RESUMEN
Excessive caloric intake without a rise in energy expenditure promotes adipocyte hyperplasia and adiposity. The rise in adipocyte number is triggered by signaling factors that induce conversion of mesenchymal stem cells (MSCs) to preadipocytes that differentiate into adipocytes. MSCs, which are recruited from the vascular stroma of adipose tissue, provide an unlimited supply of adipocyte precursors. Members of the BMP and Wnt families are key mediators of stem cell commitment to produce preadipocytes. Following commitment, exposure of growth-arrested preadipocytes to differentiation inducers [insulin-like growth factor 1 (IGF1), glucocorticoid, and cyclic AMP (cAMP)] triggers DNA replication and reentry into the cell cycle (mitotic clonal expansion). Mitotic clonal expansion involves a transcription factor cascade, followed by the expression of adipocyte genes. Critical to these events are phosphorylations of the transcription factor CCATT enhancer-binding protein ß (C/EBPß) by MAP kinase and GSK3ß to produce a conformational change that gives rise to DNA-binding activity. "Activated" C/EBPß then triggers transcription of peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which in turn coordinately activate genes whose expression produces the adipocyte phenotype.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis , Animales , Humanos , Transducción de Señal , Células Madre/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Obesity is accompanied by an increase in both adipocyte number and size. The increase in adipocyte number is the result of recruitment to the adipocyte lineage of pluripotent stem cells present in the vascular stroma of adipose tissue. These pluripotent cells have the potential to undergo commitment and then differentiate into adipocytes, as well as myocytes, osteocytes, and chondrocytes. In this article, we show that both bone morphogenetic protein (BMP)2 and BMP4 can induce commitment of C3H10T1/2 pluripotent stem cells into adipocytes. After treatment of C3H10T1/2 stem cells with these BMPs during proliferation followed by exposure to differentiation inducers at growth arrest, nearly all cells enter the adipose development pathway, express specific adipocyte markers, and acquire the adipocyte phenotype. Overexpression of constitutively active BMP receptor (CA)-BMPr1A or CA-BMPr1B induces commitment in the absence of BMP2/4, whereas overexpression of a dominant-negative receptor dominant-negative-BMPr1A suppresses commitment induced by BMP. Also, knockdown of the expression of Smad4 (coregulator in the BMP/Smad signaling pathway) with RNAi disrupts commitment by the BMPs. However, knockdown of expression of p38 MAPK (an intermediary in the BMP/MAPK signaling pathway) with RNAi had little effect on BMP-induced commitment. Together, these findings indicate that the BMP/Smad signaling pathway has a dominant role in adipocyte lineage determination. Proteomic analysis identified lysyl oxidase (LOX), a bona fide downstream target gene of the BMP signaling pathway. Expression of LOX is induced by BMP2/4 during adipocyte lineage commitment, and knockdown of its expression disrupts the commitment process.
Asunto(s)
Adipocitos/citología , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 4/farmacología , Linaje de la Célula , Células Madre Pluripotentes/citología , Transducción de Señal/fisiología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Ratones , Proteína-Lisina 6-Oxidasa/fisiología , Proteínas Smad/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Increased O-linked beta-N-acetylglucosamine (O-GlcNAc) is associated with insulin resistance in muscle and adipocytes. Upon insulin treatment of insulin-responsive adipocytes, O-GlcNAcylation of several proteins is increased. Key insulin signaling proteins, including IRS-1, IRS-2, and PDK1, are substrates for OGT, suggesting potential O-GlcNAc control points within the pathway. To elucidate the roles of O-GlcNAc in dampening insulin signaling (Vosseller, K., Wells, L., Lane, M. D., and Hart, G. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5313-5318), we focused on the pathway upstream of AKT. Increasing O-GlcNAc in 3T3-L1 adipocytes decreases phosphoinositide 3-kinase (PI3K) interactions with both IRS-1 and IRS-2. Elevated O-GlcNAc also reduces phosphorylation of the PI3K p85 binding motifs (YXXM) of IRS-1 and results in a concomitant reduction in tyrosine phosphorylation of Y(608)XXM in IRS-1, one of the two main PI3K p85 binding motifs. Additionally, insulin signaling stimulates the interaction of OGT with PDK1. We conclude that one of the steps at which O-GlcNAc contributes to insulin resistance is by inhibiting phosphorylation at the Y(608)XXM PI3K p85 binding motif in IRS-1 and possibly at PDK1 as well.
Asunto(s)
Acetilglucosamina/metabolismo , Adipocitos/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Células 3T3-L1 , Acetilglucosamina/genética , Secuencias de Aminoácidos/fisiología , Animales , Humanos , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina/genética , Resistencia a la Insulina , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Transducción de Señal/efectos de los fármacosRESUMEN
The American diet, especially that of adolescents, contains highly palatable foods of high-energy content and large amounts of high-fructose sweeteners. These factors are believed to contribute to the obesity epidemic and insulin resistance. Previous investigations revealed that the central metabolism of glucose suppresses food intake mediated by the hypothalamic AMP-kinase/malonyl-CoA signaling system. Unlike glucose, centrally administered fructose increases food intake. Evidence presented herein indicates that the more rapid initial steps of central fructose metabolism deplete hypothalamic ATP level, whereas the slower regulated steps of glucose metabolism elevate hypothalamic ATP level. Consistent with effects on the [ATP]/[AMP] ratio, fructose increases phosphorylation/activation of hypothalamic AMP kinase causing phosphorylation/inactivation of acetyl-CoA carboxylase, whereas glucose has the inverse effects. The changes provoked by central fructose administration reduce hypothalamic malonyl-CoA level and thereby increase food intake. These findings explain the paradoxical fructose effect on food intake and lend credence to the malonyl-CoA hypothesis.
Asunto(s)
Ingestión de Alimentos/fisiología , Fructosa/farmacología , Glucosa/farmacología , Hipotálamo/metabolismo , Malonil Coenzima A/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/metabolismo , Animales , Glucemia/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Fructosa/administración & dosificación , Glucosa/administración & dosificación , Hipotálamo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/metabolismo , FosforilaciónRESUMEN
Central nervous system control of energy balance affects susceptibility to obesity and diabetes, but how fatty acids, malonyl-CoA, and other metabolites act at this site to alter metabolism is poorly understood. Pharmacological inhibition of fatty acid synthase (FAS), rate limiting for de novo lipogenesis, decreases appetite independently of leptin but also promotes weight loss through activities unrelated to FAS inhibition. Here we report that the conditional genetic inactivation of FAS in pancreatic beta cells and hypothalamus produced lean, hypophagic mice with increased physical activity and impaired hypothalamic PPARalpha signaling. Administration of a PPARalpha agonist into the hypothalamus increased PPARalpha target genes and normalized food intake. Inactivation of beta cell FAS enzyme activity had no effect on islet function in culture or in vivo. These results suggest a critical role for brain FAS in the regulation of not only feeding, but also physical activity, effects that appear to be mediated through the provision of ligands generated by FAS to PPARalpha. Thus, 2 diametrically opposed proteins, FAS (induced by feeding) and PPARalpha (induced by starvation), unexpectedly form an integrative sensory module in the central nervous system to orchestrate energy balance.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Homeostasis , PPAR alfa/metabolismo , Animales , Conducta Apetitiva , Peso Corporal , Diferenciación Celular , Activación Enzimática , Ácido Graso Sintasas/deficiencia , Ácido Graso Sintasas/genética , Femenino , Hormonas/sangre , Enfermedades Hipotalámicas/enzimología , Enfermedades Hipotalámicas/patología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/enzimología , Masculino , Ratones , Ratones Noqueados , PPAR alfa/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
Hypothalamic malonyl-CoA has been shown to function in global energy homeostasis by modulating food intake and energy expenditure. Little is known, however, about the regulation of malonyl-CoA concentration in the central nervous system. To address this issue we investigated the response of putative intermediates in the malonyl-CoA pathway to metabolic and endocrine cues, notably those provoked by glucose and leptin. Hypothalamic malonyl-CoA rises in proportion to the carbohydrate content of the diet consumed after food deprivation. Malonyl-CoA concentration peaks 1 h after refeeding or after peripheral glucose administration. This response depends on the dose of glucose administered and is blocked by the i.c.v. administration of an inhibitor of glucose metabolism, 2-deoxyglucose (2-DG). The kinetics of change in hypothalamic malonyl-CoA after glucose administration is coincident with the suppression of phosphorylation of AMP kinase and acetyl-CoA carboxylase. Blockade of glucose utilization in the CNS by i.c.v. 2-DG prevented the effects of glucose on 5'AMP-activated protein kinase, malonyl-CoA, hypothalamic neuropeptide expression, and food intake. Finally, we showed that leptin can increase hypothalamic malonyl-CoA and that the increase is additive with glucose administration. Leptin-deficient ob/ob mice, however, showed no defect in the glucose- or refeeding-induced rise in hypothalamic malonyl-CoA after food deprivation, demonstrating that leptin was not required for this effect. These studies show that hypothalamic malonyl-CoA responds to the level of circulating glucose and leptin, both of which affect energy homeostasis.
Asunto(s)
Glucosa/metabolismo , Hipotálamo/metabolismo , Leptina/metabolismo , Malonil Coenzima A/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Adenilato Quinasa/metabolismo , Proteína Relacionada con Agouti/genética , Proteína Relacionada con Agouti/metabolismo , Animales , Antimetabolitos/farmacología , Glucemia/metabolismo , Desoxiglucosa/farmacología , Carbohidratos de la Dieta/administración & dosificación , Ácidos Grasos/metabolismo , Glucosa/administración & dosificación , Glucosa/antagonistas & inhibidores , Hipotálamo/química , Hipotálamo/efectos de los fármacos , Leptina/administración & dosificación , Leptina/genética , Malonil Coenzima A/análisis , Ratones , Ratones Mutantes , Fosforilación , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismoRESUMEN
In the brain malonyl-CoA serves the important function of monitoring and modulating energy balance. Because of its central role in the metabolism of higher animals, glucose acts as the principal indicator of global energy status. Specialized neuronal nuclei within the hypothalamus sense blood glucose and signal higher brain centers to adjust feeding behavior and energy expenditure accordingly. As the level of glucose entering the brain rises, food intake is suppressed. Energy status information triggered by glucose is transmitted via hypothalamic signaling intermediaries, i.e. AMPK and malonyl-CoA, to the orexigenic/anorexigenic neuropeptide system that determines hunger and energy expenditure. The central metabolism of glucose by the glycolytic pathway generates ATP which produces a compensatory decrease in AMP level and AMPK activity. Since acetyl-CoA carboxylase (ACC) is a substrate of AMPK, lowering AMP increases the catalytic activity of ACC and thereby, the level of its reaction product, malonyl-CoA. Malonyl-CoA signals the anorexigenic-orexigenic neuropeptide system to suppress food intake. Unlike glucose, however, centrally metabolized fructose increases food intake. This paradox results because fructose bypasses the rate-limiting step of glycolysis and uses a rapid ATP-requiring reaction that abruptly depletes ATP and provokes a compensatory rise in AMP. Thus, fructose has the opposite effect of glucose on the AMPK/malonyl-CoA signaling system and thereby, feeding behavior. The fact that fructose metabolism by the brain increases food intake and obesity risk raises health concerns in view of the large and increasing per capita consumption of high fructose sweeteners, especially by youth.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Fructosa/administración & dosificación , Glucosa/administración & dosificación , Hipotálamo/enzimología , Malonil Coenzima A/metabolismo , Animales , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
Previous studies showed that centrally administered glucose and fructose exert different effects on food intake--glucose decreasing and fructose increasing food intake. Because of the uncertainty of whether fructose can cross the blood-brain-barrier, the question is raised; can dietary fructose directly enter the CNS? Evidence is presented that fructose administered by intraperitoneal (ip) injection to mice is rapidly (<10 min) converted to lactate in the hypothalamus. Thus, fructose can cross the blood-brain-barrier to enter the CNS/hypothalamus for conversion to lactate without prior (slower) conversion to glucose in the liver. Fructose-derived hypothalamic lactate is not, however, responsible for the orexigenic effect of fructose. Ip lactate administered at a level equivalent to that of fructose generates a higher level of hypothalamic lactate, which rapidly triggers dephosphorylation/inactivation of AMP-kinase. Thereby, ACC--a substrate of AMP-kinase that catalyzes malonyl-CoA formation--is dephosphorylated and activated. Consistent with these findings, ip or centrally (icv) administered lactate rapidly increases (<10 min) hypothalamic malonyl-CoA. Increasing hypothalamic malonyl-CoA suppresses the expression of the orexigenic and increases the expression of the anorexigenic neuropeptides, which decrease food intake. All downstream effects of hypothalamic lactate are blocked by icv administered oxamate, a potent inhibitor of lactate dehydrogenase, thus verifying the central action of lactate.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Hipotálamo/metabolismo , Ácido Láctico/administración & dosificación , Ácido Láctico/metabolismo , Malonil Coenzima A/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Fructosa/administración & dosificación , Fructosa/metabolismo , Hipotálamo/efectos de los fármacos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/metabolismo , Ratones , Neuropéptidos/metabolismo , Transducción de SeñalRESUMEN
An intermediate in the fatty acid biosynthetic pathway, malonyl-coenzyme A (CoA), has emerged as a major regulator of energy homeostasis not only in peripheral metabolic tissues but also in regions of the central nervous system that control satiety and energy expenditure. Fluctuations in hypothalamic malonyl-CoA lead to changes in food intake and peripheral energy expenditure in a manner consistent with an anorexigenic signaling intermediate. Hypothalamic malonyl-CoA is regulated by nutritional and endocrine cues including glucose and leptin, respectively. That malonyl-CoA is an essential component in the energy homeostatic signaling system of the hypothalamus is supported by convergence of physiological, pharmacological, and genetic evidence. This review will focus on evidence implicating malonyl-CoA as a central player in the control of body weight and adiposity as well as clues to the molecular mechanism by which carbon flux through the fatty acid biosynthetic pathway is linked to the neural control of energy balance.
Asunto(s)
Hipotálamo/metabolismo , Malonil Coenzima A/metabolismo , Adiposidad/fisiología , Animales , Peso Corporal/fisiología , Carboxiliasas/genética , Carboxiliasas/metabolismo , Sistema Nervioso Central/metabolismo , Ingestión de Alimentos/fisiología , Metabolismo Energético , Ácido Graso Sintasas/deficiencia , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Homeostasis , Humanos , Leptina/metabolismo , Ratones , Modelos Biológicos , Músculo Esquelético/metabolismo , Oxidación-Reducción , Transducción de SeñalRESUMEN
While the brain does not utilize fatty acids as a primary energy source, recent evidence shows that intermediates of fatty acid metabolism serve as hypothalamic sensors of energy status. Increased hypothalamic malonyl-CoA, an intermediate in fatty acid synthesis, is indicative of energy surplus and leads to the suppression of food intake and increased energy expenditure. Malonyl-CoA functions as an inhibitor of carnitine palmitoyl-transferase 1 (CPT1), a mitochondrial outer membrane enzyme that initiates translocation of fatty acids into mitochondria for oxidation. The mammalian brain expresses a unique homologous CPT1, CPT1c, that binds malonyl-CoA tightly but does not support fatty acid oxidation in vivo, in hypothalamic explants or in heterologous cell culture systems. CPT1c knockout (KO) mice under fasted or refed conditions do not exhibit an altered CNS transcriptome of genes known to be involved in fatty acid metabolism. CPT1c KO mice exhibit normal levels of metabolites and of hypothalamic malonyl-CoA and fatty acyl-CoA levels either in the fasted or refed states. However, CPT1c KO mice exhibit decreased food intake and lower body weight than wild-type littermates. In contrast, CPT1c KO mice gain excessive body weight and body fat when fed a high-fat diet while maintaining lower or equivalent food intake. Heterozygous mice display an intermediate phenotype. These findings provide further evidence that CPT1c plays a role in maintaining energy homeostasis, but not through altered fatty acid oxidation.
Asunto(s)
Peso Corporal/fisiología , Encéfalo/metabolismo , Carnitina O-Palmitoiltransferasa/fisiología , Ingestión de Alimentos/fisiología , Ácidos Grasos/metabolismo , Animales , Peso Corporal/genética , Encéfalo/enzimología , Células CHO , Células COS , Carnitina O-Palmitoiltransferasa/genética , Sistema Nervioso Central/enzimología , Sistema Nervioso Central/metabolismo , Chlorocebus aethiops , Cricetinae , Cricetulus , Ingestión de Alimentos/genética , Ácidos Grasos/genética , Femenino , Isoenzimas/genética , Isoenzimas/fisiología , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Masculino , Ratones , Ratones Noqueados , Oxidación-ReducciónAsunto(s)
Adipocitos/fisiología , Comunicación Celular/fisiología , Neuronas/fisiología , Neuropéptido Y/fisiología , Adipocitos/citología , Adipocitos/metabolismo , Animales , Comunicación Celular/genética , Técnicas de Cocultivo , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Humanos , Neuronas/citología , Neuronas/metabolismo , Neuropéptido Y/genéticaRESUMEN
Metabolic integration of nutrient sensing in the central nervous system has been shown to be an important regulator of adiposity by affecting food intake and peripheral energy expenditure. Modulation of de novo fatty acid synthetic flux by cytokines and nutrient availability plays an important role in this process. Inhibition of hypothalamic fatty acid synthase by pharmacologic or genetic means leads to an increased malonyl-CoA level and suppression of food intake and adiposity. Conversely, the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus is sufficient to promote feeding and adiposity. Based on these and other findings, metabolic intermediates in fatty acid biogenesis, including malonyl-CoA and long-chain acyl-CoAs, have been implicated as signaling mediators in the central control of body weight. Malonyl-CoA has been hypothesized to mediate its effects in part through an allosteric interaction with an atypical and brain-specific carnitine palmitoyltransferase-1 (CPT1c). CPT1c is expressed in neurons and binds malonyl-CoA, however, it does not perform the same biochemical function as the prototypical CPT1 enzymes. Mouse knockout models of CPT1c exhibit suppressed food intake and smaller body weight, but are highly susceptible to weight gain when fed a high-fat diet. Thus, the brain can directly sense and respond to changes in nutrient availability and composition to affect body weight and adiposity.
Asunto(s)
Carnitina O-Palmitoiltransferasa/metabolismo , Hipotálamo/metabolismo , Malonil Coenzima A/metabolismo , Obesidad/metabolismo , Animales , Fármacos Antiobesidad/uso terapéutico , Humanos , Obesidad/tratamiento farmacológicoRESUMEN
The adipose tissue has important secretory and endocrine functions in humans. The regulation of adipocyte differentiation has been actively pursued using transcriptomic methods over the last several years. Quantitative proteomics has emerged as a promising approach to obtain temporal profiles of biological processes such as differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust method for labeling proteins in vivo. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine to study the nuclear proteome and the secretome during the course of adipocyte differentiation. Tandem mass spectrometry analysis using a quadrupole time-of-flight instrument resulted in identification of a total 882 proteins from these two proteomes. Of these proteins, 427 were identified on the basis of one or more arginine-containing peptides that allowed quantitation. In addition to previously reported molecules that are differentially expressed during the process of adipogenesis (e.g., adiponectin and lipoprotein lipase), we identified several proteins whose differential expression during adipocyte differentiation has not been documented previously. For example, THO complex 4, a context-dependent transcriptional activator in the T-cell receptor alpha enhancer complex, showed highest expression at middle stage of adipogenesis, while SNF2 alpha, a chromatin remodeling protein, was downregulated upon initiation of adipogenesis and remained so during subsequent time points. This study using a 5-plex SILAC to investigate dynamics illustrates the power of this approach to identify differentially expressed proteins in a temporal fashion.
Asunto(s)
Adipocitos/metabolismo , Regulación de la Expresión Génica , Proteómica/métodos , Células 3T3-L1 , Adipogénesis , Animales , Compuestos Azo/farmacología , Diferenciación Celular , Núcleo Celular/metabolismo , Cromatografía Liquida/métodos , Isótopos , Espectrometría de Masas/métodos , Ratones , Factores de Tiempo , Tripsina/farmacologíaRESUMEN
AMP-activated protein kinase (AMPK) plays an important role in regulating whole body energy homeostasis. Recently, it has been demonstrated that berberine (BBR) exerts antiobesity and antidiabetic effects in obese and diabetic rodent models through the activation of AMPK in peripheral tissues. Here we show that BBR improves lipid dysregulation and fatty liver in obese mice through central and peripheral actions. In obese db/db and ob/ob mice, BBR treatment reduced liver weight, hepatic and plasma triglyceride, and cholesterol contents. In the liver and muscle of db/db mice, BBR promoted AMPK activity and fatty acid oxidation and changed expression of genes involved in lipid metabolism. Additionally, intracerebroventricular administration of BBR decreased the level of malonyl-CoA and stimulated the expression of fatty acid oxidation genes in skeletal muscle. Together, these data suggest that BBR would improve fatty liver in obese subjects, which is probably mediated not only by peripheral AMPK activation but also by neural signaling from the central nervous system.
Asunto(s)
Adenilato Quinasa/metabolismo , Berberina/farmacología , Berberina/uso terapéutico , Dislipidemias/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Animales , Células Cultivadas , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/genética , Evaluación Preclínica de Medicamentos , Dislipidemias/complicaciones , Dislipidemias/genética , Dislipidemias/metabolismo , Activación Enzimática/efectos de los fármacos , Ácidos Grasos/metabolismo , Hígado Graso/complicaciones , Hígado Graso/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Hipolipemiantes/farmacología , Hipolipemiantes/uso terapéutico , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Oxidación-Reducción/efectos de los fármacos , Receptores de Leptina/genéticaRESUMEN
CCAAT enhancer-binding protein (C/EBP)beta is a basic leucine zipper transcription factor family member, and can be phosphorylated, acetylated, and sumoylated. C/EBPbeta undergoes sequential phosphorylation during 3T3-L1 adipocyte differentiation. Phosphorylation on Thr(188) by MAPK or cyclin A/cdk2 primes the phosphorylations on Ser(184)/Thr(179) by GSK3beta, and these phosphorylations are required for the acquisition of DNA binding activity of C/EBPbeta. Here we show that C/EBPbeta is modified by O-GlcNAc, a dynamic single sugar modification found on nucleocytoplasmic proteins. The GlcNAcylation sites are Ser(180) and Ser(181), which are in the regulation domain and are very close to the phosphorylation sites (Thr(188), Ser(184), and Thr(179)) required for the gain of DNA binding activity. Both in vitro and ex vivo experiments demonstrate that GlcNAcylation on Ser(180) and Ser(181) prevents phosphorylation on Thr(188), Ser(184), and Thr(179), as indicated by the decreased relative phosphorylation and DNA binding activity of C/EBPbeta delayed the adipocyte differentiation program. Mutation of both Ser(180) and Ser(181) to Ala significantly increase the transcriptional activity of C/EBPbeta. These data suggest that GlcNAcylation regulates both the phosphorylation and DNA binding activity of C/EBPbeta.
Asunto(s)
Acetilglucosamina/metabolismo , Adipocitos/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular , Células 3T3-L1 , Adipocitos/citología , Secuencia de Aminoácidos , Animales , Western Blotting , Proteína beta Potenciadora de Unión a CCAAT/química , Proteína beta Potenciadora de Unión a CCAAT/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Glicosilación , Luciferasas/genética , Luciferasas/metabolismo , Espectrometría de Masas/métodos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Fragmentos de Péptidos/química , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tripsina/metabolismoRESUMEN
Obesity is characterized by an increase in the number mature fat cells. These nascent adipocytes are derived from preadipocytes, which in turn are derived from mesenchymal stem cells (MSCs). Since little is known about the mechanisms controlling the commitment of MSCs into preadipocytes, this early event in adipogenesis was further investigated. C3H10T1/2 cells (10T1/2 cells) were employed as a MSC model and a committed A33 preadipocyte cell line derived from these cells served as a model of preadipocytes. Microarray technology was used to identify genes that are differentially expressed in pluripotent 10T1/2 cells when compared with A33 preadipocytes. Several key genes of the Wnt signaling pathway were differentially expressed between 10T1/2 and A33 cells as demonstrated by microarray and quantitative real-time RT-PCR analyses. Of particular interest, R-spondins-2 and -3, newly described molecules that activate the canonical Wnt signaling pathway, are markedly upregulated in proliferating A33 cells compared to 10T1/2 cells. Consistent with these findings beta-catenin accumulates in the nuclei of proliferating A33 cells, but not 10T1/2 cells. In addition, several members of the Lef/Tcf family of transcription factors involved in Wnt signaling are also differentially expressed between 10T1/2 and A33 cells. These and other findings indicate that activation of Wnt signaling is an early event in adipogenesis.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis/fisiología , Tejido Adiposo/metabolismo , Linaje de la Célula/genética , Células Madre Mesenquimatosas/metabolismo , Proteínas Wnt/metabolismo , Adipocitos/citología , Tejido Adiposo/citología , Animales , Diferenciación Celular/genética , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/genética , Células Madre Mesenquimatosas/citología , Ratones , Modelos Biológicos , Obesidad/metabolismo , Obesidad/fisiopatología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Trombospondinas/genética , Trombospondinas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genética , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMEN
O-Linked beta-N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the addition of O-linked beta-N-acetylglucosamine (O-GlcNAc) onto serine and threonine residues in response to stimuli or stress analogous to phosphorylation by Ser/Thr-kinases. Like protein phosphatases, OGT appears to be targeted to myriad specific substrates by transiently interacting with specific targeting subunits. Here, we show that OGT is activated by insulin signaling. Insulin treatment of 3T3-L1 adipocytes stimulates both tyrosine phosphorylation and catalytic activity of OGT. A subset of OGT co-immunoprecipitates with the insulin receptor. Insulin stimulates purified insulin receptor to phosphorylate OGT in vitro. OGT is a competitive substrate with reduced and carboxyamidomethylated lysozyme (RCAM-lysozyme), a well characterized insulin receptor substrate. Insulin stimulation of 3T3-L1 adipocytes results in a partial translocation of OGT from the nucleus to the cytoplasm. The insulin activation of OGT results in increased O-GlcNAc modification of OGT and other proteins including, signal transducer and activator of transcription 3 (STAT3). We conclude that insulin stimulates the tyrosine phosphorylation and activity of OGT.
Asunto(s)
Acetilglucosamina/química , Regulación Enzimológica de la Expresión Génica , Insulina/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Células 3T3-L1 , Acetilglucosamina/metabolismo , Adipocitos/citología , Animales , Insectos , Ratones , Modelos Biológicos , Muramidasa/química , Receptor de Insulina/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
Obesity is characterized by increases in the number of mature adipocytes. Nascent adipocytes arise from mesenchymal stem cells (MSCs) by a multi-step process--MSCs are recruited to the adipocyte lineage forming determined preadipocytes, these committed progenitors proliferate, undergo growth arrest, and finally differentiate into mature adipocytes. Although the genetic mechanisms that control the differentiation of preadipocytes into mature adipocytes are understood to a large extent, the earliest events in adipogenesis--especially the commitment of MSCs into preadipocytes--are largely unknown. Recently, bone morphogenetic protein-4 (BMP-4) has been implicated in the commitment of pluripotent MSCs to the adipocyte lineage by two independent lines of investigation. First, growth-arrested 10T1/2 cells do not normally respond to a hormonal cocktail that causes various growth-arrested preadipocyte cell lines to differentiate into adipocytes, but if 10T1/2 cells are first treated with BMP-4 they will respond to these hormonal inducers by undergoing terminal adipocyte differentiation. Second, a preadipocyte cell line, A33 cells, derived from 10T1/2 cells after exposing the cells to the DNA methyltransferase inhibitor 5-azacytidine was shown to express BMP-4, and this endogenous BMP-4 expression is required for acquisition of the preadipocyte phenotype of these cells. A role for the BMP-4 signaling pathway in adipogenesis is discussed.
Asunto(s)
Adipocitos/fisiología , Proteínas Morfogenéticas Óseas/fisiología , Adipocitos/citología , Adipocitos/metabolismo , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Modelos Biológicos , Transducción de SeñalRESUMEN
Microarray gene expression profiling was used to identify bone morphogenetic protein-4 (BMP-4) responsive factors involved in late stages of adipocyte commitment in C3H10T1/2 cells. The analysis revealed that the matrix metalloproteinase-3 (MMP-3) gene decreased 100-fold after BMP-4 treatment, and expression of MMP-13 decreased 19.5-fold. Uncommitted C3H10T1/2 cells exhibit dramatic up-regulation of MMP-3 and MMP-13 genes as cells become confluent. Real-time RT-PCR demonstrated that BMP-4 blocks expression of both transcripts. Likewise, a stable committed preadipocyte line derived from C3H10T1/2 cells did not express MMP-3 or MMP-13 at confluence, despite never receiving BMP-4. Active forms of both proteins were detected in media from confluent C3H10T1/2 cells but not in BMP-4 treated cells. Addition of BMP-4 to confluent C3H10T1/2 cells repressed the expression of both genes but did not induce adipocyte differentiation. The findings indicate that BMP-4-induced down-regulation of MMP-3 and MMP-13 is associated with commitment, but is insufficient to induce adipogenesis.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Células Madre Embrionarias/efectos de los fármacos , Perfilación de la Expresión Génica , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/genética , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Proteína Morfogenética Ósea 4 , Diferenciación Celular/genética , Línea Celular , Electroforesis en Gel de Poliacrilamida , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Expresión Génica/efectos de los fármacos , Immunoblotting , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
During adipocyte differentiation, CCAAT/enhancer-binding protein alpha (C/EBPalpha) functions as a pleiotropic transcriptional activator of numerous adipocyte genes. The promoter of the C/EBPalpha gene has an E-box upstream of C/EBP binding site. Deletion or mutation of the E-box decreases promoter activity, suggesting that the E-box participates in the regulation of C/EBPalpha expression. Protein binding to the E-box during the adipocyte differentiation is increased as indicated by EMSA and UV cross-linking. Purification of the E-box binding proteins from differentiated 3T3-L1 adipocytes, showed that USF and AP-4 are associated with the E-box. Supershift analysis showed that USF1 and USF2 bind to this element as heterodimers, whereas the addition of anti-AP-4 antibody enhanced the binding complex, suggesting that AP-4 negatively regulates the promoter activity. The expression of AP-4 is reciprocally regulated with USF-1 during adipocyte differentiation. These findings suggest that USF-1 and 2 play roles in C/EBPalpha expression, whereas the AP-4 represses it.