols. Among these, analysis by particle-beam LC-MS under CI conditions indicated the presence of 7-hydroperoxy-cholesta-5,8-dien-3 beta-ol and of cholesta-5,7,9(11)-trien-3 beta-ol which is known to derive from the oxidation of the 7-hydroperoxide. From these results we conclude that cholesta-5,7-dien-3 beta-ol accumulated in tissues of patients with SLOS may be oxidized by oxygen radicals giving rise to oxygenated sterols. Some of these compounds may be toxic and may contribute to worsen the pathological picture in patients with SLOS.

Asunto(s)

Colestenos/sangre , Síndrome de Smith-Lemli-Opitz/sangre , Animales , Deshidrocolesteroles/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Microsomas Hepáticos/metabolismo , NADP/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Cholesterol 7 alpha-hydroxylase plays a crucial role in cholesterol homeostasis. We investigated the regulation of this enzyme in the hamster, a suitable animal model for studying cholesterol metabolism. DNase I hypersensitivity assay revealed the presence of a hypersensitive region in the proximal promoter. Both negative (bile acids, phorbol esters and insulin) and positive (glucocorticoid hormones) effects were mediated through sequences in the region 318 bp upstream of the ATG codon. All-trans-retinoic acid, cAMP, and LDL did not affect transcriptional activity. These findings show that the hamster cholesterol 7 alpha-hydroxylase gene undergoes a predominant negative regulation, as opposed to the rat CYP7A homologous gene.

Asunto(s)

Colesterol 7-alfa-Hidroxilasa/genética , Regulación Enzimológica de la Expresión Génica , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilasa/biosíntesis , Codón , Secuencia de Consenso , Cricetinae , AMP Cíclico/farmacología , Desoxirribonucleasa I , Dexametasona/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/farmacología , Lipoproteínas LDL/farmacología , Hígado/enzimología , Luciferasas/biosíntesis , Masculino , Mesocricetus , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Bazo/enzimología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Tretinoina/farmacología , Células Tumorales Cultivadas

RESUMEN

Smith-Lemli-Opitz syndrome (SLOS) is a multiple congenital anomalies/mental retardation disorder possibly due to a defect of delta 7-sterol reductase, leading to low plasma cholesterol levels and to the accumulation of 7-dehydrocholesterol (7-DHC) and other cholesterol precursors. This study aimed to identify clinical features that could potentially be specific indicators for the clinical diagnosis of SLOS, and to test the reliability of ultraviolet spectrophotometry (UVS) as a biochemical screening procedure for the syndrome. Twenty patients with clinical suspicion of SLOS, referred to 11 Italian paediatric and clinical genetic centres, were collected during 1994. In 10 patients the diagnosis was confirmed biochemically by gas chromatography/mass spectrometry (GC/MS) analysis of serum sterols, whereas in the other 10 patients the serum sterol profiles were normal. A comparison between confirmed SLOS patients and biochemically negative subjects did not show clinical signs specific for the syndrome. UVS measurement of 7-DHC correlated well with GC/MS profiles, showing 100% sensitivity and specificity. Four out of five patients had serum bile acid concentrations below the normal range of controls.

Asunto(s)

Deshidrocolesteroles/sangre , Síndrome de Smith-Lemli-Opitz/sangre , Síndrome de Smith-Lemli-Opitz/diagnóstico , Adolescente , Ácidos y Sales Biliares/sangre , Biomarcadores/sangre , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Sensibilidad y Especificidad , Espectrofotometría Ultravioleta

RESUMEN

The purpose of this study was to characterize the lipoprotein profile and cholesterol metabolism in Yoshida rats, a strain of inbred genetically hyperlipemic animals. For comparison, Brown Norway rats were used as control animals. Plasma cholesterol and triglycerides were higher in Yoshida as compared to Brown Norway, the elevation of cholesterol being due to a rise in HDL fraction. Triglyceride distribution among lipoproteins showed an increase in VLDL fraction. Hyperlipemia was not related to diabetes, hypothyroidism or nephropathy. Plasma triglycerides production was increased in Yoshida rats, while lipoprotein and hepatic lipases were similar in the two groups. Hypercholesterolemia was associated with a defect of lipoprotein receptor activity and with elevated HMG-CoA reductase and cholesterol 7 alpha - hydroxylase; conversely ACAT activity was lower in Yoshida as compared to Brown Norway rats. Sterol fecal excretion was comparable in the two groups and hypercholesterolemia in Yoshida rats was not associated to an increase of cholesterol saturation of the bile. We suggest that lipoprotein overproduction is the main cause for hyperlipidemia in this strain of rats.

Asunto(s)

Colesterol/metabolismo , Hiperlipidemias/metabolismo , Lipoproteínas/sangre , Animales , Glucemia/metabolismo , Peso Corporal/fisiología , Colesterol/sangre , Modelos Animales de Enfermedad , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hiperlipidemias/sangre , Hiperlipidemias/enzimología , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/enzimología , Hiperlipoproteinemia Tipo II/metabolismo , Hígado/anatomía & histología , Hígado/enzimología , Hígado/metabolismo , Masculino , Tamaño de los Órganos/fisiología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas , Hormonas Tiroideas/sangre , Triglicéridos/sangre , Triglicéridos/metabolismo

RESUMEN

The effect of ursodeoxycholic acid analogues bearing modifications at the side-chain moiety of the molecule was tested on cholesterol 7 alpha-hydroxylase and HMG-CoA reductase in rat liver microsomes. The compounds included 23 R,S mixture and the single isomers 23R and 23S of 23 methylursodeoxycholic acid (23-methyl UDCA), the isomeric mixture (cis + trans) of 3 alpha,7 beta-dihydroxy-20,22-methylen-5 beta-cholan-23-oic acid (norcypro-UDCA) and the corresponding single isomers. Each steroid was added to liver microsomes as the sodium salt, at concentrations ranging from 25 to 200 microM. Isomers 23R and 23S of 23-methyl-UDCA inhibited cholesterol 7 alpha-hydroxylase in a concentration-dependent manner. The inhibitory capacity was similar for the two isomers. The extent of inhibition of the analogues was greater than that of the parent compound UDCA. Shortening of the side-chain in norcypro-UDCA resulted in a partial loss of the inhibitory effect, as compared to cypro-UDCA (3 alpha,7 beta-dihydroxy-22,23-methylen-5 beta-cholan-24-oic acid). None of these bile acid derivatives affected the activity of the enzyme HMG-CoA reductase.

Asunto(s)

Ácidos y Sales Biliares/farmacología , Colesterol 7-alfa-Hidroxilasa/antagonistas & inhibidores , Colesterol/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Microsomas Hepáticos/enzimología , Animales , Colesterol 7-alfa-Hidroxilasa/metabolismo , Regulación hacia Abajo , Cinética , Masculino , Ratas , Ratas Endogámicas , Ácido Ursodesoxicólico/análogos & derivados , Ácido Ursodesoxicólico/farmacología

RESUMEN

The effect of chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), tauroursodeoxycholic (TUDCA), cholic (CA), ursocholic (UCA) acids, analogues of CDCA and UDCA with a cyclopropyl ring at C22, C23 (cypro-CDCA and cypro-UDCA) and 23-methylursodeoxycholic acid (MUDCA) on cholesterol 7 alpha-hydroxylase was studied in rat liver microsomes. Cypro-analogues consisted of a mixture of four diasteroisomers, while MUDCA was the racemic mixture of two enantiomers. Each steroid was added to liver microsomes at concentrations ranging from 10 to 200 microM. With the exception of UCA and CA, all the bile acids inhibited cholesterol 7 alpha-hydroxylase activity. The inhibition shown by cypro-CDCA and cypro-UDCA was stronger than that observed with the corresponding natural compounds. 22S,23S cypro-UDCA exhibited an inhibitory effect which was more pronounced than that of the diasteroisomer mixture. The isomer 22R,23S was less effective and decreased cholesterol 7 alpha-hydroxylase activity in a manner comparable to that of UDCA. The effect of CDCA, UDCA and the cyclopropyl analogues was also tested with respect to HMG-CoA reductase and acylCoA cholesterol acyltransferase (ACAT) activities. ACAT was stimulated by the isomer 22S,23S cypro-UDCA but not affected by the other bile acids. No effect was observed as regards HMG-CoA reductase.

Asunto(s)

Ácidos y Sales Biliares/farmacología , Colesterol/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Animales , Ácidos y Sales Biliares/fisiología , Colesterol 7-alfa-Hidroxilasa/antagonistas & inhibidores , Hidroximetilglutaril-CoA Reductasas/metabolismo , Técnicas In Vitro , Masculino , Microsomas Hepáticos/enzimología , Ratas , Ratas Endogámicas , Esterol O-Aciltransferasa/metabolismo , Relación Estructura-Actividad

RESUMEN

Hamsters were given etofibrate at a dose of 300 mg/kg body wt, by gavage for 5 days, while being fed a chow diet. After treatment, serum cholesterol levels were 27% lower compared to those of the control animals. A similar trend was observed for triglyceride levels. Hepatic lipid levels were unchanged by the treatment. HMG-CoA reductase and acylCoA cholesterol acyltransferase were decreased while cholesterol 7 alpha-hydroxylase was not significantly modified by etofibrate. A choleretic effect and an increase of cholesterol excretion into hepatic bile was observed in treated animals. Nevertheless, composition and cholesterol saturation index of gallbladder bile were similar in control and treated animals. With respect to controls, hepatic bile of treated hamsters contained a lesser amount of cholic and deoxycholic acid and a greater amount of ursodeoxycholic acid.

Asunto(s)

Anticolesterolemiantes/farmacología , Ácidos y Sales Biliares/metabolismo , Colesterol/sangre , Clofibrato/análogos & derivados , Ácido Clofíbrico/análogos & derivados , Animales , Bilis/metabolismo , Peso Corporal/efectos de los fármacos , Ácido Clofíbrico/farmacología , Cricetinae , Vesícula Biliar/metabolismo , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Mesocricetus , Tamaño de los Órganos/efectos de los fármacos

RESUMEN

The kinetics of unchanged aminoglutethimide and of its major metabolite N-acetylaminoglutethimide were investigated in healthy volunteers with a new multiple selected ion monitoring (SIM) technique. This method allows a rapid detection of both the unchanged drug and of its metabolite with a single injection and after minimal handling of the samples. This rapid method provided similar kinetic findings, compared to those described with the more time consuming high pressure liquid chromatographic (HPLC) procedure. Moreover, the SIM method allowed the detection of the N-acetylamino metabolite in plasma at longer time intervals vs. the HPLC method. Some typical features of the kinetic behavior (e.g., a discontinuity in the plasma die-away curve for both unchanged drug and metabolite), attributable to partial liver extraction, could also be more clearly observed with the new procedure. This new, rapid technique confirms that aminoglutethimide and N-acetylaminoglutethimide have very similar plasma die-away curves in subjects with a normal conjugating capacity, and that kinetic patterns or individual blood levels can be readily obtained by SIM with minimal acquisition of supplementary equipment.

Asunto(s)

Aminoglutetimida/farmacocinética , Adulto , Aminoglutetimida/análogos & derivados , Aminoglutetimida/sangre , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Femenino , Humanos , Masculino

RESUMEN

1. Serum lipid and apolipoprotein levels, distribution and composition of high-density lipoprotein (HDL) subfractions and lecithin:cholesterol acryltransferase activity were analysed in nine normolipidaemic subjects, in whom a hypertriglyceridaemic state was induced by the acute administration of ethanol (40 g) plus fat (70 g) or of fat only. 2. Triglyceride (TG) levels increased by 180% 4-6 h after fat plus ethanol intake, the hypertriglyceridaemic response being inversely correlated with the basal HDL2 mass (r = -0.82). Serum apolipoprotein (apo) B levels rose by 8%, HDL-cholesterol decreased by 10% and HDL-TG increased by 57% at 6-8 h. 3. When ethanol was omitted, serum cholesterol and TG rose by 6% and 70%, respectively; both apo AI and apo B levels went up by 8%, whereas HDL-cholesterol rose progressively (15%) at 12 h. 4. The flotation rates of both HDL2 and HDL3 increased, reaching a maximum 6-8 h after ethanol plus fat intake. These changes were due to an increase in TG and phospholipid contents, whereas cholesteryl esters and proteins decreased. 5. The alterations in HDL are attributable to the increase in TG-rich lipoproteins, to the stimulated cholesterol esterification (+15%) and to an enhanced transfer of newly formed cholesteryl esters to apo-B-containing lipoproteins in exchange for TG. 6. Changes in HDL properties were evident only when ethanol was given concomitantly with fat. 7. These findings suggest that in the postprandial phase lipoprotein changes may occur, which facilitate an improved removal of cholesterol from tissues.

Asunto(s)

Grasas de la Dieta/administración & dosificación , Etanol/administración & dosificación , Hiperlipidemias/sangre , Lipoproteínas HDL/sangre , Adolescente , Adulto , Apolipoproteínas/sangre , Femenino , Humanos , Hiperlipidemias/etiología , Lipoproteínas HDL/clasificación , Masculino , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Triglicéridos/sangre