RESUMEN
My graduate and postdoctoral training in metabolism and enzymology eventually led me to study the short- and long-term regulation of glucose and lipid metabolism. In the early phase of my career, my trainees and I identified, purified, and characterized a variety of phosphofructokinase enzymes from mammalian tissues. These studies led us to discover fructose 2,6-P2, the most potent activator of phosphofructokinase and glycolysis. The discovery of fructose 2,6-P2 led to the identification and characterization of the tissue-specific bifunctional enzyme 6-phosphofructo-2-kinase:fructose 2,6-bisphosphatase. We discovered a glucose signaling mechanism by which the liver maintains glucose homeostasis by regulating the activities of this bifunctional enzyme. With a rise in glucose, a signaling metabolite, xylulose 5-phosphate, triggers rapid activation of a specific protein phosphatase (PP2ABδC), which dephosphorylates the bifunctional enzyme, thereby increasing fructose 2,6-P2 levels and upregulating glycolysis. These endeavors paved the way for us to initiate the later phase of my career in which we discovered a new transcription factor termed the carbohydrate response element binding protein (ChREBP). Now ChREBP is recognized as the masterregulator controlling conversion of excess carbohydrates to storage of fat in the liver. ChREBP functions as a central metabolic coordinator that responds to nutrients independently of insulin. The ChREBP transcription factor facilitates metabolic adaptation to excess glucose, leading to obesity and its associated diseases.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Bioquímica/historia , Fructosadifosfatos/metabolismo , Fosfofructoquinasa-2/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Gluconeogénesis/fisiología , Glucosa/metabolismo , Glucólisis , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Masculino , Ratones , Fosfofructoquinasa-2/química , Fosfofructoquinasas/química , Fosfofructoquinasas/metabolismo , Fosforilación , Estados UnidosRESUMEN
The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5â Å crystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Señales de Localización Nuclear/química , alfa Carioferinas/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Cristalografía por Rayos X , Células Hep G2 , Humanos , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismoRESUMEN
Lipogenesis in liver is highest in the postprandial state; insulin activates SREBP-1c, which transcriptionally activates genes involved in FA synthesis, whereas glucose activates carbohydrate-responsive element-binding protein (ChREBP), which activates both glycolysis and FA synthesis. Whether SREBP-1c and ChREBP act independently of one another is unknown. Here, we characterized mice with liver-specific deletion of ChREBP (L-Chrebp-/- mice). Hepatic ChREBP deficiency resulted in reduced mRNA levels of glycolytic and lipogenic enzymes, particularly in response to sucrose refeeding following fasting, a dietary regimen that elicits maximal lipogenesis. mRNA and protein levels of SREBP-1c, a master transcriptional regulator of lipogenesis, were also reduced in L-Chrebp-/- livers. Adeno-associated virus-mediated restoration of nuclear SREBP-1c in L-Chrebp-/- mice normalized expression of a subset of lipogenic genes, while not affecting glycolytic genes. Conversely, ChREBP overexpression alone failed to support expression of lipogenic genes in the livers of mice lacking active SREBPs as a result of Scap deficiency. Together, these data show that SREBP-1c and ChREBP are both required for coordinated induction of glycolytic and lipogenic mRNAs. Whereas SREBP-1c mediates insulin's induction of lipogenic genes, ChREBP mediates glucose's induction of both glycolytic and lipogenic genes. These overlapping, but distinct, actions ensure that the liver synthesizes FAs only when insulin and carbohydrates are both present.
Asunto(s)
Glucólisis , Lipogénesis , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/deficiencia , Factores de Transcripción/deficienciaRESUMEN
The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.
Asunto(s)
Adenosina Monofosfato/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Regulación Alostérica , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Núcleo Celular/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Dieta Alta en Grasa , Sacarosa en la Dieta/administración & dosificación , Hepatocitos/metabolismo , Carioferinas/metabolismo , Cuerpos Cetónicos/metabolismo , Masculino , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína Exportina 1RESUMEN
The carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in converting excess carbohydrate to storage fat in liver. In response to changing glucose levels, ChREBP activity is regulated by nucleo-cytoplasmic shuttling of ChREBP via interactions with 14-3-3 proteins and importins. The nuclear/cytosol trafficking is regulated partly by phosphorylation/dephosphorylation of serine 196 mediated by cAMP-dependent protein kinase and protein phosphatase. We show here that protein-free extracts of starved and high fat-fed livers contain metabolites that activate interaction of ChREBP·14-3-3 and inhibit the ChREBP/importin α interaction, resulting in cytosolic localization. These metabolites were identified as ß-hydroxybutyrate and acetoacetate. Nuclear localization of GFP-ChREBP is rapidly inhibited in hepatocytes incubated in ß-hydroxybutyrate or fatty acids, and the observed inhibition is closely correlated with the production of ketone bodies. These observations show that ketone bodies play an important role in the regulation of ChREBP activity by restricting ChREBP localization to the cytoplasm, thus inhibiting fat synthesis during periods of ketosis.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Regulación de la Expresión Génica , Cuerpos Cetónicos/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Transporte Biológico , Metabolismo de los Hidratos de Carbono , Núcleo Celular/metabolismo , Citosol/metabolismo , Hepatocitos/citología , Humanos , Lipogénesis , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratas , Transducción de SeñalRESUMEN
Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3ß bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3ß·ChREBP α2 complex.
Asunto(s)
Proteínas 14-3-3/química , Complejos Multiproteicos/química , Proteínas Nucleares/química , Factores de Transcripción/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Cristalografía por Rayos X , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Señales de Exportación Nuclear , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mapeo Peptídico , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. Circulating blood glucose levels affect ChREBP activity in hepatocytes largely by post-translational mechanisms that include phosphorylation-dependent subcellular localization. Previously, we showed that ChREBP is retained in the cytosol by phosphorylation-dependent binding to 14-3-3 protein dimers and identified the α2 helix (residues 125-135) phospho-Ser(140) domain as the primary 14-3-3 binding site (Sakiyama, H., Wynn, R. M., Lee, W. R., Fukasawa, M., Mizuguchi, H., Gardner, K. H., Repa, J. J., and Uyeda, K. (2008) J. Biol. Chem. 283, 24899-24908). To enter the nucleus in response to high glucose, ChREBP must bind importin-α; this heterodimer then forms a complex with importin-ß to interact with the nuclear pore complex. In this work, we recharacterized the importin-α binding nuclear localization signal (NLS) of rat ChREBP, identifying it as an extended classical bipartite NLS encompassing minimally residues 158-190. Replacing Lys(159)/Lys(190) residues of ChREBP with alanine resulted in loss of importin-α binding, glucose-stimulated transcriptional activity and nuclear localization. A secondary 14-3-3 protein binding site also was identified, the α3 helix (residues 170-190) phospho-Ser(196) domain. Importin-α and 14-3-3 were found to bind competitively to this secondary site. These results suggest an important mechanism by which importin-α and 14-3-3 control movement of ChREBP in and out of the nucleus in response to changes in glucose levels in liver and thus further suggest that the extended NLS of ChREBP is a critical glucose-sensing, glucose-responsive site.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Núcleo Celular/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , alfa Carioferinas/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Sitios de Unión , Glucemia/metabolismo , Núcleo Celular/genética , Células Cultivadas , Hepatocitos/citología , Hígado/citología , Masculino , Ratones , Señales de Localización Nuclear/genética , Proteínas Nucleares/genética , Fosforilación/fisiología , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genética , alfa Carioferinas/genéticaRESUMEN
G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma ß-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.
Asunto(s)
Ácidos Grasos/metabolismo , Proteínas Nucleares/fisiología , Proteínas RGS/fisiología , Factores de Transcripción/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Ácidos Grasos/biosíntesis , Ácidos Grasos/genética , Gluconeogénesis , Glucosa/biosíntesis , Glucosa/fisiología , Hepatocitos/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Oxidación-Reducción , Receptores Acoplados a Proteínas G/metabolismo , Transcripción GenéticaRESUMEN
The ability of an organism to sense and store nutrients is vital to survival. The liver is the major organ responsible for converting excess dietary carbohydrate to lipid for storage. An elegant molecular pathway has evolved that allows increased glucose flux into hepatocytes to generate a signaling molecule, xylulose 5-phosphate, that triggers rapid changes in glycolytic enzyme activities and nuclear import of a transcription factor, ChREBP, which coordinates the transcriptional regulation of enzymes that channel the glycolytic end-products into lipogenesis. Further understanding of this metabolic cascade should provide insights on conditions such as fatty liver, obesity, and the metabolic syndrome.
Asunto(s)
Glucosa/metabolismo , Lípidos/biosíntesis , Hígado/metabolismo , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Carbohydrate response element binding protein (ChREBP) is responsible for conversion of dietary carbohydrate to storage fat in liver by coordinating expression of the enzymes that channel glycolytic pyruvate into lipogenesis. The activation of ChREBP in response to high glucose is nuclear localization and transcription, and the inactivation of ChREBP under low glucose involves export from the nucleus to the cytosol. Here we report a new nuclear export signal site ("NES1") of ChREBP. Together these signals provide ChREBP with two NES sequences, both the previously reported NES2 and now the new NES1 coordinate to interact together with CRM1 (exportin) for nuclear export of the carbohydrate response element binding protein.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Glucosa/metabolismo , Carioferinas/metabolismo , Señales de Exportación Nuclear , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células Cultivadas , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Ratas , Proteína Exportina 1RESUMEN
The carbohydrate response element-binding protein (ChREBP) functions as a transcription factor in mediating the glucose-activated gene expression of multiple liver enzymes, which are responsible for converting excess carbohydrate to storage fat. ChREBP is translocated into the nucleus in response to high glucose levels, and then up-regulates transcriptional activity. Although this glucose activation of ChREBP is generally observed only in liver cells, overexpression of wild type max-like protein X (Mlx), but not an inactive mutant Mlx, resulted in the exhibition of the ChREBP functions also in a human kidney cell line. Because high glucose conditions induce the glycosylation of cellular proteins, the effect of O-linked GlcNAc modification on ChREBP functions was examined. Treatment with an O-GlcNAcase inhibitor (PUGNAc), which increases the O-linked GlcNAc modification of cellular proteins, caused an increase in the glucose response of ChREBP. In contrast, treatment with a glutamine fructose amidotransferase inhibitor (DON), which decreases O-GlcNAcylation by inhibiting the hexosamine biosynthetic pathway, completely blocked the glucose response of ChREBP. These results suggest that the O-linked glycosylation of ChREBP itself or other proteins that regulate ChREBP is essential for the production of functional ChREBP.
Asunto(s)
Acetilglucosamina/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Glucosa/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Línea Celular , Glucosa/farmacología , Glicosilación , Humanos , Mutación , Oximas/farmacología , Fenilcarbamatos/farmacología , Transcripción Genética/efectos de los fármacos , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Previously, we found a novel protein factor in the livers of rats fed a high-carbohydrate diet, which binds to the major late transcription factor (MLTF)-like site within the glucose response element (GRE) of the liver-type pyruvate kinase (L-PK) gene [J. Biol. Chem. 274 (1999) 1100]. This factor, termed glucose response element binding protein (GRBP), exists in both liver cytosol and nucleus. In order to identify GRBP, we purified to homogeneity cytosolic GRBP from rat liver extract and identified it as a Translin/Trax heteromeric complex. Based on partial amino acid sequences, we have cloned full-length rat cDNAs of both Translin and Trax. The nuclear and the cytosolic Translin/Trax complex were both large polymers of 240 and 420 kDa, respectively. The molar ratio of Translin/Trax in the polymers was 2:1 in the liver cytosols. The nuclear and cytosolic Translin/Trax complexes as well as expressed His-tagged Translin bound to double- and single-stranded MLTF sites of the GRE of L-PK gene more avidly than to single-stranded Bcl-CL1, which was initially thought to be specific for Translin. Our findings indicate that the Translin/Trax complex constitutes the previously described GRBP, and that this complex binds the GRE of the L-PK gene with high affinity. The precise physiologic role of GRBP, however, remains unclear.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Glucosa/farmacología , Hígado/química , Proteínas Nucleares/aislamiento & purificación , Piruvato Quinasa/genética , Elementos de Respuesta , Animales , Peso Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
Feeding a high carbohydrate diet induces transcription of more than 15 genes involved in the metabolic conversion of glucose to fat. A new transcription factor binding to a glucose response element of the pyruvate kinase and lipogenesis enzyme genes was discovered recently. This factor, termed carbohydrate responsive element-binding protein (ChREBP), is activated in response to high glucose and up-regulates these genes. Cyclic AMP and a high fat diet inhibit ChREBP and slow down glucose utilization. ChREBP is able to control transcription of lipogenic enzyme genes in response to nutritional and hormonal inputs, and may play an important role in disease states such as diabetes, obesity, and hypertension.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Grasas/metabolismo , Glucosa/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Ácidos Grasos/farmacología , Glucagón/fisiología , Humanos , Fenómenos Fisiológicos de la Nutrición/fisiología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Esteroles/biosíntesis , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K, identical to PFKFB3) is expressed in a variety of cells and tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, primary blood mononuclear cells and cancer cells. We observed previously that the enhancer region of the HP2K gene, which has been identified in the 5'-flanking region between -1265 and -1329, could respond to serum stimulation following the transfection of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. The HP2K enhancer region also contains two copies of the hypoxia-inducible factor-1 (HIF-1) binding motif (5'-ACGTG-3'). In this study we performed characterization of the HP2K gene expression in response to hypoxic conditions. Both electrophoretic mobility shift and co-transfection assays of the HP2K promoter-luciferase reporter with HIF-1 expression vectors indicated that HIF-1 binds to the hypoxia-responsive element (HRE) of HP2K, thereby upregulating its gene expression. In addition, we demonstrated using site-directed mutagenesis that a complete tandem repeat of the HIF-1 binding motif with a 4-bp interruption is required for full induction of HP2K expression (up to 22-fold) under hypoxic conditions, and that this response is much stronger than that of the erythropoietin (EPO) gene. These results suggest that the sequence 5'-ACGTGNNNNACGTG-3' in the HP2K enhancer is the authentic HRE consensus motif that mediates increased transcription, under hypoxic conditions, via HIF-1.
Asunto(s)
Hipoxia , Fosfofructoquinasa-2/química , Placenta/enzimología , Factores de Transcripción/genética , Secuencias de Aminoácidos , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Elementos de Facilitación Genéticos , Eritropoyetina/genética , Eritropoyetina/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos , Glucólisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Luciferasas/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/química , Fosfofructoquinasa-2/fisiología , Regiones Promotoras Genéticas , Isoformas de Proteínas , Estructura Terciaria de Proteína , Factores de Tiempo , Distribución Tisular , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Regulación hacia ArribaRESUMEN
Livers from mice lacking the carbohydrate-responsive element-binding protein (ChREBP) were compared with wild type (WT) mice to determine the effect of this transcription factor on hepatic energy metabolism. The pyruvate dehydrogenase complex was considerably more active in ChREBP(-/-) mice because of diminished pyruvate dehydrogenase kinase activity. Greater pyruvate dehydrogenase complex activity caused a stimulation of lactate and pyruvate oxidation, and it significantly impaired fatty acid oxidation in perfused livers from ChREBP(-/-) mice. This shift in mitochondrial substrate utilization led to a 3-fold reduction of the free cytosolic [NAD(+)]/[NADH] ratio, a 1.7-fold increase in the free mitochondrial [NAD(+)]/[NADH] ratio, and a 2-fold decrease in the free cytosolic [ATP]/[ADP][P(i)] ratio in the ChREBP(-/-) liver compared with control. Hepatic pyruvate carboxylase flux was impaired with ChREBP deletion secondary to decreased fatty acid oxidation, increased pyruvate oxidation, and limited pyruvate availability because of reduced activity of liver pyruvate kinase and malic enzyme, which replenish pyruvate via glycolysis and pyruvate cycling. Overall, the shift from fat utilization to pyruvate and lactate utilization resulted in a decrease in the energy of ATP hydrolysis and a hypo-energetic state in the livers of ChREBP(-/-) mice.
Asunto(s)
Metabolismo Energético , Eliminación de Gen , Hígado/metabolismo , Proteínas Nucleares/deficiencia , Factores de Transcripción/deficiencia , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Western Blotting , Citosol/metabolismo , Ácidos Grasos/metabolismo , Hígado/enzimología , Espectroscopía de Resonancia Magnética , Ratones , Mitocondrias/metabolismo , Nucleótidos/metabolismo , Oxidación-Reducción , Fosforilación Oxidativa , Consumo de Oxígeno , Perfusión , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Complejo Piruvato Deshidrogenasa/metabolismo , Piruvatos/metabolismo , Especificidad por SustratoRESUMEN
Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an alpha-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin alpha. Notably, 14-3-3 appears to compete with importin alpha for ChREBP binding. 14-3-3beta bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 microm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3beta. These results suggest that interactions with importin alpha and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.
Asunto(s)
Proteínas 14-3-3/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas 14-3-3/genética , Transporte Activo de Núcleo Celular/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Línea Celular , Núcleo Celular/genética , Citosol/metabolismo , Glucosa/genética , Glucosa/metabolismo , Glucólisis/fisiología , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Lipogénesis/fisiología , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/genética , Proteína Exportina 1RESUMEN
The transcription factor carbohydrate response element-binding protein (ChREBP) mediates insulin-independent, glucose-stimulated gene expression of multiple liver enzymes responsible for converting excess carbohydrate to fatty acids for long-term storage. To investigate ChREBP's role in the development of obesity and obesity-associated metabolic dysregulation, ChREBP-deficient mice were intercrossed with ob/ob mice. As a result of deficient leptin expression, ob/ob mice overeat, become obese and resistant to insulin, and display marked elevations in hepatic lipogenesis, gluconeogenesis, and plasma glucose and triglycerides. mRNA expression of all hepatic lipogenic enzymes was significantly lower in ob/ob-ChREBP-/- than in ob/ob mice, resulting in decreased hepatic fatty acid synthesis and normalization of plasma free fatty acid and triglyceride levels. Overall weight gain in addition to adiposity was reduced in the doubly deficient mice. The former was largely attributable to decreased food intake and may result from decreased hypothalamic expression of the appetite-stimulating neuropeptide agouti-related protein. mRNA expression and activity of gluconeogenic enzymes also was lower in the doubly deficient mice, contributing to significantly lower blood glucose levels. The results of this study suggest that inactivation of ChREBP expression not only reduces fat synthesis and obesity in ob/ob mice but also results in improved glucose tolerance and appetite control.
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Tejido Adiposo/metabolismo , Glucemia/metabolismo , Conducta Alimentaria , Hiperinsulinismo/metabolismo , Leptina/metabolismo , Proteínas Nucleares/metabolismo , Obesidad/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Retroalimentación , Hiperinsulinismo/complicaciones , Leptina/deficiencia , Hígado/metabolismo , Ratones , Ratones Noqueados , Proteínas Nucleares/deficiencia , Obesidad/complicaciones , Factores de Transcripción/deficienciaRESUMEN
Carbohydrate response element (ChRE)-binding protein (ChREBP) is a recently discovered transcription factor that is activated in response to high glucose concentrations in liver independently of insulin. ChREBP was first identified by its ability to bind the ChRE of the liver pyruvate kinase (LPK) gene. We recently reported that the increase in expression of multiple liver lipogenic enzyme mRNAs elicited by feeding a high-carbohydrate diet as well as that of LPK mRNA is markedly reduced in mice lacking ChREBP gene expression (ChREBP(-/-)) in comparison to WT mice. The present study provides evidence for a direct and dominant role of ChREBP in the glucose regulation of two key liver lipogenic enzymes, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). ACC, FAS, and LPK mRNA levels were higher in WT hepatocytes cultured with high (25 mM) rather than low (5.5 mM) glucose medium, but there was no effect of glucose concentration on these mRNA levels in ChREBP(-/-) hepatocytes. Similarly, reporter constructs containing ACC, FAS, or LPK gene ChREs were responsive to glucose when transfected into WT but not ChREBP(-/-) hepatocytes, and glucose transactivation of the constructs in ChREBP(-/-) hepatocytes was restored by cotransfection with a ChREBP expression plasmid. ChREBP binding to ACC, FAS, and LPK ChRE sequences in vitro was demonstrated by electrophoretic mobility super shift assays. In vivo binding of ChREBP to ACC, FAS, and LPK gene promoters in intact liver nuclei from rats fed a high-carbohydrate diet was demonstrated by using a formaldehyde crosslinking and chromatin immunoprecipitation procedure.
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Proteínas de Unión al ADN/metabolismo , Lípidos/biosíntesis , Factores de Transcripción/metabolismo , Acetil-CoA Carboxilasa/genética , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión/genética , Inmunoprecipitación de Cromatina , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Ácido Graso Sintasas/genética , Glucosa/metabolismo , Glucosa/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Técnicas In Vitro , Ratones , Ratones Noqueados , Proteínas Nucleares , Regiones Promotoras Genéticas , Piruvato Quinasa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética , TransfecciónRESUMEN
Carbohydrate-responsive element binding protein (ChREBP) is a transcription factor that activates lipogenic genes in liver in response to excess carbohydrate in the diet. ChREBP is regulated in a reciprocal manner by glucose and cAMP. cAMP-dependent protein kinase (protein kinase A) phosphorylates two physiologically important sites in ChREBP, Ser-196, which is located near nuclear localization signal sequence (NLS), and Thr-666, within the basic helix-loop-helix (bHLH) site, resulting in inactivation of nuclear translocation of ChREBP and of the DNA-binding activity, respectively. We demonstrate here that crude cytosolic extracts from livers of rats fed a high carbohydrate diet contained protein phosphatase (PPase) activity that dephosphorylated a peptide containing Ser-196, whereas a PPase in the nuclear extract catalyzed dephosphorylation of Ser-568 and Thr-666. All these PPases are activated specifically by xylulose 5-phosphate (Xu5P), but not by other sugar phosphates. Furthermore, addition of Xu5P elevated PPase activity to the level observed in extracts of fed liver cells. These partially purified PPases were characterized as PP2A-AB delta C by immunoblotting with specific antibodies. These results suggest that (ia) Xu5P-dependent PPase is responsible for activation of transcription of the L-type pyruvate kinase gene and lipogenic enzyme genes, and (ii) Xu5P is the glucose signaling compound. Thus, we propose that the same Xu5P-activated PPase controls both acute and long-term regulation of glucose metabolism and fat synthesis.
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Glucosa/farmacología , Lípidos/biosíntesis , Hígado/enzimología , Pentosafosfatos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Citosol/enzimología , Citosol/metabolismo , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Cinética , Masculino , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/metabolismoRESUMEN
The liver provides for long-term energy needs of the body by converting excess carbohydrate into fat for storage. Insulin is one factor that promotes hepatic lipogenesis, but there is increasing evidence that glucose also contributes to the coordinated regulation of carbohydrate and fat metabolism in liver by mechanisms that are independent of insulin. In this study, we show that the transcription factor, carbohydrate response element-binding protein (ChREBP), is required both for basal and carbohydrate-induced expression of several liver enzymes essential for coordinated control of glucose metabolism, fatty acid, and the synthesis of fatty acids and triglycerides in vivo.