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1.
Am J Physiol Gastrointest Liver Physiol ; 319(3): G345-G360, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32755310

RESUMEN

The pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the progression to nonalcoholic steatohepatitis (NASH) and increased risk of hepatocellular carcinoma remain poorly understood. Additionally, there is increasing recognition of the extrahepatic manifestations associated with NAFLD and NASH. We demonstrate that intervention with the American lifestyle-induced obesity syndrome (ALIOS) diet in male and female mice recapitulates many of the clinical and transcriptomic features of human NAFLD and NASH. Male and female C57BL/6N mice were fed either normal chow (NC) or ALIOS from 11 to 52 wk and underwent comprehensive metabolic analysis throughout the duration of the study. From 26 wk, ALIOS-fed mice developed features of hepatic steatosis, inflammation, and fibrosis. ALIOS-fed mice also had an increased incidence of hepatic tumors at 52 wk compared with those fed NC. Hepatic transcriptomic analysis revealed alterations in multiple genes associated with inflammation and tissue repair in ALIOS-fed mice. Ingenuity Pathway Analysis confirmed dysregulation of metabolic pathways as well as those associated with liver disease and cancer. In parallel the development of a robust hepatic phenotype, ALIOS-fed mice displayed many of the extrahepatic manifestations of NAFLD, including hyperlipidemia, increased fat mass, sarcopenia, and insulin resistance. The ALIOS diet in mice recapitulates many of the clinical features of NAFLD and, therefore, represents a robust and reproducible model for investigating the pathogenesis of NAFLD and its progression.NEW & NOTEWORTHY Nonalcoholic fatty liver disease (NAFLD) affects 30% of the general population and can progress to nonalcoholic steatohepatitis (NASH) and potentially hepatocellular carcinoma. Preclinical models rely on mouse models that often display hepatic characteristics of NAFLD but rarely progress to NASH and seldom depict the multisystem effects of the disease. We have conducted comprehensive metabolic analysis of both male and female mice consuming a Western diet of trans fats and sugar, focusing on both their hepatic phenotype and extrahepatic manifestations.


Asunto(s)
Dieta Occidental/efectos adversos , Hígado Graso/genética , Estilo de Vida , Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad/metabolismo , Alimentación Animal , Animales , Composición Corporal , Hígado Graso/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Prueba de Tolerancia a la Glucosa , Resistencia a la Insulina , Lípidos/sangre , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Pruebas de Función Hepática , Neoplasias Hepáticas/epidemiología , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Síndrome
2.
Clin Endocrinol (Oxf) ; 91(1): 72-81, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30667079

RESUMEN

OBJECTIVE AND CONTEXT: Increasing adiposity, ageing and tissue-specific regeneration of cortisol through the activity of 11ß-hydroxysteroid dehydrogenase type 1 have been associated with deterioration in glucose tolerance. We undertook a longitudinal, prospective clinical study to determine if alterations in local glucocorticoid metabolism track with changes in glucose tolerance. DESIGN, PATIENTS, AND MEASUREMENTS: Sixty-five overweight/obese individuals (mean age 50.3 ± 7.3 years) underwent oral glucose tolerance testing, body composition assessment, subcutaneous adipose tissue biopsy and urinary steroid metabolite analysis annually for up to 5 years. Participants were categorized into those in whom glucose tolerance deteriorated ("deteriorators") or improved ("improvers"). RESULTS: Deteriorating glucose tolerance was associated with increasing total and trunk fat mass and increased subcutaneous adipose tissue expression of lipogenic genes. Subcutaneous adipose tissue 11ß-HSD1 gene expression decreased in deteriorators, and at study completion, it was highest in the improvers. There was a significant negative correlation between change in area under the curve glucose and 11ß-HSD1 expression. Global 11ß-HSD1 activity did not change and was not different between deteriorators and improvers at baseline or follow-up. CONCLUSION: Longitudinal deterioration in metabolic phenotype is not associated with increased 11ß-HSD1 activity, but decreased subcutaneous adipose tissue gene expression. These changes may represent a compensatory mechanism to decrease local glucocorticoid exposure in the face of an adverse metabolic phenotype.


Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Adiposidad/fisiología , Grasa Subcutánea/metabolismo , Adiposidad/genética , Corticoesteroides/metabolismo , Corticoesteroides/orina , Adulto , Femenino , Glucocorticoides/metabolismo , Glucocorticoides/orina , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa
3.
Proc Natl Acad Sci U S A ; 111(24): E2482-91, 2014 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-24889609

RESUMEN

The adverse metabolic effects of prescribed and endogenous glucocorticoid (GC) excess, Cushing syndrome, create a significant health burden. We found that tissue regeneration of GCs by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), rather than circulating delivery, is critical to developing the phenotype of GC excess; 11ß-HSD1 KO mice with circulating GC excess are protected from the glucose intolerance, hyperinsulinemia, hepatic steatosis, adiposity, hypertension, myopathy, and dermal atrophy of Cushing syndrome. Whereas liver-specific 11ß-HSD1 KO mice developed a full Cushingoid phenotype, adipose-specific 11ß-HSD1 KO mice were protected from hepatic steatosis and circulating fatty acid excess. These data challenge our current view of GC action, demonstrating 11ß-HSD1, particularly in adipose tissue, is key to the development of the adverse metabolic profile associated with circulating GC excess, offering 11ß-HSD1 inhibition as a previously unidentified approach to treat Cushing syndrome.


Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Tejido Adiposo/metabolismo , Síndrome de Cushing/sangre , Síndrome de Cushing/genética , Glucocorticoides/sangre , Hidrocortisona/sangre , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Animales , Antiinflamatorios/química , Presión Sanguínea , Modelos Animales de Enfermedad , Ácidos Grasos no Esterificados/sangre , Regulación de la Expresión Génica , Intolerancia a la Glucosa , Prueba de Tolerancia a la Glucosa , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regeneración/efectos de los fármacos , Triglicéridos/sangre
4.
J Hepatol ; 64(2): 399-408, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26394161

RESUMEN

BACKGROUND & AIMS: Insulin resistance and lipotoxicity are pathognomonic in non-alcoholic steatohepatitis (NASH). Glucagon-like peptide-1 (GLP-1) analogues are licensed for type 2 diabetes, but no prospective experimental data exists in NASH. This study determined the effect of a long-acting GLP-1 analogue, liraglutide, on organ-specific insulin sensitivity, hepatic lipid handling and adipose dysfunction in biopsy-proven NASH. METHODS: Fourteen patients were randomised to 1.8mg liraglutide or placebo for 12-weeks of the mechanistic component of a double-blind, randomised, placebo-controlled trial (ClinicalTrials.gov-NCT01237119). Patients underwent paired hyperinsulinaemic euglycaemic clamps, stable isotope tracers, adipose microdialysis and serum adipocytokine/metabolic profiling. In vitro isotope experiments on lipid flux were performed on primary human hepatocytes. RESULTS: Liraglutide reduced BMI (-1.9 vs. +0.04kg/m(2); p<0.001), HbA1c (-0.3 vs. +0.3%; p<0.01), cholesterol-LDL (-0.7 vs. +0.05mmol/L; p<0.01), ALT (-54 vs. -4.0IU/L; p<0.01) and serum leptin, adiponectin, and CCL-2 (all p<0.05). Liraglutide increased hepatic insulin sensitivity (-9.36 vs. -2.54% suppression of hepatic endogenous glucose production with low-dose insulin; p<0.05). Liraglutide increased adipose tissue insulin sensitivity enhancing the ability of insulin to suppress lipolysis both globally (-24.9 vs. +54.8pmol/L insulin required to ½ maximally suppress serum non-esterified fatty acids; p<0.05), and specifically within subcutaneous adipose tissue (p<0.05). In addition, liraglutide decreased hepatic de novo lipogenesis in vivo (-1.26 vs. +1.30%; p<0.05); a finding endorsed by the effect of GLP-1 receptor agonist on primary human hepatocytes (24.6% decrease in lipogenesis vs. untreated controls; p<0.01). CONCLUSIONS: Liraglutide reduces metabolic dysfunction, insulin resistance and lipotoxicity in the key metabolic organs in the pathogenesis of NASH. Liraglutide may offer the potential for a disease-modifying intervention in NASH.


Asunto(s)
Péptido 1 Similar al Glucagón/análogos & derivados , Metabolismo de los Lípidos/efectos de los fármacos , Liraglutida , Enfermedad del Hígado Graso no Alcohólico , Adulto , Anciano , Índice de Masa Corporal , Método Doble Ciego , Monitoreo de Drogas/métodos , Femenino , Técnica de Clampeo de la Glucosa/métodos , Hemoglobina Glucada/análisis , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Resistencia a la Insulina , Liraglutida/administración & dosificación , Liraglutida/farmacocinética , Hígado/metabolismo , Hígado/patología , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Resultado del Tratamiento
5.
Liver Int ; 36(11): 1704-1712, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27178444

RESUMEN

BACKGROUND AND AIMS: Alström syndrome (AS) is a recessive monogenic syndrome characterized by obesity, extreme insulin resistance and multi-organ fibrosis. Despite phenotypically being high risk of non-alcoholic fatty liver disease (NAFLD), there is a lack of data on the extent of fibrosis in the liver and its close links to adipose in patients with AS. Our aim was to characterize the hepatic and adipose phenotype in patients with AS. METHODS: Observational cohort study with comprehensive assessment of metabolic liver phenotype including liver elastography (Fibroscan® ), serum Enhanced Liver Fibrosis (ELF) Panel and liver histology. In addition, abdominal adipose histology and gene expression was assessed. We recruited 30 patients from the UK national AS clinic. A subset of six patients underwent adipose biopsies which was compared with control tissue from nine healthy participants. RESULTS: Patients were overweight/obese (BMI 29.3 (25.95-34.05) kg/m2 ). A total of 80% (24/30) were diabetic; 74% (20/27) had liver ultrasound scanning suggestive of NAFLD. As judged by the ELF panel, 96% (24/25) were categorized as having fibrosis and 10/21 (48%) had liver elastography consistent with advanced liver fibrosis/cirrhosis. In 7/8 selected cases, there was evidence of advanced NAFLD on liver histology. Adipose tissue histology showed marked fibrosis as well as disordered pro-inflammatory and fibrotic gene expression profiles. CONCLUSIONS: NAFLD and adipose dysfunction are common in patients with AS. The severity of liver disease in our cohort supports the need for screening of liver fibrosis in AS.


Asunto(s)
Tejido Adiposo/patología , Síndrome de Alstrom/complicaciones , Cirrosis Hepática/epidemiología , Enfermedad del Hígado Graso no Alcohólico/epidemiología , Adulto , Estudios de Cohortes , Femenino , Fibrosis , Expresión Génica , Humanos , Resistencia a la Insulina , Hígado/patología , Cirrosis Hepática/patología , Masculino , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/complicaciones , Reino Unido , Adulto Joven
6.
Am J Physiol Endocrinol Metab ; 305(9): E1134-44, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-24022868

RESUMEN

Glucocorticoids increase adipocyte proliferation and differentiation, a process underpinned by the local reactivation of inactive cortisone to active cortisol within adipocytes catalyzed by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). The adrenal sex steroid precursor dehydroepiandrosterone (DHEA) has been shown to inhibit 11ß-HSD1 in murine adipocytes; however, rodent adrenals do not produce DHEA physiologically. Here, we aimed to determine the effects and underlying mechanisms of the potential antiglucocorticoid action of DHEA and its sulfate ester DHEAS in human preadipocytes. Utilizing a human subcutaneous preadipocyte cell line, Chub-S7, we examined the metabolism and effects of DHEA in human adipocytes, including adipocyte proliferation, differentiation, 11ß-HSD1 expression, and activity and glucose uptake. DHEA, but not DHEAS, significantly inhibited preadipocyte proliferation via cell cycle arrest in the G1 phase independent of sex steroid and glucocorticoid receptor activation. 11ß-HSD1 oxoreductase activity in differentiated adipocytes was inhibited by DHEA. DHEA coincubated with cortisone significantly inhibited preadipocyte differentiation, which was assessed by the expression of markers of early (LPL) and terminal (G3PDH) adipocyte differentiation. Coincubation with cortisol, negating the requirement for 11ß-HSD1 oxoreductase activity, diminished the inhibitory effect of DHEA. Further consistent with glucocorticoid-opposing effects of DHEA, insulin-independent glucose uptake was significantly enhanced by DHEA treatment. DHEA increases basal glucose uptake and inhibits human preadipocyte proliferation and differentiation, thereby exerting an antiglucocorticoid action. DHEA inhibition of the amplification of glucocorticoid action mediated by 11ß-HSD1 contributes to the inhibitory effect of DHEA on human preadipocyte differentiation.


Asunto(s)
Adipocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Deshidroepiandrosterona/farmacología , Glucocorticoides/antagonistas & inhibidores , Glucosa/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Células Cultivadas , Colorimetría , Cartilla de ADN , Deshidroepiandrosterona/metabolismo , Humanos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Timidina/metabolismo
7.
J Endocrinol ; 255(3): 117-129, 2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36205523

RESUMEN

The aged phenotype shares several metabolic similarities with that of circulatory glucocorticoid excess (Cushing's syndrome), including type 2 diabetes, obesity, hypertension, and myopathy. We hypothesise that local tissue generation of glucocorticoids by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which converts 11-dehydrocorticosterone to active corticosterone in rodents (corticosterone to cortisol in man), plays a role in driving age-related chronic disease. In this study, we have examined the impact of ageing on glucocorticoid metabolism, insulin tolerance, adiposity, muscle strength, and blood pressure in both wildtype (WT) and transgenic male mice with a global deletion of 11ß-HSD1 (11ß-HSD1-/-) following 4 months high-fat feeding. We found that high fat-fed 11ß-HSD1-/- mice were protected from age-related glucose intolerance and hyperinsulinemia when compared to age/diet-matched WTs. By contrast, aged 11ß-HSD1-/- mice were not protected from the onset of sarcopenia observed in the aged WTs. Young 11ß-HSD1-/- mice were partially protected from diet-induced obesity; however, this partial protection was lost with age. Despite greater overall obesity, the aged 11ß-HSD1-/- animals stored fat in more metabolically safer adipose depots as compared to the aged WTs. Serum analysis revealed both WT and 11ß-HSD1-/- mice had an age-related increase in morning corticosterone. Surprisingly, 11ß-HSD1 oxo-reductase activity in the liver and skeletal muscle was unchanged with age in WT mice and decreased in gonadal adipose tissue. These data suggest that deletion of 11ß-HSD1 in high fat-fed, but not chow-fed, male mice protects from age-related insulin resistance and supports a metabolically favourable fat distribution.


Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1 , Diabetes Mellitus Tipo 2 , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Animales , Corticosterona/metabolismo , Glucocorticoides/metabolismo , Hidrocortisona , Insulina , Masculino , Ratones , Ratones Transgénicos , Obesidad/genética
8.
Metabol Open ; 14: 100177, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35313531

RESUMEN

Background and aims: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver condition. It is tightly associated with an adverse metabolic phenotype (including obesity and type 2 diabetes) as well as with obstructive sleep apnoea (OSA) of which intermittent hypoxia is a critical component. Hepatic de novo lipogenesis (DNL) is a significant contributor to hepatic lipid content and the pathogenesis of NAFLD and has been proposed as a key pathway to target in the development of pharmacotherapies to treat NAFLD. Our aim is to use experimental models to investigate the impact of hypoxia on hepatic lipid metabolism independent of obesity and metabolic disease. Methods: Human and rodent studies incorporating stable isotopes and hyperinsulinaemic euglycaemic clamp studies were performed to assess the regulation of DNL and broader metabolic phenotype by intermittent hypoxia. Cell-based studies, including pharmacological and genetic manipulation of hypoxia-inducible factors (HIF), were used to examine the underlying mechanisms. Results: Hepatic DNL increased in response to acute intermittent hypoxia in humans, without alteration in glucose production or disposal. These observations were endorsed in a prolonged model of intermittent hypoxia in rodents using stable isotopic assessment of lipid metabolism. Changes in DNL were paralleled by increases in hepatic gene expression of acetyl CoA carboxylase 1 and fatty acid synthase. In human hepatoma cell lines, hypoxia increased both DNL and fatty acid uptake through HIF-1α and -2α dependent mechanisms. Conclusions: These studies provide robust evidence linking intermittent hypoxia and the regulation of DNL in both acute and sustained in vivo models of intermittent hypoxia, providing an important mechanistic link between hypoxia and NAFLD.

9.
J Endocrinol ; 253(3): 97-113, 2022 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-35318963

RESUMEN

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.


Asunto(s)
Glucocorticoides , Oxidorreductasas , Animales , Ácidos y Sales Biliares , Dieta Alta en Grasa , Femenino , Glucocorticoides/metabolismo , Insulina/metabolismo , Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas/genética , Fenotipo
10.
J Mol Endocrinol ; 66(3): 181-194, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33502336

RESUMEN

Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5ß-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were overexpressed in HEK293 cells, and successful overexpression confirmed by qPCR and Western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following overexpression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action.


Asunto(s)
Empalme Alternativo/genética , Oxidorreductasas/genética , Secuencia de Aminoácidos , Andrógenos/metabolismo , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glucocorticoides/metabolismo , Células HEK293 , Humanos , Hígado/metabolismo , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/genética , Oxidorreductasas/química , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Testículo/metabolismo
11.
J Endocrinol ; 245(2): 207-218, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32106090

RESUMEN

Steroid 5ß-reductase (AKR1D1) is highly expressed in human liver where it inactivates endogenous glucocorticoids and catalyses an important step in bile acid synthesis. Endogenous and synthetic glucocorticoids are potent regulators of metabolic phenotype and play a crucial role in hepatic glucose metabolism. However, the potential of synthetic glucocorticoids to be metabolised by AKR1D1 as well as to regulate its expression and activity has not been investigated. The impact of glucocorticoids on AKR1D1 activity was assessed in human liver HepG2 and Huh7 cells; AKR1D1 expression was assessed by qPCR and Western blotting. Genetic manipulation of AKR1D1 expression was conducted in HepG2 and Huh7 cells and metabolic assessments were made using qPCR. Urinary steroid metabolite profiling in healthy volunteers was performed pre- and post-dexamethasone treatment, using gas chromatography-mass spectrometry. AKR1D1 metabolised endogenous cortisol, but cleared prednisolone and dexamethasone less efficiently. In vitro and in vivo, dexamethasone decreased AKR1D1 expression and activity, further limiting glucocorticoid clearance and augmenting action. Dexamethasone enhanced gluconeogenic and glycogen synthesis gene expression in liver cell models and these changes were mirrored by genetic knockdown of AKR1D1 expression. The effects of AKR1D1 knockdown were mediated through multiple nuclear hormone receptors, including the glucocorticoid, pregnane X and farnesoid X receptors. Glucocorticoids down-regulate AKR1D1 expression and activity and thereby reduce glucocorticoid clearance. In addition, AKR1D1 down-regulation alters the activation of multiple nuclear hormone receptors to drive changes in gluconeogenic and glycogen synthesis gene expression profiles, which may exacerbate the adverse impact of exogenous glucocorticoids.


Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Gluconeogénesis/efectos de los fármacos , Hígado/enzimología , Oxidorreductasas/efectos de los fármacos , Adulto , Células Cultivadas , Voluntarios Sanos , Hepatocitos , Humanos , Masculino
12.
Chem Biol Interact ; 305: 163-170, 2019 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-30928400

RESUMEN

Mammalian steroid 5ß-reductases belong to the Aldo-Keto Reductase 1D sub-family and are essential for the formation of A-ring 5ß-reduced steroids. Steroid 5ß-reduction is required for the biosynthesis of bile-acids and the metabolism of all steroid hormones that contain a Δ4-3-ketosteroid functionally to yield the 5ß-reduced metabolites. In mammalian AKR1D enzymes the conserved catalytic tetrad found in all AKRs (Y55, H117, K84 and D50) has changed in that the conserved H117 is replaced with a glutamic acid (E120). E120 may act as a "superacid" to facilitate enolization of the Δ4-ketosteroid. In addition, the absence of the bulky imidazole side chain of histidine in E120 permits the steroid to penetrate deeper into the active site so that hydride transfer can occur to the steroid C5 position. In murine steroid 5ß-reductase AKR1D4, we find that there is a long-form, with an 18 amino-acid extension at the N-terminus (AKR1D4L) and a short-form (AKR1D4S), where the latter is recognized as AKR1D4 by the major data-bases. Both enzymes were purified to homogeneity and product profiling was performed. With progesterone and cortisol, AKR1D4L and AKR1D4S catalyzed smooth conversion to the 5ß-dihydrosteroids. However, with Δ4-androstene-3,17-dione as substrate, a mixture of products was observed which included, 5ß-androstane-3,17-dione (expected) but 3α-hydroxy-5ß- androstan-17-one was also formed. The latter compound was distinguished from its isomeric 3ß-hydroxy-5ß-androstan-17-one by forming picolinic acid derivatives followed by LC-MS. These data show that AKR1D4L and AKR1D4S also act as 3α-hydroxysteroid dehydrogenases when presented with Δ4-androstene-3,17-dione and suggest that E120 alters the position the steroid to enable a correct trajectory for hydride transfer and may not act as a "superacid".


Asunto(s)
Ácido Glutámico/química , Oxidorreductasas/metabolismo , Androstanos/análisis , Androstanos/química , Androstanos/metabolismo , Animales , Biocatálisis , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Ácido Glutámico/metabolismo , Humanos , Isomerismo , Cinética , Hígado/metabolismo , Ratones , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esteroides/química , Esteroides/metabolismo , Especificidad por Sustrato , Espectrometría de Masas en Tándem
13.
J Steroid Biochem Mol Biol ; 189: 218-227, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30769091

RESUMEN

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.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Glucocorticoides/metabolismo , Neoplasias Hepáticas/metabolismo , Oxidorreductasas/metabolismo , Receptores de Glucocorticoides/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Hígado/metabolismo
14.
Metabolism ; 99: 67-80, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31330134

RESUMEN

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. Steroid hormones and bile acids are potent regulators of hepatic carbohydrate and lipid metabolism. Steroid 5ß-reductase (AKR1D1) is highly expressed in human liver where it inactivates steroid hormones and catalyzes a fundamental step in bile acid synthesis. METHODS: Human liver biopsies were obtained from 34 obese patients and AKR1D1 mRNA expression levels were measured using qPCR. Genetic manipulation of AKR1D1 was performed in human HepG2 and Huh7 liver cell lines. Metabolic assessments were made using transcriptome analysis, western blotting, mass spectrometry, clinical biochemistry, and enzyme immunoassays. RESULTS: In human liver biopsies, AKR1D1 expression decreased with advancing steatosis, fibrosis and inflammation. Expression was decreased in patients with type 2 diabetes. In human liver cell lines, AKR1D1 knockdown decreased primary bile acid biosynthesis and steroid hormone clearance. RNA-sequencing identified disruption of key metabolic pathways, including insulin action and fatty acid metabolism. AKR1D1 knockdown increased hepatocyte triglyceride accumulation, insulin sensitivity, and glycogen synthesis, through increased de novo lipogenesis and decreased ß-oxidation, fueling hepatocyte inflammation. Pharmacological manipulation of bile acid receptor activation prevented the induction of lipogenic and carbohydrate genes, suggesting that the observed metabolic phenotype is driven through bile acid rather than steroid hormone availability. CONCLUSIONS: Genetic manipulation of AKR1D1 regulates the metabolic phenotype of human hepatoma cell lines, driving steatosis and inflammation. Taken together, the observation that AKR1D1 mRNA is down-regulated with advancing NAFLD suggests that it may have a crucial role in the pathogenesis and progression of the disease.


Asunto(s)
Hepatocitos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Oxidorreductasas/fisiología , Fenotipo , Ácidos y Sales Biliares/metabolismo , Células Hep G2 , Humanos , Inflamación/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad , Oxidorreductasas/genética , ARN Mensajero/metabolismo
15.
J Clin Endocrinol Metab ; 92(11): 4332-9, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17711920

RESUMEN

CONTEXT: Glucocorticoid (GC) excess is characterized by central obesity, insulin resistance, and in some cases, type 2 diabetes. However, the impact of GC upon insulin signaling in human adipose tissue has not been fully explored. OBJECTIVE: We have examined the effect of GC upon insulin signaling in both human sc primary preadipocyte cultures and a novel human immortalized sc adipocyte cell line (Chub-S7) and contrasted this with observations in primary cultures of human skeletal muscle. DESIGN AND SETTING: This is an in vitro study characterizing the impact of GC upon insulin signaling in human tissues. PATIENTS: Biopsy specimens were from healthy volunteers who gave their full and informed written consent. INTERVENTIONS: Combinations of treatments, including GC, RU38486, and wortmannin, were used. MAIN OUTCOME MEASURES: Insulin signaling cascade gene and protein expression and insulin-stimulated glucose uptake were determined. RESULTS: In human adipocytes, pretreatment with GC induced a dose-dependent [1.0 (control); 1.2 +/- 0.1 (50 nm); 2.2 +/- 0.2 (250 nm), P < 0.01 vs. control; 3.4 +/- 0.2 (1000 nm), P < 0.001 vs. control] and time-dependent [1.0 (1 h); 3.2 +/- 2.0 (6 h); 9.1 +/- 5.9 (24 h), P < 0.05 vs. 1 h; 4.5 +/- 2.2 (48 h)] increase in insulin-stimulated protein kinase B/akt phosphorylation. In addition, whereas insulin receptor substrate (IRS)-1 protein expression did not change, IRS-1 tyrosine phosphorylation increased. Furthermore, GC induced IRS-2 mRNA expression (2.8-fold; P < 0.05) and increased insulin-stimulated glucose uptake [1.0 (control) 1.8 +/- 0.1 (insulin) vs. 2.8 +/- 0.2 (insulin + GC); P < 0.05]. In contrast, in primary cultures of human muscle, GC decreased insulin-stimulated glucose uptake [1.0 (control) 1.9 +/- 0.2 (insulin) vs. GC 1.3 +/- 0.1 (insulin + GC); P < 0.05]. CONCLUSIONS: We have demonstrated tissue-specific regulation of insulin signaling by GC. Within sc adipose tissue, GCs augment insulin signaling, yet in muscle GCs cause insulin resistance. We propose that enhanced insulin action in adipose tissue increases adipocyte differentiation, thereby contributing to GC-induced obesity.


Asunto(s)
Glucocorticoides/farmacología , Insulina/fisiología , Transducción de Señal/fisiología , Grasa Subcutánea/fisiología , Adipocitos/fisiología , Línea Celular , Relación Dosis-Respuesta a Droga , Glucosa/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Receptores de Glucocorticoides/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Células Madre/fisiología , Grasa Subcutánea/citología
16.
Endocrinology ; 157(9): 3493-504, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27384305

RESUMEN

Nonalcoholic fatty liver disease (NAFLD) defines a spectrum of conditions from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis and is regarded as the hepatic manifestation of the metabolic syndrome. Glucocorticoids can promote steatosis by stimulating lipolysis within adipose tissue, free fatty acid delivery to liver and hepatic de novo lipogenesis. Glucocorticoids can be reactivated in liver through 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) enzyme activity. Inhibition of 11ß-HSD1 has been suggested as a potential treatment for NAFLD. To test this, male mice with global (11ß-HSD1 knockout [KO]) and liver-specific (LKO) 11ß-HSD1 loss of function were fed the American Lifestyle Induced Obesity Syndrome (ALIOS) diet, known to recapitulate the spectrum of NAFLD, and metabolic and liver phenotypes assessed. Body weight, muscle and adipose tissue masses, and parameters of glucose homeostasis showed that 11ß-HSD1KO and LKO mice were not protected from systemic metabolic disease. Evaluation of hepatic histology, triglyceride content, and blinded NAFLD activity score assessment indicated that levels of steatosis were similar between 11ß-HSD1KO, LKO, and control mice. Unexpectedly, histological analysis revealed significantly increased levels of immune foci present in livers of 11ß-HSD1KO but not LKO or control mice, suggestive of a transition to NASH. This was endorsed by elevated hepatic expression of key immune cell and inflammatory markers. These data indicate that 11ß-HSD1-deficient mice are not protected from metabolic disease or hepatosteatosis in the face of a NAFLD-inducing diet. However, global deficiency of 11ß-HSD1 did increase markers of hepatic inflammation and suggests a critical role for 11ß-HSD1 in restraining the transition to NASH.


Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Jarabe de Maíz Alto en Fructosa/efectos adversos , Síndrome Metabólico/etiología , Enfermedad del Hígado Graso no Alcohólico/etiología , Ácidos Grasos trans/efectos adversos , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Fibrosis , Hígado/patología , Masculino , Síndrome Metabólico/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología
17.
Endocrinology ; 156(8): 2863-71, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25974403

RESUMEN

Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux.


Asunto(s)
3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/fisiología , Glucocorticoides/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Lipogénesis/genética , Proteínas de la Membrana/fisiología , Inhibidores de 5-alfa-Reductasa/farmacología , Adulto , Anciano , Andrógenos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Células Cultivadas , Femenino , Finasterida/farmacología , Glucocorticoides/metabolismo , Humanos , Insulina/farmacología , Lipogénesis/efectos de los fármacos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Persona de Mediana Edad , Fenotipo
18.
Vitam Horm ; 91: 1-27, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23374710

RESUMEN

Obesity has reached epidemic proportions with severe heath consequences including type 2 diabetes, nonalcoholic fatty liver disease, and premature cardiovascular mortality. Understanding the biological processes that govern fat deposition in a tissue-specific manner is therefore crucial if we are to try to design novel and efficacious treatments that might limit fat accumulation and improve metabolic phenotype and clinical prognosis. Lipid accumulation within a given cell type represents a balance between synthesis, mobilization, and utilization. Common endocrine conditions characterized by hormonal excess and deficiency are often associated with profound abnormalities in body composition and fat deposition. This undoubtedly reflects the complex regulation of lipid metabolism by endocrine factors. In this review, we will outline the current literature that has investigated the hormonal regulation of lipogenesis. This is a complex field, and in many studies, its assessment has been oversimplified with a focus on individual hormones acting in isolation and this bears little relationship to the in vivo situation where multiple hormones act in concert. Further, regulation may be different between rodents and humans and this will be explored. Limitation of lipid accumulation still represents a valid therapeutic target, and it is possible that manipulation of hormonal action has the potential to offer a new therapeutic horizon.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Hormonas/metabolismo , Metabolismo de los Lípidos/fisiología , Lipogénesis/fisiología , Animales , Humanos
19.
Endocr Rev ; 34(4): 525-55, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23612224

RESUMEN

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) interconverts the inactive glucocorticoid cortisone and its active form cortisol. It is widely expressed and, although bidirectional, in vivo it functions predominantly as an oxoreductase, generating active glucocorticoid. This allows glucocorticoid receptor activation to be regulated at a prereceptor level in a tissue-specific manner. In this review, we will discuss the enzymology and molecular biology of 11ß-HSD1 and the molecular basis of cortisone reductase deficiencies. We will also address how altered 11ß-HSD1 activity has been implicated in a number of disease states, and we will explore its role in the physiology and pathologies of different tissues. Finally, we will address the current status of selective 11ß-HSD1 inhibitors that are in development and being tested in phase II trials for patients with the metabolic syndrome. Although the data are preliminary, therapeutic inhibition of 11ß-HSD1 is also an exciting prospect for the treatment of a variety of other disorders such as osteoporosis, glaucoma, intracranial hypertension, and cognitive decline.


Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Glándulas Endocrinas/enzimología , Enfermedades del Sistema Endocrino/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Animales , Drogas en Investigación/uso terapéutico , Glándulas Endocrinas/efectos de los fármacos , Glándulas Endocrinas/metabolismo , Enfermedades del Sistema Endocrino/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Variación Genética , Humanos , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/enzimología , Investigación Biomédica Traslacional
20.
J Clin Endocrinol Metab ; 98(4): 1631-40, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23426618

RESUMEN

CONTEXT: It is widely believed that glucocorticoids cause insulin resistance in all tissues. We have previously demonstrated that glucocorticoids cause insulin sensitization in human adipose tissue in vitro and induce insulin resistance in skeletal muscle. OBJECTIVE: Our aim was to determine whether glucocorticoids have tissue-specific effects on insulin sensitivity in vivo. DESIGN: Fifteen healthy volunteers were recruited into a double-blind, randomized, placebo-controlled, crossover study, receiving both an overnight hydrocortisone and saline infusion. The tissue-specific actions of insulin were determined using paired 2-step hyperinsulinemic euglycemic clamps incorporating stable isotopes with concomitant adipose tissue microdialysis. SETTING: The study was performed in the Wellcome Trust Clinical Research Facility, Queen Elizabeth Hospital, Birmingham, United Kingdom. MAIN OUTCOME MEASURES: The sensitivity of sc adipose tissue to insulin action was measured. RESULTS: Hydrocortisone induced systemic insulin resistance but failed to cause sc adipose tissue insulin resistance as measured by suppression of adipose tissue lipolysis and enhanced insulin-stimulated pyruvate generation. In primary cultures of human hepatocytes, glucocorticoids increased insulin-stimulated p-ser473akt/protein kinase B. Similarly, glucocorticoids enhanced insulin-stimulated p-ser473akt/protein kinase B and increased Insulin receptor substrate 2 mRNA expression in sc, but not omental, intact human adipocytes, suggesting a depot-specificity of action. CONCLUSIONS: This study represents the first description of sc adipose insulin sensitization by glucocorticoids in vivo and demonstrates tissue-specific actions of glucocorticoids to modify insulin action. It defines an important advance in our understanding of the actions of both endogenous and exogenous glucocorticoids and may have implications for the development and targeting of future glucocorticoid therapies.


Asunto(s)
Glucocorticoides/farmacología , Resistencia a la Insulina , Grasa Subcutánea/efectos de los fármacos , Grasa Subcutánea/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Estudios Cruzados , Método Doble Ciego , Femenino , Glucocorticoides/administración & dosificación , Humanos , Hidrocortisona/administración & dosificación , Hidrocortisona/farmacología , Bombas de Infusión , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Masculino , Persona de Mediana Edad , Placebos
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