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1.
Genes Dev ; 31(7): 660-673, 2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-28428261

RESUMEN

The transcription factor early B-cell factor 2 (EBF2) is an essential mediator of brown adipocyte commitment and terminal differentiation. However, the mechanisms by which EBF2 regulates chromatin to activate brown fat-specific genes in adipocytes were unknown. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by deep sequencing) analyses in brown adipose tissue showed that EBF2 binds and regulates the activity of lineage-specific enhancers. Mechanistically, EBF2 physically interacts with the chromatin remodeler BRG1 and the BAF chromatin remodeling complex in brown adipocytes. We identified the histone reader protein DPF3 as a brown fat-selective component of the BAF complex that was required for brown fat gene programming and mitochondrial function. Loss of DPF3 in brown adipocytes reduced chromatin accessibility at EBF2-bound enhancers and led to a decrease in basal and catecholamine-stimulated expression of brown fat-selective genes. Notably, Dpf3 is a direct transcriptional target of EBF2 in brown adipocytes, thereby establishing a regulatory module through which EBF2 activates and also recruits DPF3-anchored BAF complexes to chromatin. Together, these results reveal a novel mechanism by which EBF2 cooperates with a tissue-specific chromatin remodeling complex to activate brown fat identity genes.


Asunto(s)
Adipogénesis/genética , Tejido Adiposo Pardo/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Histonas/metabolismo , Factores de Transcripción/genética , Tejido Adiposo Pardo/metabolismo , Animales , Linaje de la Célula/genética , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transcripción Genética
2.
Arterioscler Thromb Vasc Biol ; 43(7): 1081-1092, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37259866

RESUMEN

There is overwhelming clinical and genetic evidence supporting the concept that low-density-lipoprotein cholesterol should be as low as possible for as long as possible in patients at very high cardiovascular risk. Despite the wide availability of effective lipid-lowering therapies, the majority of patients still fail to reach guideline-based lipid goals. Advances in novel approaches targeting PCSK9 (proprotein convertase subtilisin/kexin type 9) through small-interfering RNA and genome editing hold the potential to bridge this gap, by offering long-acting alternatives, which may overcome adherence and other challenges in the current chronic care model. In this review, we discuss the history of targeting PCSK9 with the use of mRNA and small-interfering ribonucleic acid. We also shed light on targeting PCSK9 with genome editing, including discussion of the VERVE-101 clustered regularly interspaced short palindromic repeats-base editing medicine currently being evaluated in a clinical trial and others in development.


Asunto(s)
Edición Génica , Proproteína Convertasa 9 , Humanos , Proproteína Convertasa 9/genética , LDL-Colesterol , ARN Interferente Pequeño/genética
3.
Genes Dev ; 29(3): 298-307, 2015 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25644604

RESUMEN

PR (PRD1-BF1-RIZ1 homologous) domain-containing 16 (PRDM16) drives a brown fat differentiation program, but the mechanisms by which PRDM16 activates brown fat-selective genes have been unclear. Through chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) analyses in brown adipose tissue (BAT), we reveal that PRDM16 binding is highly enriched at a broad set of brown fat-selective genes. Importantly, we found that PRDM16 physically binds to MED1, a component of the Mediator complex, and recruits it to superenhancers at brown fat-selective genes. PRDM16 deficiency in BAT reduces MED1 binding at PRDM16 target sites and causes a fundamental change in chromatin architecture at key brown fat-selective genes. Together, these data indicate that PRDM16 controls chromatin architecture and superenhancer activity in BAT.


Asunto(s)
Tejido Adiposo Pardo/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Subunidad 1 del Complejo Mediador/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Animales , Cromatina/química , Cromatina/genética , Elementos de Facilitación Genéticos , Ratones
4.
EMBO J ; 36(11): 1528-1542, 2017 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-28408438

RESUMEN

Brown adipose has the potential to counteract obesity, and thus, identifying signaling pathways that regulate the activity of this tissue is of great clinical interest. PRDM16 is a transcription factor that activates brown fat-specific genes while repressing white fat and muscle-specific genes in adipocytes. Whether PRDM16 also controls other gene programs to regulate adipocyte function was unclear. Here, we identify a novel role for PRDM16 in suppressing type I interferon (IFN)-stimulated genes (ISGs), including Stat1, in adipocytes in vitro and in vivo Ectopic activation of type I IFN signaling in brown adipocytes induces mitochondrial dysfunction and reduces uncoupling protein 1 (UCP1) expression. Prdm16-deficient adipose displays an exaggerated response to type I IFN, including higher STAT1 levels and reduced mitochondrial gene expression. Mechanistically, PRDM16 represses ISGs through binding to promoter regions of these genes and blocking the activating function of IFN regulatory factor 1 (IRF1). Together, these data indicate that PRDM16 diminishes responsiveness to type I IFN in adipose cells to promote thermogenic and mitochondrial function.


Asunto(s)
Adipocitos/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factor 1 Regulador del Interferón/antagonistas & inhibidores , Interferón Tipo I/metabolismo , Mitocondrias/metabolismo , Termogénesis , Factores de Transcripción/metabolismo , Animales , Ratones , Factor de Transcripción STAT1/antagonistas & inhibidores , Proteína Desacopladora 1/metabolismo
5.
Arterioscler Thromb Vasc Biol ; 32(2): 264-72, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22053073

RESUMEN

OBJECTIVE: We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. METHODS AND RESULTS: We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoe(h/h)Ldlr(-/-)) were compared to Apoe(-/-)Ldlr(-/-) mice. Despite 4-fold more plasma apoE than WT mice, Apoe(h/h)Ldlr(-/-) mice displayed similar plasma cholesterol as Apoe(-/-) Ldlr(-/-) mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoe(h/h)Ldlr(-/-) mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoe(h/h)Ldlr(-/-) mice had less circulating leukocytes and proinflammatory Ly6C(high) monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoe(h/h)Ldlr(-/-) mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. CONCLUSIONS: Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.


Asunto(s)
Apolipoproteínas E/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Endotelio Vascular/metabolismo , Mediadores de Inflamación/metabolismo , Metabolismo de los Lípidos , Monocitos/metabolismo , Animales , Apolipoproteínas E/deficiencia , Moléculas de Adhesión Celular/metabolismo , Colesterol/metabolismo , Modelos Animales de Enfermedad , Integrina alfa4beta1/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Selectina L/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/metabolismo
6.
Nat Med ; 27(11): 1941-1953, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34608330

RESUMEN

Obesity is considered an important factor for many chronic diseases, including diabetes, cardiovascular disease and cancer. The expansion of adipose tissue in obesity is due to an increase in both adipocyte progenitor differentiation and mature adipocyte cell size. Adipocytes, however, are thought to be unable to divide or enter the cell cycle. We demonstrate that mature human adipocytes unexpectedly display a gene and protein signature indicative of an active cell cycle program. Adipocyte cell cycle progression associates with obesity and hyperinsulinemia, with a concomitant increase in cell size, nuclear size and nuclear DNA content. Chronic hyperinsulinemia in vitro or in humans, however, is associated with subsequent cell cycle exit, leading to a premature senescent transcriptomic and secretory profile in adipocytes. Premature senescence is rapidly becoming recognized as an important mediator of stress-induced tissue dysfunction. By demonstrating that adipocytes can activate a cell cycle program, we define a mechanism whereby mature human adipocytes senesce. We further show that by targeting the adipocyte cell cycle program using metformin, it is possible to influence adipocyte senescence and obesity-associated adipose tissue inflammation.


Asunto(s)
Adipocitos/metabolismo , Ciclo Celular/fisiología , Senescencia Celular/fisiología , Hiperinsulinismo/patología , Obesidad/patología , Tejido Adiposo/metabolismo , Diferenciación Celular/fisiología , Ciclina D1/metabolismo , Humanos , Hipoglucemiantes/farmacología , Metformina/farmacología
7.
J Vis Exp ; (156)2020 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-32116300

RESUMEN

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.


Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo Blanco/metabolismo , Técnicas de Cultivo de Célula/métodos , Humanos
8.
Theranostics ; 10(2): 585-601, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31903139

RESUMEN

Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an in vivo model of obesity-associated dysmetabolism. Methods: Male leptin-deficient (ob/ob) mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells. Results: Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery. Conclusions: These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations in vivo. These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.


Asunto(s)
Alcanosulfonatos/farmacología , Riñón/efectos de los fármacos , Liposomas/administración & dosificación , Hígado/efectos de los fármacos , Macrófagos/efectos de los fármacos , Obesidad/tratamiento farmacológico , PPAR alfa/agonistas , PPAR gamma/agonistas , Fenilpropionatos/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Inflamación/metabolismo , Riñón/metabolismo , Liposomas/química , Hígado/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , Obesidad/patología
9.
Cell Rep ; 27(1): 213-225.e5, 2019 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-30943403

RESUMEN

White adipose tissue (WAT) is a central factor in the development of type 2 diabetes, but there is a paucity of translational models to study mature adipocytes. We describe a method for the culture of mature white adipocytes under a permeable membrane. Compared to existing culture methods, MAAC (membrane mature adipocyte aggregate cultures) better maintain adipogenic gene expression, do not dedifferentiate, display reduced hypoxia, and remain functional after long-term culture. Subcutaneous and visceral adipocytes cultured as MAAC retain depot-specific gene expression, and adipocytes from both lean and obese patients can be cultured. Importantly, we show that rosiglitazone treatment or PGC1α overexpression in mature white adipocytes induces a brown fat transcriptional program, providing direct evidence that human adipocytes can transdifferentiate into brown-like adipocytes. Together, these data show that MAAC are a versatile tool for studying phenotypic changes of mature adipocytes and provide an improved translational model for drug development.


Asunto(s)
Adipocitos Marrones/fisiología , Adipocitos Blancos/citología , Adipocitos Blancos/fisiología , Adipogénesis/fisiología , Transdiferenciación Celular , Cultivo Primario de Células/métodos , Adipocitos Marrones/citología , Animales , Transdiferenciación Celular/fisiología , Células Cultivadas , Femenino , Humanos , Membranas Artificiales , Ratones , Células RAW 264.7
10.
Cell Rep ; 24(10): 2746-2756.e5, 2018 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-30184507

RESUMEN

Adipocytes, once considered simple lipid-storing cells, are rapidly emerging as complex cells with many biologically diverse functions. A powerful high-throughput method for analyzing single cells is flow cytometry. Several groups have attempted to analyze and sort freshly isolated adipocytes; however, using an adipocyte-specific reporter mouse, we demonstrate that these studies fail to detect the majority of white adipocytes. We define critical settings required for adipocyte flow cytometry and provide a rigid strategy for analyzing and sorting white and brown adipocyte populations. The applicability of our protocol is shown by sorting mouse adipocytes based on size or UCP1 expression and demonstrating that a subset of human adipocytes lacks the ß2-adrenergic receptor, particularly in the insulin-resistant state. In conclusion, the present study confers key technological insights for analyzing and sorting mature adipocytes, opening up numerous downstream research applications.


Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Citometría de Flujo/métodos , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Humanos , Ratones , Proteína Desacopladora 1/metabolismo
11.
Cell Metab ; 19(4): 593-604, 2014 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-24703692

RESUMEN

Prdm16 is a transcription factor that regulates the thermogenic gene program in brown and beige adipocytes. However, whether Prdm16 is required for the development or physiological function of brown adipose tissue (BAT) in vivo has been unclear. By analyzing mice that selectively lacked Prdm16 in the brown adipose lineage, we found that Prdm16 was dispensable for embryonic BAT development. However, Prdm16 was required in young mice to suppress the expression of white-fat-selective genes in BAT through recruitment of the histone methyltransferase Ehmt1. Additionally, Prdm16 deficiency caused a severe adult-onset decline in the thermogenic character of interscapular BAT. This resulted in BAT dysfunction and cold sensitivity but did not predispose the animals to obesity. Interestingly, the loss of brown fat identity due to ablation of Prdm16 was accelerated by concurrent deletion of the closely related Prdm3 gene. Together, these results show that Prdm16 and Prdm3 control postnatal BAT identity and function.


Asunto(s)
Adipocitos Marrones/fisiología , Envejecimiento/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Factores de Transcripción/metabolismo , Adipocitos Marrones/metabolismo , Análisis de Varianza , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Citometría de Flujo , Técnicas Histológicas , Ratones , Ratones Noqueados , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética
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