RESUMEN
Obesity is a major risk factor for the development of type II diabetes. Increases in adipose tissue mass trigger insulin resistance via the release of pro-inflammatory cytokines from adipocytes and macrophages. CREB and the CRTC coactivators have been found to promote insulin resistance in obesity, although the mechanism is unclear. Here we show that high fat diet feeding activates the CREB/CRTC pathway in adipocytes by decreasing the expression of SIK2, a Ser/Thr kinase that phosphorylates and inhibits CRTCs. SIK2 levels are regulated by the adipogenic factor C/EBPα, whose expression is reduced in obesity. Exposure to PPARγ agonist rescues C/EBPα expression and restores SIK2 levels. CRTC2/3 promote insulin resistance via induction of the chemokines CXCL1/2. Knockout of CRTC2/3 in adipocytes reduces CXCL1/2 expression and improves insulin sensitivity. As administration of CXCL1/2 reverses salutary effects of CRTC2/3 depletion, our results demonstrate the importance of the CREB/CRTC pathway in modulating adipose tissue function.
Asunto(s)
Adipocitos/metabolismo , Obesidad/metabolismo , Transducción de Señal , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Ratones , Factores de Transcripción/fisiologíaRESUMEN
CREB mediates effects of cyclic AMP on cellular gene expression. Ubiquitous CREB target genes are induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell-specific genes by targeting CBP/p300 to promoter-distal enhancer regions. Subsequent increases in histone acetylation facilitate recruitment of the coactivators CRTC2 and BRD4, leading to release of RNA polymerase II over the target gene body. Indeed, CREB-induced hyperacetylation of chromatin over superenhancers promoted beta cell-restricted gene expression, which is sensitive to inhibitors of CBP/p300 and BRD4 activity. Neurod1 appears critical in establishing nucleosome-free regions for recruitment of CREB to beta cell-specific enhancers. Deletion of a CREB-Neurod1-bound enhancer within the Lrrc10b-Syt7 superenhancer disrupted the expression of both genes and decreased beta cell function. Our results demonstrate how cross talk between signal-dependent and lineage-determining factors promotes the expression of cell-type-specific gene programs in response to extracellular cues.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Línea Celular , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Masculino , Ratones , Especificidad de Órganos , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , RatasRESUMEN
In response to cold exposure, placental mammals maintain body temperature by increasing sympathetic nerve activity in brown adipose tissue (BAT). Triggering of ß-adrenergic receptors on brown adipocytes stimulates thermogenesis via induction of the cAMP/PKA pathway. Although cAMP response element-binding protein (CREB) and its coactivators-the cAMP-regulated transcriptional coactivators (CRTCs)-mediate transcriptional effects of cAMP in most tissues, other transcription factors such as ATF2 appear critical for induction of thermogenic genes by cAMP in BAT. Brown adipocytes arise from Myf5-positive mesenchymal cells under the control of PRDM16, a coactivator that concurrently represses differentiation along the skeletal muscle lineage. Here, we show that the CREB coactivator CRTC3 is part of an inhibitory feedback pathway that antagonizes PRDM16-dependent differentiation. Mice with a knockout of CRTC3 in BAT (BKO) have increased cold tolerance and reduced adiposity, whereas mice overexpressing constitutively active CRTC3 in adipose tissue are more cold sensitive and have greater fat mass. CRTC3 reduced sympathetic nerve activity in BAT by up-regulating the expression of miR-206, a microRNA that promotes differentiation along the myogenic lineage and that we show here decreases the expression of VEGFA and neurotrophins critical for BAT innervation and vascularization. Sympathetic nerve activity to BAT was enhanced in BKO mice, leading to increases in catecholamine signaling that stimulated energy expenditure. As reexpression of miR-206 in BAT from BKO mice reversed the salutary effects of CRTC3 depletion on cold tolerance, our studies suggest that small-molecule inhibitors against this coactivator may provide therapeutic benefit to overweight individuals.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Termogénesis/fisiología , Factores de Transcripción/metabolismo , Adipocitos Marrones/metabolismo , Adiposidad/genética , Adiposidad/fisiología , Animales , Diferenciación Celular/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Metabolismo Energético , Ratones , Ratones Noqueados , MicroARNs/genética , Transducción de Señal , Sistema Nervioso Simpático/metabolismo , Factores de Transcripción/genéticaRESUMEN
Although persistent elevations in circulating glucose concentrations promote compensatory increases in pancreatic islet mass, unremitting insulin resistance causes deterioration in beta cell function that leads to the progression to diabetes. Here, we show that mice with a knockout of the CREB coactivator CRTC2 in beta cells have impaired oral glucose tolerance due to decreases in circulating insulin concentrations. CRTC2 was found to promote beta cell function in part by stimulating the expression of the transcription factor MafA. Chronic hyperglycemia disrupted cAMP signaling in pancreatic islets by activating the hypoxia inducible factor (HIF1)-dependent induction of the protein kinase A inhibitor beta (PKIB), a potent inhibitor of PKA catalytic activity. Indeed, disruption of the PKIB gene improved islet function in the setting of obesity. These results demonstrate how crosstalk between nutrient and hormonal pathways contributes to loss of pancreatic islet function.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Resistencia a la Insulina , Animales , Línea Celular Tumoral , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Prueba de Tolerancia a la Glucosa , Factor 1 Inducible por Hipoxia/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Islotes Pancreáticos/metabolismo , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Under feeding conditions, the incretin hormone GLP-1 promotes pancreatic islet viability by triggering the cAMP pathway in beta cells. Increases in PKA activity stimulate the phosphorylation of CREB, which in turn enhances beta cell survival by upregulating IRS2 expression. Although sustained GLP-1 action appears important for its salutary effects on islet function, the transient nature of CREB activation has pointed to the involvement of additional nuclear factors in this process. Following the acute induction of CREB-regulated genes, cAMP triggers a second delayed phase of gene expression that proceeds via the HIF transcription factor. Increases in cAMP promote the accumulation of HIF1α in beta cells by activating the mTOR pathway. As exposure to rapamycin disrupts GLP-1 effects on beta cell viability, these results demonstrate how a pathway associated with tumor growth also mediates salutary effects of an incretin hormone on pancreatic islet function.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Incretinas/metabolismo , Células Secretoras de Insulina/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Secuencia de Bases , Línea Celular , Supervivencia Celular/fisiología , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Células Secretoras de Insulina/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Ratas , Transducción de SeñalRESUMEN
Under specific environmental conditions, the yeast Saccharomyces cerevisiae can undergo a morphological switch to a pseudohyphal growth pattern. Pseudohyphal differentiation is generally studied upon induction by nitrogen limitation in the presence of glucose. It is known to be controlled by several signaling pathways, including mitogen-activated protein kinase, cyclic AMP-protein kinase A (cAMP-PKA), and Snf1 kinase pathways. We show that the alpha-glucoside sugars maltose and maltotriose, and especially sucrose, are more potent inducers of filamentation than glucose. Sucrose even induces filamentation in nitrogen-rich media and in the mep2Delta/mep2Delta ammonium permease mutant on ammonium-limiting medium. We demonstrate that glucose also inhibits filamentation by means of a pathway parallel to the cAMP-PKA pathway. Deletion of HXK2 shifted the pseudohyphal growth pattern on glucose to that of sucrose, while deletion of SNF4 abrogated filamentation on both sugars, indicating a negative role of glucose repression and a positive role for Snf1 activity in the control of filamentation. In all strains and in all media, sucrose induction of filamentation is greatly diminished by deletion of the sucrose/glucose-sensing G-protein-coupled receptor Gpr1, whereas it has no effect on induction by maltose and maltotriose. The competence of alpha-glucoside sugars to induce filamentation is reflected in the increased expression of the cell surface flocculin gene FLO11. In addition, sucrose is the only alpha-glucoside sugar capable of rapidly inducing FLO11 expression in a Gpr1-dependent manner, reflecting the sensitivity of Gpr1 for this sugar and its involvement in rapid sucrose signaling. Our study identifies sucrose as the most potent nutrient inducer of pseudohyphal growth and shows that glucose inactivation of Snf1 kinase signaling is responsible for the lower potency of glucose.
Asunto(s)
Diferenciación Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Transducción de Señal , Sacarosa/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Several examples of G protein-coupled receptors have recently been suggested to respond to common sugars in millimolar concentrations. This low affinity has made it difficult to demonstrate direct receptor-ligand interaction. In the yeast Saccharomyces cerevisiae, rapid activation of the cAMP pathway by glucose and sucrose requires the GPCR Gpr1. Our results obtained by cysteine scanning mutagenesis and SCAM (substituted cysteine accessibility method) of residues in TMD VI provide strong evidence that glucose and sucrose directly interact as ligands with Gpr1. The affinity for sucrose is much higher. Structurally similar sugars such as galactose, mannose, and fructose do not act as agonists, but mannose acts as an antagonist for both sucrose and glucose. These results support the idea that Gpr1 directly senses sugars and that sugars can effectively bind GPCRs with a low affinity in a binding pocket formed by the transmembrane domains. The ligand repertoire of GPCRs can thus be extended to common sugars in millimolar concentrations.
Asunto(s)
Glucosa/metabolismo , Manosa/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/agonistas , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/metabolismo , Sacarosa/metabolismo , AMP Cíclico/metabolismo , Cisteína/genética , Cisteína/metabolismo , Ligandos , Mutagénesis Sitio-Dirigida , Mutación , Receptores Acoplados a Proteínas G/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
In eukaryotic cells, G-protein-coupled receptors (GPCRs), non-transporting nutrient carrier homologues and active nutrient carriers have been recently shown to function as sensors that directly monitor the level of nutrients in the extracellular environment. The plasma membrane is not only the cellular boundary at which signalling molecules that govern metabolism and proliferation are detected, but also the boundary across which nutrients that sustain the generation of energy and building blocks are transported. Nutrient sensors combine these functions in various ways. Classical receptor proteins detect the presence of nutrients, carriers combine the functions of nutrient transporters and receptors, and carrier homologues have lost their transport capacity and become pure receptors. The activation of signal transduction pathways by nutrients adds a new layer to the regulatory network that controls metabolism and proliferation. Nutrient sensors highlight the importance of both nutrients as signalling molecules and nutrient carriers as receptors for signalling pathways.
Asunto(s)
Membrana Celular/metabolismo , Células Eucariotas/metabolismo , Transporte Biológico , Glucosa/genética , Glucosa/metabolismo , Modelos Biológicos , Fenómenos Fisiológicos de la Nutrición , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
The yeast Saccharomyces cerevisiae shows a great variety of cellular responses to glucose via several glucose-sensing and signaling pathways. Recent microarray analysis has revealed multiple levels of genomic sensitivity to glucose and highlighted the power of genome-wide analysis to detect cellular responses to minute environmental changes.