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OBJECTIVE: The lactation-suckling period is critical for white adipose tissue (WAT) development. Early postnatal nutrition influences later obesity risk but underlying mechanisms remain elusive. Here, we tested whether altered postnatal nutrition specifically during suckling impacts epigenetic regulation of key metabolic genes in WAT and alter long-term adiposity set point. METHODS: We analyzed the effects of maternal high-fat (HF) feeding in rats exclusively during lactation-suckling on breast milk composition and its impact on male offspring visceral epidydimal (eWAT) and subcutaneous inguinal (iWAT) depots during suckling and in adulthood. RESULTS: Maternal HF feeding during lactation had no effect on mothers' body weight (BW) or global breast milk composition, but induced qualitative changes in breast milk fatty acid (FA) composition (high n-6/n-3 polyunsaturated FA ratio and low medium-chain FA content). During suckling, HF neonates showed increased BW and mass of both eWAT and iWAT depot but only eWAT displayed an enhanced adipogenic transcriptional signature. In adulthood, HF offspring were predisposed to weight gain and showed increased hyperplastic growth only in eWAT. This specific eWAT expansion was associated with increased expression and activity of stearoyl-CoA desaturase-1 (SCD1), a key enzyme of FA metabolism. SCD1 converts saturated FAs, e.g. palmitate and stearate, to monounsaturated FAs, palmitoleate and oleate, which are the predominant substrates for triglyceride synthesis. Scd1 upregulation in eWAT was associated with reduced DNA methylation in Scd1 promoter surrounding a PPARγ-binding region. Conversely, changes in SCD1 levels and methylation were not observed in iWAT, coherent with a depot-specific programming. CONCLUSIONS: Our data reveal that maternal HF feeding during suckling programs long-term eWAT expansion in part by SCD1 epigenetic reprogramming. This programming events occurred with drastic changes in breast milk FA composition, suggesting that dietary FAs are key metabolic programming factors in the early postnatal period.
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Tejido Adiposo Blanco , Dieta Alta en Grasa , Epigénesis Genética/genética , Lactancia/genética , Estearoil-CoA Desaturasa , Tejido Adiposo Blanco/química , Tejido Adiposo Blanco/enzimología , Tejido Adiposo Blanco/metabolismo , Animales , Animales Recién Nacidos , Peso Corporal/genética , Femenino , Grasa Intraabdominal/química , Grasa Intraabdominal/enzimología , Grasa Intraabdominal/metabolismo , Masculino , Leche/química , Ratas Wistar , Estearoil-CoA Desaturasa/análisis , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismoRESUMEN
According to the Developmental Origin of Health and Disease (DOHaD) concept, maternal obesity and accelerated growth in neonates program obesity later in life. White adipose tissue (WAT) has been the focus of developmental programming events, although underlying mechanisms remain elusive. In rodents, WAT development primarily occurs during lactation. We previously reported that adult rat offspring from dams fed a high-fat (HF) diet exhibited fat accumulation and decreased peroxisome proliferator-activated receptor γ (PPARγ) mRNA levels in WAT. We hypothesized that PPARγ down-regulation occurs via epigenetic malprogramming which takes place during adipogenesis. We therefore examined epigenetic modifications in the PPARγ1 and PPARγ2 promoters in perirenal (pWAT) and inguinal fat pads of HF offspring at weaning (postnatal d 21) and in adulthood. Postnatal d 21 is a period characterized by active epigenomic remodeling in the PPARγ2 promoter (DNA hypermethylation and depletion in active histone modification H3ac and H3K4me3) in pWAT, consistent with increased DNA methyltransferase and DNA methylation activities. Adult HF offspring exhibited sustained hypermethylation and histone modification H3ac of the PPARγ2 promoter in both deposits, correlated with persistent decreased PPARγ2 mRNA levels. Consistent with the DOHaD hypothesis, retained epigenetic marks provide a mechanistic basis for the cellular memory linking maternal obesity to a predisposition for later adiposity.-Lecoutre, S., Pourpe, C., Butruille, L., Marousez, L., Laborie, C., Guinez, C., Lesage, J., Vieau, D., Eeckhoute, J., Gabory, A., Oger, F., Eberlé, D., Breton, C. Reduced PPARγ2 expression in adipose tissue of male rat offspring from obese dams is associated with epigenetic modifications.
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Tejido Adiposo/metabolismo , Metilación de ADN , Epigénesis Genética , Obesidad/metabolismo , PPAR gamma/biosíntesis , Regiones Promotoras Genéticas , Tejido Adiposo/patología , Adiposidad/genética , Animales , Femenino , Histonas/genética , Histonas/metabolismo , Masculino , Obesidad/genética , PPAR gamma/genética , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
CCCTC-binding factor (CTCF) is a ubiquitously expressed multifunctional transcription factor characterized by chromatin binding patterns often described as largely invariant. In this context, how CTCF chromatin recruitment and functionalities are used to promote cell type-specific gene expression remains poorly defined. Here, we show that, in addition to constitutively bound CTCF binding sites (CTS), the CTCF cistrome comprises a large proportion of sites showing highly dynamic binding patterns during the course of adipogenesis. Interestingly, dynamic CTCF chromatin binding is positively linked with changes in expression of genes involved in biological functions defining the different stages of adipogenesis. Importantly, a subset of these dynamic CTS are gained at cell type-specific regulatory regions, in line with a requirement for CTCF in transcriptional induction of adipocyte differentiation. This relates to, at least in part, CTCF requirement for transcriptional activation of both the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) and its target genes. Functionally, we show that CTCF interacts with TET methylcytosine dioxygenase (TET) enzymes and promotes adipogenic transcriptional enhancer DNA hydroxymethylation. Our study reveals a dynamic CTCF chromatin binding landscape required for epigenomic remodeling of enhancers and transcriptional activation driving cell differentiation.
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Adipogénesis/genética , Cromatina/metabolismo , Proteínas Represoras/metabolismo , Activación Transcripcional , Animales , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular , Células Cultivadas , Metilación de ADN , Dioxigenasas/metabolismo , Elementos de Facilitación Genéticos , Células HEK293 , Humanos , Ratones Endogámicos C57BL , PPAR gamma/metabolismoRESUMEN
The nuclear receptor peroxisome proliferator-activated receptor (PPAR) is a transcription factor whose expression is induced during adipogenesis and that is required for the acquisition and control of mature adipocyte functions. Indeed, PPAR induces the expression of genes involved in lipid synthesis and storage through enhancers activated during adipocyte differentiation. Here, we show that PPAR also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is more restricted to adipocytes. Thus, PPAR utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of genes involved in the adipogenic process.
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Adipogénesis/genética , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Metabolismo de los Lípidos/genética , PPAR gamma/metabolismo , Transducción de Señal/genética , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Insulina/metabolismo , Ratones , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Somatomedinas/metabolismo , TranscriptomaRESUMEN
Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes.
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Diferenciación Celular/genética , Metilación de ADN , Elementos de Facilitación Genéticos , Células 3T3-L1 , 5-Metilcitosina/análogos & derivados , Animales , Sitios de Unión , Línea Celular Tumoral , Cromatina/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/metabolismo , Neurogénesis/genética , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Type 2 diabetes (T2D) is a metabolic disorder characterized by loss of pancreatic ß-cell function, decreased insulin secretion and increased insulin resistance, that affects more than 537 million people worldwide. Although several treatments are proposed to patients suffering from T2D, long-term control of glycemia remains a challenge. Therefore, identifying new potential drugs and targets that positively affect ß-cell function and insulin secretion remains crucial. Here, we developed an automated approach to allow the identification of new compounds or genes potentially involved in ß-cell function in a 384-well plate format, using the murine ß-cell model Min6. By using MALDI-TOF mass spectrometry, we implemented a high-throughput screening (HTS) strategy based on the automation of a cellular assay allowing the detection of insulin secretion in response to glucose, i.e., the quantitative detection of insulin, in a miniaturized system. As a proof of concept, we screened siRNA targeting well-know ß-cell genes and 1600 chemical compounds and identified several molecules as potential regulators of insulin secretion and/or synthesis, demonstrating that our approach allows HTS of insulin secretion in vitro.
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Diabetes Mellitus Tipo 2 , Insulina , Humanos , Animales , Ratones , Insulina/metabolismo , Secreción de Insulina , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/farmacología , Glucosa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Ensayos Analíticos de Alto Rendimiento , Insulina Regular Humana/metabolismoRESUMEN
Human induced pluripotent stem cells (hiPSCs) have the potential to be differentiated into any cell type, making them a relevant tool for therapeutic purposes such as cell-based therapies. In particular, they show great promise for obesity treatment as they represent an unlimited source of brown/beige adipose progenitors (hiPSC-BAPs). However, the low brown/beige adipocyte differentiation potential in 2D cultures represents a strong limitation for clinical use. In adipose tissue, besides its cell cycle regulator functions, the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus modulates the commitment of stem cells to the brown-like type fate, mature adipocyte energy metabolism and the browning of adipose tissue. Here, using a new method of hiPSC-BAPs 3D culture, via the formation of an organoid-like structure, we silenced CDKN2A expression during hiPSC-BAP adipogenic differentiation and observed that knocking down CDKN2A potentiates adipogenesis, oxidative metabolism and the browning process, resulting in brown-like adipocytes by promoting UCP1 expression and beiging markers. Our results suggest that modulating CDKN2A levels could be relevant for hiPSC-BAPs cell-based therapies.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina , Células Madre Pluripotentes Inducidas , Humanos , Adipocitos Marrones/metabolismo , Diferenciación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Células Madre Pluripotentes Inducidas/metabolismo , Obesidad/metabolismo , Estrés OxidativoRESUMEN
The loss of pancreatic ß-cell identity has emerged as an important feature of type 2 diabetes development, but the molecular mechanisms are still elusive. Here, we explore the cell-autonomous role of the cell-cycle regulator and transcription factor E2F1 in the maintenance of ß-cell identity, insulin secretion, and glucose homeostasis. We show that the ß-cell-specific loss of E2f1 function in mice triggers glucose intolerance associated with defective insulin secretion, altered endocrine cell mass, downregulation of many ß-cell genes, and concomitant increase of non-ß-cell markers. Mechanistically, epigenomic profiling of the promoters of these non-ß-cell upregulated genes identified an enrichment of bivalent H3K4me3/H3K27me3 or H3K27me3 marks. Conversely, promoters of downregulated genes were enriched in active chromatin H3K4me3 and H3K27ac histone marks. We find that specific E2f1 transcriptional, cistromic, and epigenomic signatures are associated with these ß-cell dysfunctions, with E2F1 directly regulating several ß-cell genes at the chromatin level. Finally, the pharmacological inhibition of E2F transcriptional activity in human islets also impairs insulin secretion and the expression of ß-cell identity genes. Our data suggest that E2F1 is critical for maintaining ß-cell identity and function through sustained control of ß-cell and non-ß-cell transcriptional programs. ARTICLE HIGHLIGHTS: ß-Cell-specific E2f1 deficiency in mice impairs glucose tolerance. Loss of E2f1 function alters the ratio of α- to ß-cells but does not trigger ß-cell conversion into α-cells. Pharmacological inhibition of E2F activity inhibits glucose-stimulated insulin secretion and alters ß- and α-cell gene expression in human islets. E2F1 maintains ß-cell function and identity through control of transcriptomic and epigenetic programs.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animales , Humanos , Ratones , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Histonas/metabolismo , Homeostasis/genética , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones NoqueadosRESUMEN
Histone deacetylases enzymes (HDACs) are chromatin modifiers that regulate gene expression through deacetylation of lysine residues within specific histone and non-histone proteins. A cell-specific gene expression pattern defines the identity of insulin-producing pancreatic ß cells, yet molecular networks driving this transcriptional specificity are not fully understood. Here, we investigated the HDAC-dependent molecular mechanisms controlling pancreatic ß-cell identity and function using the pan-HDAC inhibitor trichostatin A through chromatin immunoprecipitation assays and RNA sequencing experiments. We observed that TSA alters insulin secretion associated with ß-cell specific transcriptome programming in both mouse and human ß-cell lines, as well as on human pancreatic islets. We also demonstrated that this alternative ß-cell transcriptional program in response to HDAC inhibition is related to an epigenome-wide remodeling at both promoters and enhancers. Our data indicate that HDAC activity could be required to protect against loss of ß-cell identity with unsuitable expression of genes associated with alternative cell fates.
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Type 2 diabetes is characterized by chronic hyperglycemia associated with impaired insulin action and secretion. Although the heritability of type 2 diabetes is high, the environment, including blood components, could play a major role in the development of the disease. Amongst environmental effects, epitranscriptomic modifications have been recently shown to affect gene expression and glucose homeostasis. The epitranscriptome is characterized by reversible chemical changes in RNA, with one of the most prevalent being the m6A methylation of RNA. Since pancreatic ß cells fine tune glucose levels and play a major role in type 2 diabetes physiopathology, we hypothesized that the environment, through variations in blood glucose or blood free fatty acid concentrations, could induce changes in m6A methylation of RNAs in pancreatic ß cells. Here we observe a significant decrease in m6A methylation upon high glucose concentration, both in mice and human islets, associated with altered expression levels of m6A demethylases. In addition, the use of siRNA and/or specific inhibitors against selected m6A enzymes demonstrate that these enzymes modulate the expression of genes involved in pancreatic ß-cell identity and glucose-stimulated insulin secretion. Our data suggest that environmental variations, such as glucose, control m6A methylation in pancreatic ß cells, playing a key role in the control of gene expression and pancreatic ß-cell functions. Our results highlight novel causes and new mechanisms potentially involved in type 2 diabetes physiopathology and may contribute to a better understanding of the etiology of this disease.
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Adenosina/análogos & derivados , Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , ARN/metabolismo , Adenosina/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Glucosa/farmacología , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Palmitatos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The glucagon-like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential in type 2 diabetes (T2D) treatment, notably by improving ß cell functions. The cell-cycle regulator and transcription factor E2f1 is involved in glucose homeostasis by modulating ß cell mass and function. Here, we report that ß cell-specific genetic ablation of E2f1 (E2f1ß-/-) impairs glucose homeostasis associated with decreased expression of the Glp-1 receptor (Glp1r) in E2f1ß-/- pancreatic islets. Pharmacological inhibition of E2F1 transcriptional activity in nondiabetic human islets decreases GLP1R levels and blunts the incretin effect of GLP1R agonist exendin-4 (ex-4) on insulin secretion. Overexpressing E2f1 in pancreatic ß cells increases Glp1r expression associated with enhanced insulin secretion mediated by ex-4. Interestingly, ex-4 induces retinoblastoma protein (pRb) phosphorylation and E2f1 transcriptional activity. Our findings reveal critical roles for E2f1 in ß cell function and suggest molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Diabetes Mellitus Tipo 2/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Exenatida/farmacología , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismoRESUMEN
Alzheimer's disease (AD) is the leading cause of dementia. While impaired glucose homeostasis has been shown to increase AD risk and pathological loss of tau function, the latter has been suggested to contribute to the emergence of the glucose homeostasis alterations observed in AD patients. However, the links between tau impairments and glucose homeostasis, remain unclear. In this context, the present study aimed at investigating the metabolic phenotype of a new tau knock-in (KI) mouse model, expressing, at a physiological level, a human tau protein bearing the P301L mutation under the control of the endogenous mouse Mapt promoter. Metabolic investigations revealed that, while under chow diet tau KI mice do not exhibit significant metabolic impairments, male but not female tau KI animals under High-Fat Diet (HFD) exhibited higher insulinemia as well as glucose intolerance as compared to control littermates. Using immunofluorescence, tau protein was found colocalized with insulin in the ß cells of pancreatic islets in both mouse (WT, KI) and human pancreas. Isolated islets from tau KI and tau knock-out mice exhibited impaired glucose-stimulated insulin secretion (GSIS), an effect recapitulated in the mouse pancreatic ß-cell line (MIN6) following tau knock-down. Altogether, our data indicate that loss of tau function in tau KI mice and, particularly, dysfunction of pancreatic ß cells might promote glucose homeostasis impairments and contribute to metabolic changes observed in AD.
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Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.
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Cromatina/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Desacetilasas/metabolismo , Schistosoma/efectos de los fármacos , Animales , Cromatina/metabolismo , Diseño de Fármacos , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Schistosoma/enzimologíaRESUMEN
Besides its role as a cell cycle and proliferation regulator, the INK4a/ARF (CDKN2A) locus and its associated pathways are thought to play additional functions in the control of energy homeostasis. Genome-wide association studies in humans and rodents have revealed that single nucleotide polymorphisms in this locus are risk factors for obesity and related metabolic diseases including cardiovascular complications and type-2 diabetes (T2D). Recent studies showed that both p16INK4a-CDK4-E2F1/pRB and p19ARF-P53 (p14ARF in humans) related pathways regulate adipose tissue (AT) physiology and adipocyte functions such as lipid storage, inflammation, oxidative activity, and cellular plasticity (browning). Targeting these metabolic pathways in AT emerged as a new putative therapy to alleviate the effects of obesity and prevent T2D. This review aims to provide an overview of the literature linking the INK4a/ARF locus with AT functions, focusing on its mechanisms of action in the regulation of energy homeostasis.
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Tejido Adiposo/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Obesidad/metabolismo , Tejido Adiposo/citología , Animales , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Regulación de la Expresión Génica , Humanos , Resistencia a la Insulina/genética , Obesidad/diagnóstico , Obesidad/genética , Polimorfismo de Nucleótido SimpleRESUMEN
Nuclear receptors are ligand-activated transcription factors involved in all major physiological functions of complex organisms. In this respect, they are often described as drugable targets for a number of pathological states including hypercholesterolemia and atherosclerosis. HNF4alpha (NR2A1) is a recently 'deorphanized' nuclear receptor which is bound in vivo by linoleic acid, although this natural ligand does not seem to promote transcriptional activation. In mouse, HNF4alpha is a major regulator of liver development and hepatic lipid metabolism and mutations in human have been linked to diabetes. Here, we have used a yeast one-hybrid system to identify small molecule activators of HNF4alpha in a library of synthetic compounds and found one hit bearing a methoxy group branched on a nitronaphthofuran backbone. A collection of molecules deriving from the discovered hit was generated and tested for activity toward HNF4alpha in yeast one-hybrid system. It was found that both the nitro group and a complete naphthofuran backbone were required for full activity of the compounds. Furthermore, adding a hydroxy group at position 7 of the minimal backbone led to the most active compound of the collection. Accordingly, a direct interaction of the hydroxylated compound with the ligand binding domain of HNF4alpha was detected by NMR and thermal denaturation assays. When used in mammalian cell culture systems, these compounds proved to be highly toxic, except when methylated on the furan ring. One such compound was able to modulate HNF4alpha-driven transcription in transfected HepG2C3A cells. These data indicate that HNF4alpha activity can be modulated by small molecules and suggest new routes for targeting the receptor in humans.
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Furanos/química , Factor Nuclear 4 del Hepatocito/efectos de los fármacos , Animales , Línea Celular Tumoral , Furanos/farmacología , Humanos , Ligandos , Naftalenos , Receptores Citoplasmáticos y Nucleares , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacosRESUMEN
Histone deacetylases (HDAC) form a conserved enzyme family that control gene expression via the removal of acetyl residues from histones and other proteins and are under increasing investigation as therapeutic targets, notably in cancer and parasitic diseases. To investigate the conservation of these enzymes in the platyhelminth parasite Schistosoma mansoni, we cloned and characterized three class I HDACs, orthologues of mammalian HDAC1, 3 and 8, and confirmed their identities by phylogenetic analysis. The identification of an HDAC8 orthologue showed that it is not vertebrate-specific as previously thought and insertions in its catalytic domain suggest specific enzymatic properties. SmHDAC1, 3, and 8 mRNAs are expressed at all schistosome life-cycle stages. SmHDAC1 repressed transcriptional activity in a mammalian cell line and this activity was dependent on its catalytic activity since transcription was partially restored by treatment with trichostatin A and a catalytic site mutant failed to repress transcription.
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Regulación de la Expresión Génica , Proteínas del Helminto/clasificación , Proteínas del Helminto/metabolismo , Histona Desacetilasas/clasificación , Histona Desacetilasas/metabolismo , Schistosoma mansoni/enzimología , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Clonación Molecular , Secuencia Conservada , Inhibidores Enzimáticos/farmacología , Proteínas del Helminto/genética , Histona Desacetilasas/genética , Ácidos Hidroxámicos/farmacología , Datos de Secuencia Molecular , Filogenia , Schistosoma mansoni/genética , Transcripción GenéticaRESUMEN
OBJECTIVES: Genome-wide association studies have reported that DNA polymorphisms at the CDKN2A locus modulate fasting glucose in human and contribute to type 2 diabetes (T2D) risk. Yet the causal relationship between this gene and defective energy homeostasis remains elusive. Here we sought to understand the contribution of Cdkn2a to metabolic homeostasis. METHODS: We first analyzed glucose and energy homeostasis from Cdkn2a-deficient mice subjected to normal or high fat diets. Subsequently Cdkn2a-deficient primary adipose cells and human-induced pluripotent stem differentiated into adipocytes were further characterized for their capacity to promote browning of adipose tissue. Finally CDKN2A levels were studied in adipocytes from lean and obese patients. RESULTS: We report that Cdkn2a deficiency protects mice against high fat diet-induced obesity, increases energy expenditure and modulates adaptive thermogenesis, in addition to improving insulin sensitivity. Disruption of Cdkn2a associates with increased expression of brown-like/beige fat markers in inguinal adipose tissue and enhances respiration in primary adipose cells. Kinase activity profiling and RNA-sequencing analysis of primary adipose cells further demonstrate that Cdkn2a modulates gene networks involved in energy production and lipid metabolism, through the activation of the Protein Kinase A (PKA), PKG, PPARGC1A and PRDM16 signaling pathways, key regulators of adipocyte beiging. Importantly, CDKN2A expression is increased in adipocytes from obese compared to lean subjects. Moreover silencing CDKN2A expression during human-induced pluripotent stem cells adipogenic differentiation promoted UCP1 expression. CONCLUSION: Our results offer novel insight into brown/beige adipocyte functions, which has recently emerged as an attractive therapeutic strategy for obesity and T2D. Modulating Cdkn2a-regulated signaling cascades may be of interest for the treatment of metabolic disorders.
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Adipocitos Marrones/metabolismo , Adipogénesis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Obesidad/metabolismo , Adipocitos Marrones/citología , Animales , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Redes Reguladoras de Genes , Glucosa/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , TermogénesisRESUMEN
OBJECTIVE: According to the Developmental Origin of Health and Disease (DOHaD) concept, maternal obesity and accelerated growth in neonates predispose offspring to white adipose tissue (WAT) accumulation. In rodents, adipogenesis mainly develops during lactation. The mechanisms underlying the phenomenon known as developmental programming remain elusive. We previously reported that adult rat offspring from high-fat diet-fed dams (called HF) exhibited hypertrophic adipocyte, hyperleptinemia and increased leptin mRNA levels in a depot-specific manner. We hypothesized that leptin upregulation occurs via epigenetic malprogramming, which takes place early during development of WAT. METHODS: As a first step, we identified in silico two potential enhancers located upstream and downstream of the leptin transcription start site that exhibit strong dynamic epigenomic remodeling during adipocyte differentiation. We then focused on epigenetic modifications (methylation, hydroxymethylation, and histone modifications) of the promoter and the two potential enhancers regulating leptin gene expression in perirenal (pWAT) and inguinal (iWAT) fat pads of HF offspring during lactation (postnatal days 12 (PND12) and 21 (PND21)) and in adulthood. RESULTS: PND12 is an active period for epigenomic remodeling in both deposits especially in the upstream enhancer, consistent with leptin gene induction during adipogenesis. Unlike iWAT, some of these epigenetic marks were still observable in pWAT of weaned HF offspring. Retained marks were only visible in pWAT of 9-month-old HF rats that showed a persistent "expandable" phenotype. CONCLUSIONS: Consistent with the DOHaD hypothesis, persistent epigenetic remodeling occurs at regulatory regions especially within intergenic sequences, linked to higher leptin gene expression in adult HF offspring in a depot-specific manner.
Asunto(s)
Epigénesis Genética , Leptina/genética , Obesidad/genética , Complicaciones del Embarazo/genética , Tejido Adiposo Blanco/metabolismo , Animales , Metilación de ADN , Femenino , Código de Histonas , Leptina/metabolismo , Masculino , Embarazo , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan member of the nuclear receptor family of transcription factors whose activities are modulated upon binding of small molecules into an hydrophobic ligand-binding pocket (LBP). Although the LBP of COUP-TFII is filled with aromatic amino-acid side chains, alternative modes of ligand binding could potentially lead to regulation of the orphan receptor. Here, we screened a synthetic and natural compound library in a yeast one-hybrid assay and identified 4-methoxynaphthol as an inhibitor of COUP-TFII. This synthetic inhibitor was able to counteract processes either positively or negatively regulated by COUP-TFII in different mammalian cell systems. Hence, we demonstrate that the true orphan receptor COUP-TFII can be targeted by small chemicals which could be used to study the physiological functions of COUP-TFII or to counteract detrimental COUP-TFII activities in various pathological conditions.
Asunto(s)
Factor de Transcripción COUP II/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Sitios de Unión , Factor de Transcripción COUP II/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Hep G2 , Humanos , RatonesRESUMEN
Adipocyte differentiation and function relies on a network of transcription factors, which is disrupted in obesity-associated low grade, chronic inflammation leading to adipose tissue dysfunction. In this context, there is a need for a thorough understanding of the transcriptional regulatory network involved in adipose tissue pathophysiology. Recent advances in the functional annotation of the genome has highlighted the role of non-coding RNAs in cellular differentiation processes in coordination with transcription factors. Using an unbiased genome-wide approach, we identified and characterized a novel long intergenic non-coding RNA (lincRNA) strongly induced during adipocyte differentiation. This lincRNA favors adipocyte differentiation and coactivates the master adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARγ) through interaction with the paraspeckle component and hnRNP-like RNA binding protein 14 (RBM14/NCoAA), and was therefore called PPARγ-activator RBM14-associated lncRNA (Paral1). Paral1 expression is restricted to adipocytes and decreased in humans with increasing body mass index. A decreased expression was also observed in diet-induced or genetic mouse models of obesity and this down-regulation was mimicked in vitro by TNF treatment. In conclusion, we have identified a novel component of the adipogenic transcriptional regulatory network defining the lincRNA Paral1 as an obesity-sensitive regulator of adipocyte differentiation and function.