RESUMEN
Interest in the biology of adipose tissue has undergone a revival in recent years with the discovery of a host of genes that contribute to the regulation of satiety and metabolic rate. The catecholamines have long been known to be key modulators of adipose tissue lipolysis and the hydrolysis of triglyceride energy stores. However, more recent efforts to understand the role of individual adrenergic receptor subtypes expressed in adipocytes and their signal transduction pathways have revealed a complexity not previously appreciated. Combined with this interest in the modulation of adipocyte metabolism is a renewed focus upon brown adipose tissue and the mechanisms of whole body thermogenesis in general. The discovery of novel homologs of the brown fat uncoupling protein (UCP) such as UCP2 and UCP3 has provoked intensive study of these mitochondrial proteins and the role that they play in fuel metabolism. The story of the novel UCPs has proven to be intriguing and still incompletely understood. Here, we review the status of adipose tissue from inert storage depot to endocrine organ, interesting signal transduction pathways triggered by beta-adrenergic receptors in adipocytes, the potential of these receptors for discriminating and coordinated metabolic regulation, and current views on the role of UCP2 and UCP3 based on physiological studies and gene knockout models.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Metabolismo Energético , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Receptores Adrenérgicos/fisiología , Adipocitos/metabolismo , Adipocitos/fisiología , Tejido Adiposo/metabolismo , Animales , Humanos , Canales Iónicos , Ratones , Ratones Noqueados , Ratones Obesos , Modelos Biológicos , Proteínas/metabolismo , Transducción de Señal , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
The beta(3)-adrenergic receptor (beta(3)AR) is expressed predominantly in adipocytes, and it plays a major role in regulating lipolysis and adaptive thermogenesis. Its expression in a variety of adipocyte cell models is preceded by the appearance of CCAAT/enhancer-binding protein alpha (C/EBP alpha), which has been shown to regulate a number of other adipocyte-specific genes. Importantly, it has been demonstrated that several adipocyte cell lines that fail to express C/EBP alpha exhibit reduced insulin sensitivity, despite an apparent adipogenic phenotype. Here we show that transcription and function of the beta(3)AR correlates with C/EBP alpha expression in these adipocyte models. A 5.13-kilobase pair fragment of the mouse beta(3)AR promoter was isolated and sequenced. This fragment conferred a 50-fold increase in luciferase reporter gene expression in adipocytes. Two putative C/EBP binding sites exist at -3306 to -3298 and at -1462 to -1454, but only the more distal site is functional. Oligonucleotides corresponding to both the wild-type and mutated -3306 element were inserted upstream of a thymidine kinase luciferase construct. When cotransfected in fibroblasts with a C/EBP alpha expression vector, reporter gene expression increased 3-fold only in the wild-type constructs. The same mutation, when placed into the intact 5.13-kilobase pair promoter, reduced promoter activity in adipocytes from 50-fold to <10-fold. Electrophoretic mobility shift analysis demonstrated that the site at -3306 generated a specific protein-oligonucleotide complex that was supershifted by C/EBP alpha antibody, while a probe corresponding to a putative site at -1462 did not. These results define C/EBP alpha as a key transcriptional regulator of the mouse beta(3)AR gene during adipogenesis.
Asunto(s)
Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Receptores Adrenérgicos beta 3/genética , Células 3T3 , Adenilil Ciclasas/metabolismo , Tejido Adiposo/efectos de los fármacos , Agonistas de Receptores Adrenérgicos beta 3 , Animales , Células COS , ADN/genética , ADN/metabolismo , Dioxoles/farmacología , Activación Enzimática/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Ratones , Datos de Secuencia Molecular , Mutación , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta/genética , Análisis de Secuencia de ADN , TransfecciónRESUMEN
Both beta(2)- and beta(3)-adrenergic receptors (ARs) are able to activate the extracellular signal-regulated kinase (ERK) pathway. We previously showed that c-Src is required for ERK activation by beta(2)AR and that it is recruited to activated beta(2)AR through binding of the Src homology 3 (SH3) domain to proline-rich regions of the adapter protein beta-arrestin1. Despite the absence of sites for phosphorylation and beta-arrestin binding, ERK activation by beta(3)AR still requires c-Src. Agonist activation of beta(2)AR, but not beta(3)AR, led to redistribution of green fluorescent protein-tagged beta-arrestin to the plasma membrane. In beta-arrestin-deficient COS-7 cells, beta-agonist-dependent co-precipitation of c-Src with the beta(2)AR required exogenous beta-arrestin, but activated beta(3)AR co-precipitated c-Src in the absence or presence of beta-arrestin. ERK activation and Src co-precipitation with beta(3)AR also occurred in adipocytes in an agonist-dependent and pertussis toxin-sensitive manner. Protein interaction studies show that the beta(3)AR interacts directly with the SH3 domain of Src through proline-rich motifs (PXXP) in the third intracellular loop and the carboxyl terminus. ERK activation and Src co-precipitation were abolished in cells expressing point mutations in these PXXP motifs. Together, these data describe a novel mechanism of ERK activation by a G protein-coupled receptor in which the intracellular domains directly recruit c-Src.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Adipocitos/citología , Adipocitos/fisiología , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Animales , Arrestinas/farmacología , Sitios de Unión , Células COS , Diferenciación Celular , Línea Celular , Chlorocebus aethiops , Dioxoles/farmacología , Activación Enzimática , Isoproterenol/farmacología , Ratones , Datos de Secuencia Molecular , Prolina , Propranolol/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/química , Receptores Adrenérgicos beta 3/química , Receptores Adrenérgicos beta 3/efectos de los fármacos , Transfección , beta-ArrestinasRESUMEN
We previously demonstrated that the expression and function of the adipocyte-specific beta3-adrenergic receptor (beta3AR) are significantly depressed in single gene and diet-induced rodent models of obesity. Furthermore, these models are relatively unresponsive to the anti-obesity effects of beta3AR agonists. Because all of these models are hyperinsulinemic, we hypothesized that hyperinsulinemia could be responsible for this abnormality in beta3AR function. The goal of this study was to determine whether lowering insulin with the K-ATP channel agonist, diazoxide (Dz) would reverse the depressed expression and function of the beta3AR found in a model of diet-induced diabetes and obesity in C57BL/6J (B6) mice. B6 male mice were placed on either high fat (HF) or low fat experimental diets. After 4 weeks, HF-fed mice were assigned to a group: HF or HF containing disodium (R,R)-5- [2-( [2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl-1,3-benzodioxole-2,2-di carboxylate (CL; 0.001%, wt/wt), Dz (0.32%, wt/wt), or their combination (CLDz). Dz animals exhibited significantly reduced plasma insulin levels as well as increased 3pAR expression and agonist-stimulated adenylyl cyclase activity in adipocytes. CLDz was more effective in reducing percent body fat, lowering nonesterified fatty acids, improving glucose tolerance, and reducing feed efficiency than either treatment alone.