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1.
Circ Res ; 112(5): 771-80, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23307819

RESUMEN

RATIONALE: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. OBJECTIVE: The role of vaspin in the diabetic vascular complications remains elusive, and we investigated the effects of vaspin on the vascular function under the diabetic milieu. METHODS AND RESULTS: Adenovirus carrying the full length of the vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon-injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb, and Pdgfrb genes was significantly reduced by the treatment of Vaspin-Ad. In cuff-injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells. Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to human aortic endothelial cells, subjected to tandem tag purification and liquid chromatography-tandem mass spectrometry, and we identified GRP78 (78-kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78, and voltage-dependent anion channel on the plasma membrane was confirmed by the immunoprecipitation studies using aortas of Vaspin Tg mice. The binding assay using (125)I-vaspin in human aortic endothelial cells revealed high-affinity binding (dissociation constant = 0.565×10(-9) m) by the treatment of 5 µM thapsigargin, which recruited GRP78 from the endoplasmic reticulum to plasma membrane by inducing endoplasmic reticulum stress. In human aortic endothelial cells, vaspin induced phosphorylation of Akt and inhibited the kringle 5-induced Ca(2+) influx and subsequent apoptosis. CONCLUSIONS: Vaspin is a novel ligand for the cell-surface GRP78/voltage-dependent anion channel complex in endothelial cells and promotes proliferation, inhibits apoptosis, and protects vascular injuries in diabetes mellitus.


Asunto(s)
Adipoquinas/metabolismo , Apoptosis/fisiología , Endotelio Vascular/patología , Proteínas de Choque Térmico/metabolismo , Serpinas/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Adenoviridae/genética , Adipoquinas/genética , Animales , Membrana Celular/metabolismo , Proliferación Celular , Células Cultivadas , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ratas , Ratas Wistar , Serpinas/genética , Estreptozocina/efectos adversos
2.
Cancer Sci ; 105(5): 560-7, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24602018

RESUMEN

Targeting tumor angiogenesis is an established strategy for cancer therapy. Because angiogenesis is not limited to pathological conditions such as cancer, molecular markers that can distinguish between physiological and pathological angiogenesis are required to develop more effective and safer approaches for cancer treatment. To identify such molecules, we determined the gene expression profiles of murine tumor endothelial cells (mTEC) and murine normal endothelial cells using DNA microarray analysis followed by quantitative reverse transcription-polymerase chain reaction analysis. We identified 131 genes that were differentially upregulated in mTEC. Functional analysis using siRNA-mediated gene silencing revealed five novel tumor endothelial cell markers that were involved in the proliferation or migration of mTEC. The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma. Comparative gene expression analysis revealed molecular differences between tumor endothelial cells and normal endothelial cells and identified novel tumor endothelial cell markers that may be exploited to target tumor angiogenesis for cancer treatment.


Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Biomarcadores de Tumor/genética , Endotelio Vascular/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Animales , Carcinoma de Células Renales/irrigación sanguínea , Línea Celular Tumoral , Movimiento Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Persona de Mediana Edad , Trasplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba
3.
J Biol Chem ; 286(43): 37458-69, 2011 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-21862590

RESUMEN

Krüppel-like factor 15 (KLF15), a member of the Krüppel-like factor family of transcription factors, has been found to play diverse roles in adipocytes in vitro. However, little is known of the function of KLF15 in adipocytes in vivo. We have now found that the expression of KLF15 in adipose tissue is down-regulated in obese mice, and we therefore generated adipose tissue-specific KLF15 transgenic (aP2-KLF15 Tg) mice to investigate the possible contribution of KLF15 to various pathological conditions associated with obesity in vivo. The aP2-KLF15 Tg mice manifest insulin resistance and are resistant to the development of obesity induced by maintenance on a high fat diet. However, they also exhibit improved glucose tolerance as a result of enhanced insulin secretion. Furthermore, this enhancement of insulin secretion was shown to result from down-regulation of the expression of stearoyl-CoA desaturase 1 (SCD1) in white adipose tissue and a consequent reduced level of oxidative stress. This is supported by the findings that restoration of SCD1 expression in white adipose tissue of aP2-KLF15 Tg mice exhibited increased oxidative stress in white adipose tissue and reduced insulin secretion with hyperglycemia. Our data thus provide an example of cross-talk between white adipose tissue and pancreatic ß cells mediated through modulation of oxidative stress.


Asunto(s)
Adipocitos/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Insulina/metabolismo , Estearoil-CoA Desaturasa/biosíntesis , Factores de Transcripción/metabolismo , Adipocitos/patología , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Comunicación Celular/genética , Línea Celular , Proteínas de Unión al ADN/genética , Glucosa/genética , Insulina/genética , Resistencia a la Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Transgénicos , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Estrés Oxidativo/genética , Ratas , Estearoil-CoA Desaturasa/genética , Factores de Transcripción/genética
4.
Cell Metab ; 3(4): 267-75, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16581004

RESUMEN

STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. The mechanism by which hepatic STAT3 is regulated by nutritional or hormonal status has remained unknown, however. Here, we show that an increase in the plasma insulin concentration, achieved either by glucose administration or by intravenous insulin infusion, stimulates tyrosine phosphorylation of STAT3 in the liver. This effect of insulin was mediated by the hormone's effects in the brain, and the increase in hepatic IL-6 induced by the brain-insulin action is essential for the activation of STAT3. The inhibition of hepatic glucose production and of expression of gluconeogenic genes induced by intracerebral ventricular insulin infusion was impaired in mice with liver-specific STAT3 deficiency or in mice with IL-6 deficiency. These results thus indicate that IL-6-STAT3 signaling in the liver contributes to insulin action in the brain, leading to the suppression of hepatic glucose production.


Asunto(s)
Encéfalo/metabolismo , Glucosa/metabolismo , Insulina/fisiología , Hígado/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Activación Enzimática , Gluconeogénesis , Glucosa/farmacología , Técnica de Clampeo de la Glucosa , Glucosa-6-Fosfatasa/fisiología , Homeostasis , Insulina/administración & dosificación , Insulina/sangre , Insulina/farmacología , Resistencia a la Insulina , Interleucina-6/análisis , Interleucina-6/fisiología , Macrófagos del Hígado/química , Macrófagos del Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfoenolpiruvato Carboxilasa/fisiología , Fosforilación , Receptor de Insulina/fisiología , Transducción de Señal
5.
J Biol Chem ; 285(23): 17442-52, 2010 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-20353945

RESUMEN

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.


Asunto(s)
MicroARNs/metabolismo , Compuestos de Anilina/farmacología , Animales , Compuestos de Bencilideno/farmacología , Transporte Biológico , Biomarcadores de Tumor/metabolismo , Células COS , Chlorocebus aethiops , Medios de Cultivo Condicionados/metabolismo , Exosomas/metabolismo , Silenciador del Gen , Humanos , Neoplasias/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo
6.
J Clin Invest ; 118(8): 2808-21, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18654663

RESUMEN

White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPARgamma in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity.


Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo Blanco/metabolismo , Metabolismo Energético/fisiología , Lipólisis/fisiología , Proteínas/metabolismo , Células 3T3-L1 , Adipocitos/citología , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/ultraestructura , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/ultraestructura , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Cruzamientos Genéticos , Regulación de la Expresión Génica , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Peso Molecular , Proteínas/química , Proteínas/genética , ARN Interferente Pequeño/metabolismo
7.
Biochem Biophys Res Commun ; 379(1): 98-103, 2009 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-19094967

RESUMEN

KLF15 (Krüppel-like factor 15) plays a key role in adipocyte differentiation and glucose transport in adipocytes through activation of its target genes. We have now identified six target genes regulated directly by KLF15 in 3T3-L1 mouse adipocytes with the use of a combination of microarray-based chromatin immunoprecipitation and gene expression analyses. We confirmed the direct regulation by KLF15 of one of these genes, that for adrenomedullin, with the use of a luciferase reporter assay in 3T3-L1 preadipocytes and adipocytes. Such analysis revealed that the most proximal CACCC element in the promoter of the human adrenomedullin gene (located in the region spanning nucleotides -70 and -29) is required for trans-inhibition by KLF15. Furthermore, chromatin immunoprecipitation showed that KLF15 binds to this region of the human adrenomedullin gene promoter in cultured human adipocytes. These results thus implicate KLF15 in the regulation of adrenomedullin expression in adipose tissue.


Asunto(s)
Adipocitos/metabolismo , Adrenomedulina/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Células 3T3 , Adrenomedulina/antagonistas & inhibidores , Animales , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Humanos , Factores de Transcripción de Tipo Kruppel , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Transcripción Genética
8.
Biochem Biophys Res Commun ; 378(3): 399-403, 2009 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-19032942

RESUMEN

In patients with various catabolic conditions, glucocorticoid excess induces skeletal muscle wasting by accelerating protein degradation via the ubiquitin-proteasome pathway. Although the transcriptional coactivator p300 has been implicated in this pathological process, regulatory mechanisms and molecular targets of its action remain unclear. Here we show that CREB-binding protein (CBP)/p300-interacting transactivator with ED-rich tail 2 (Cited2), which binds to the cysteine-histidine-rich region 1 of p300 and CBP, regulates muscle mass in vitro. Adenovirus-mediated overexpression of wild-type Cited2 significantly blocked morphological alterations of C2C12 myotubes with a concomitant decrease in myosin heavy chain protein in response to synthetic glucocorticoid dexamethasone, which were attributable to the reduced induction of atrophy-related ubiquitin ligases MuRF1 and MAFbx. These myotube-sparing effects were less pronounced, however, with a carboxyl-terminally truncated mutant of Cited2 that lacked the ability to bind p300. These results suggest that the gain of Cited2 function counteracts glucocorticoid-induced muscle atrophy through inhibition of proteolysis mediated by p300-dependent gene transcription.


Asunto(s)
Dexametasona/toxicidad , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/inducido químicamente , Atrofia Muscular/metabolismo , Proteínas Represoras/fisiología , Transactivadores/fisiología , Animales , Glucocorticoides/toxicidad , Humanos , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/metabolismo , Atrofia Muscular/patología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Ligasas SKP Cullina F-box/antagonistas & inhibidores , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Factores de Transcripción p300-CBP/metabolismo
9.
Biochem Biophys Res Commun ; 379(2): 249-54, 2009 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-19109928

RESUMEN

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) complex. Genetic ablation of p27(Kip1) in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27(Kip1) by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2(-/-) MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), largely restored lipid accumulation and PPARgamma gene expression in Skp2(-/-) MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27(Kip1) expression.


Asunto(s)
Adipocitos/fisiología , Adipogénesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/citología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Animales , Cromanos/farmacología , Embrión de Mamíferos/citología , Ratones , Ratones Noqueados , Proteínas Quinasas Asociadas a Fase-S/genética , Tiazolidinedionas/farmacología , Troglitazona
10.
Biochem Biophys Res Commun ; 381(4): 537-43, 2009 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-19233136

RESUMEN

The transcriptional regulator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1alpha expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1alpha transcript (designated PGC-1alpha-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1alpha-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca(2+)- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1alpha-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1alpha expression in contracting muscle.


Asunto(s)
Proteínas de Choque Térmico/genética , Contracción Muscular/genética , Músculo Esquelético/fisiología , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Transcripción Genética , Activación Transcripcional , Secuencia de Bases , Calcio/farmacología , AMP Cíclico/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción p300-CBP/metabolismo
11.
Diabetes ; 56(4): 1000-9, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17267763

RESUMEN

Phosphoinositide-dependent kinase-1 (PDK1) is implicated in the metabolic effects of insulin as a key mediator of phosphoinositide 3-kinase-dependent signaling. Here we show that mice with liver-specific PDK1 deficiency manifest various defects in the metabolic actions of insulin in the liver as well as a type 2 diabetes-like phenotype characterized by marked hyperinsulinemia and postprandial hyperglycemia. The hepatic abundance of glucokinase, an important determinant of glucose flux and glucose-evoked signaling in hepatocytes, was substantially reduced in these mice. Restoration of hepatic glucokinase expression, with the use of an adenoviral vector, induced insulin-like effects in the liver and almost completely normalized the fasting hyperinsulinemia and postprandial hyperglycemia in these animals. These results indicate that, if the hepatic abundance of glucokinase is maintained, ingested glucose is normally disposed of even in the absence of acute activation of proximal insulin signaling, such as the activation of Akt, in the liver.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glucoquinasa/genética , Hígado/enzimología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Cartilla de ADN , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Insulina/farmacología , Ratones , Ratones Transgénicos , Periodo Posprandial , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
12.
J Clin Invest ; 112(6): 935-44, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12975478

RESUMEN

PKClambda is implicated as a downstream effector of PI3K in insulin action. We show here that mice that lack PKClambda specifically in the liver (L-lambdaKO mice), produced with the use of the Cre-loxP system, exhibit increased insulin sensitivity as well as a decreased triglyceride content and reduced expression of the sterol regulatory element-binding protein-1c (SREBP-1c) gene in the liver. Induction of the hepatic expression of Srebp1c and of its target genes involved in fatty acid/triglyceride synthesis by fasting and refeeding or by hepatic expression of an active form of PI3K was inhibited in L-lambdaKO mice compared with that in control animals. Expression of Srebp1c induced by insulin or by active PI3K in primary cultured rat hepatocytes was inhibited by a dominant-negative form of PKClambda and was mimicked by overexpression of WT PKClambda. Restoration of PKClambda expression in the liver of L-lambdaKO mice with the use of adenovirus-mediated gene transfer corrected the metabolic abnormalities of these animals. Hepatic PKClambda is thus a determinant of hepatic lipid content and whole-body insulin sensitivity.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Insulina/metabolismo , Metabolismo de los Lípidos , Hígado/fisiología , Proteína Quinasa C/metabolismo , Animales , Glucemia/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Isoenzimas , Hígado/química , Masculino , Ratones , Ratones Noqueados , Fenotipo , Proteína Quinasa C/genética , Ratas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Distribución Tisular , Factores de Transcripción/metabolismo
13.
Nat Med ; 18(4): 612-7, 2012 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-22426420

RESUMEN

During fasting, induction of hepatic gluconeogenesis is crucial to ensure proper energy homeostasis. Such induction is dysregulated in type 2 diabetes, resulting in the development of fasting hyperglycemia. Hormonal and nutrient regulation of metabolic adaptation during fasting is mediated predominantly by the transcriptional coactivator peroxisome proliferative activated receptor γ coactivator 1α (PGC-1α) in concert with various other transcriptional regulators. Although CITED2 (CBP- and p300-interacting transactivator with glutamic acid- and aspartic acid-rich COOH-terminal domain 2) interacts with many of these molecules, the role of this protein in the regulation of hepatic gluconeogenesis was previously unknown. Here we show that CITED2 is required for the regulation of hepatic gluconeogenesis through PGC-1α. The abundance of CITED2 was increased in the livers of mice by fasting and in cultured hepatocytes by glucagon-cAMP-protein kinase A (PKA) signaling, and the amount of CITED2 in liver was higher in mice with type 2 diabetes than in non-diabetic mice. CITED2 inhibited the acetylation of PGC-1α by blocking its interaction with the acetyltransferase general control of amino acid synthesis 5-like 2 (GCN5). The consequent downregulation of PGC-1α acetylation resulted in an increase in its transcriptional coactivation activity and an increased expression of gluconeogenic genes. The interaction of CITED2 with GCN5 was disrupted by insulin in a manner that was dependent on phosphoinositide 3-kinase (PI3K)-thymoma viral proto-oncogene (Akt) signaling. Our results show that CITED2 functions as a transducer of glucagon and insulin signaling in the regulation of PGC-1α activity that is associated with the transcriptional control of gluconeogenesis and that this function is mediated through the modulation of GCN5-dependent PGC-1α acetylation. We also found that loss of hepatic CITED2 function suppresses gluconeogenesis in diabetic mice, suggesting it as a therapeutic target for hyperglycemia.


Asunto(s)
Gluconeogénesis/fisiología , Glucosa/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Acetilación/efectos de los fármacos , Acetiltransferasas/metabolismo , Adenoviridae/genética , Animales , Células Cultivadas , Cromonas/farmacología , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Gluconeogénesis/genética , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores de Leptina/deficiencia , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirtuinas/genética , Sirtuinas/metabolismo , Transactivadores/genética , Factores de Transcripción , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismo
14.
Diabetes ; 61(11): 2823-32, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22837305

RESUMEN

It is unknown whether adipokines derived from adipose tissues modulate endoplasmic reticulum (ER) stress induced in obesity. Here, we show that visceral adipose tissue-derived serine protease inhibitor (vaspin) binds to cell-surface 78-kDa glucose-regulated protein (GRP78), which is recruited from ER to plasma membrane under ER stress. Vaspin transgenic mice were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis, while vaspin-deficient mice developed glucose intolerance associated with upregulation of ER stress markers. With tandem affinity tag purification using HepG2 cells, we identified GRP78 as an interacting molecule. The complex formation of vaspin, GRP78, and murine tumor cell DnaJ-like protein 1 (MTJ-1) (DnaJ homolog, subfamily C, member 1) on plasma membrane was confirmed by cell-surface labeling with biotin and immunoprecipitation in liver tissues and H-4-II-E-C3 cells. The addition of recombinant human vaspin in the cultured H-4-II-E-C3 cells also increased the phosphorylation of Akt and AMP-activated protein kinase (AMPK) in a dose-dependent manner, and anti-GRP78 antibodies completely abrogated the vaspin-induced upregulation of pAkt and pAMPK. Vaspin is a novel ligand for cell-surface GRP78/MTJ-1 complex, and its subsequent signals exert beneficial effects on ER stress-induced metabolic dysfunctions.


Asunto(s)
Adipoquinas/metabolismo , Estrés del Retículo Endoplásmico , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Grasa Intraabdominal/metabolismo , Proteínas de Neoplasias/metabolismo , Obesidad/metabolismo , Serpinas/metabolismo , Adipoquinas/química , Adipoquinas/genética , Adipoquinas/aislamiento & purificación , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Chaperón BiP del Retículo Endoplásmico , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Ligandos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Obesidad/patología , Transporte de Proteínas , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Serpinas/química , Serpinas/genética , Serpinas/aislamiento & purificación , Transducción de Señal , Regulación hacia Arriba
15.
Diabetes ; 59(7): 1608-15, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20393151

RESUMEN

OBJECTIVE: An increase in the rate of gluconeogenesis is largely responsible for the hyperglycemia in individuals with type 2 diabetes, with the antidiabetes action of metformin being thought to be achieved at least in part through suppression of gluconeogenesis. RESEARCH DESIGN AND METHODS: We investigated whether the transcription factor KLF15 has a role in the regulation of gluconeogenesis and whether KLF15 participates in the antidiabetes effect of metformin. RESULTS: Here we show that KLF15 regulates the expression of genes for gluconeogenic or amino acid-degrading enzymes in coordination with the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha. Liver-specific ablation of KLF15 in diabetic mice resulted in downregulation of the expression of genes for gluconeogenic or amino acid catabolic enzymes and in amelioration of hyperglycemia. Exposure of cultured hepatocytes to metformin reduced the abundance of KLF15 through acceleration of its degradation and downregulation of its mRNA. Metformin suppressed the expression of genes for gluconeogenic or amino acid-degrading enzymes in cultured hepatocytes, and these effects of metformin were attenuated by restoration of KLF15 expression. Administration of metformin to mice inhibited both the expression of KLF15 and glucose production in the liver, the latter effect also being attenuated by restoration of hepatic KLF15 expression. CONCLUSIONS: KLF15 plays an important role in regulation of the expression of genes for gluconeogenic and amino acid-degrading enzymes and that the inhibitory effect of metformin on gluconeogenesis is mediated at least in part by downregulation of KLF15 and consequent attenuation of the expression of such genes.


Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Gluconeogénesis/genética , Hepatocitos/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Hígado/metabolismo , Metformina/metabolismo , Animales , Glucemia/metabolismo , Western Blotting , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Expresión Génica/efectos de los fármacos , Gluconeogénesis/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hiperglucemia/genética , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacología , Hígado/efectos de los fármacos , Masculino , Metformina/farmacología , Ratones , Ratones Transgénicos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
16.
J Mol Endocrinol ; 42(2): 161-9, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19060180

RESUMEN

Gene related to anergy in lymphocytes (GRAIL) is an E3 ubiquitin ligase that regulates energy in T-lymphocytes. Whereas, the relevance of GRAIL to T lymphocyte function is well established, the role of this protein in other cell types remains unknown. Given that GRAIL is abundant in the liver, we investigated the potential function of GRAIL in nutrient metabolism by generating mice in which the expression of GRAIL is reduced specifically in the liver. Adenovirus-mediated transfer of a short hairpin RNA specific for GRAIL mRNA markedly reduced the amounts of GRAIL mRNA and protein in the liver. Blood glucose levels of the mice with hepatic GRAIL deficiency did not differ from those of control animals in the fasted or fed states. However, these mice manifested glucose intolerance in association with a normal increase in plasma insulin levels during glucose challenge. The mice also manifested an increase in the serum concentration of free fatty acids, whereas the serum levels of cholesterol and triglyceride were unchanged. The hepatic abundance of mRNAs for glucose-6-phosphatase, catalytic (a key enzyme in hepatic glucose production) and for sterol regulatory element-binding transcription factor 1 (an important transcriptional regulator of lipogenesis) was increased in the mice with hepatic GRAIL deficiency, possibly contributing to the metabolic abnormalities of these animals. Our results thus demonstrate that GRAIL in the liver is essential for maintenance of normal glucose and lipid metabolism in living animals.


Asunto(s)
Anergia Clonal/inmunología , Glucosa/metabolismo , Metabolismo de los Lípidos , Hígado/enzimología , Linfocitos/enzimología , Linfocitos/inmunología , Ubiquitina-Proteína Ligasas/genética , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo
17.
Endocrinology ; 150(6): 2683-91, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19264873

RESUMEN

Physical exercise ameliorates metabolic disorders such as type 2 diabetes mellitus and obesity, but the molecular basis of these effects remains elusive. In the present study, we found that exercise up-regulates heparin-binding epidermal growth factor-like growth factor (HB-EGF) in skeletal muscle. To address the metabolic consequences of such gain of HB-EGF function, we generated mice that overexpress this protein specifically in muscle. The transgenic animals exhibited a higher respiratory quotient than did wild-type mice during indirect calorimetry, indicative of their selective use of carbohydrate rather than fat as an energy substrate. They also showed substantial increases in glucose tolerance, insulin sensitivity, and glucose uptake by skeletal muscle. These changes were accompanied by increased kinase activity of Akt in skeletal muscle and consequent inhibition of Forkhead box O1-dependent expression of the pyruvate dehydrogenase kinase 4 gene. Furthermore, mice with a high level of transgene expression were largely protected from obesity, hepatic steatosis, and insulin resistance, even when maintained on a high-fat diet. Our results suggest that HB-EGF produced by contracting muscle acts as an insulin sensitizer that facilitates peripheral glucose disposal.


Asunto(s)
Glucosa/metabolismo , Resistencia a la Insulina/fisiología , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Modelos Animales de Enfermedad , Metabolismo Energético/fisiología , Hígado Graso/prevención & control , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Factor de Crecimiento Similar a EGF de Unión a Heparina , Homeostasis/fisiología , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Transgénicos , Obesidad/prevención & control , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo
18.
Kobe J Med Sci ; 54(4): E200-8, 2008 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-19258740

RESUMEN

Regulation of hepatic gene expression is largely responsible for the control of nutrient metabolism. We previously showed that the transcription factor STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. However, the role of STAT3 in the control of lipid metabolism has remained unknown. We have now investigated the effects of hepatic overexpression of STAT3, achieved by adenovirus-mediated gene transfer, on glucose and lipid metabolism in insulin-resistant diabetic mice. Forced expression of STAT3 reduced blood glucose and plasma insulin concentrations as well as the hepatic abundance of mRNA for phosphoenolpyruvate carboxykinase. However, it also increased the plasma levels of triglyceride and total cholesterol without affecting those of low density lipoprotein- or high density lipoprotein-cholesterol. The hepatic abundance of mRNAs for fatty acid synthase and acetyl-CoA carboxylase, both of which catalyze the synthesis of fatty acids, was increased by overexpression of STAT3, whereas that of mRNAs for sterol regulatory element-binding proteins 1a, 1c, or 2 was unaffected. Moreover, the amount of mRNA for acyl-CoA oxidase, which contributes to beta-oxidation, was decreased by forced expression of STAT3. These results indicate that forced activation of STAT3 signaling in the liver of insulin-resistant diabetic mice increased the circulating levels of atherogenic lipids through changes in the hepatic expression of genes involved in lipid metabolism. Furthermore, these alterations in hepatic gene expression likely occurred through a mechanism independent of sterol regulatory element-binding proteins.


Asunto(s)
Metabolismo de los Lípidos , Hígado/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Glucemia/metabolismo , Regulación de la Expresión Génica , Gluconeogénesis/genética , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/genética , Receptores de Leptina/deficiencia , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Factor de Transcripción STAT3/genética
19.
J Biol Chem ; 283(25): 17702-11, 2008 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-18430722

RESUMEN

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).


Asunto(s)
Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Adenoviridae/metabolismo , Animales , Apoptosis , Ciclo Celular , Proliferación Celular , ADN/química , Fibroblastos/metabolismo , Homocigoto , Ratones , Ratones Noqueados , Modelos Biológicos , Oligonucleótidos/química , Proteínas Serina-Treonina Quinasas/genética , Factores de Tiempo
20.
Nat Med ; 14(2): 188-93, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18204460

RESUMEN

Insulin receptor substrate (IRS)-1 and IRS-2 have dominant roles in the action of insulin, but other substrates of the insulin receptor kinase, such as Gab1, c-Cbl, SH2-B and APS, are also of physiological relevance. Although the protein downstream of tyrosine kinases-1 (Dok1) is known to function as a multisite adapter molecule in insulin signaling, its role in energy homeostasis has remained unclear. Here we show that Dok1 regulates adiposity. Expression of Dok1 in white adipose tissue was markedly increased in mice fed a high-fat diet, whereas adipocytes lacking this adapter were smaller and showed a reduced hypertrophic response to this dietary manipulation. Dok1-deficient mice were leaner and showed improved glucose tolerance and insulin sensitivity compared with wild-type mice. Embryonic fibroblasts from Dok1-deficient mice were impaired in adipogenic differentiation, and this defect was accompanied by an increased activity of the protein kinase ERK and a consequent increase in the phosphorylation of peroxisome proliferator-activated receptor (PPAR)-gamma on Ser112. Mutation of this negative regulatory site for the transactivation activity of PPAR-gamma blocked development of the lean phenotype caused by Dok1 ablation. These results indicate that Dok1 promotes adipocyte hypertrophy by counteracting the inhibitory effect of ERK on PPAR-gamma and may thus confer predisposition to diet-induced obesity.


Asunto(s)
Adipocitos/patología , Proteínas de Unión al ADN/metabolismo , Dieta , Ácidos Grasos/farmacología , Obesidad/patología , PPAR gamma/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/patología , Adiposidad/efectos de los fármacos , Animales , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Glucosa/metabolismo , Hipertrofia , Insulina/metabolismo , Ratones , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Proteínas de Unión al ARN/genética , Regulación hacia Arriba/efectos de los fármacos
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