RESUMEN
More than one-half billion people are obese, and despite progress in genetic research, much of the heritability of obesity remains enigmatic. Here, we identify a Trim28-dependent network capable of triggering obesity in a non-Mendelian, "on/off" manner. Trim28(+/D9) mutant mice exhibit a bi-modal body-weight distribution, with isogenic animals randomly emerging as either normal or obese and few intermediates. We find that the obese-"on" state is characterized by reduced expression of an imprinted gene network including Nnat, Peg3, Cdkn1c, and Plagl1 and that independent targeting of these alleles recapitulates the stochastic bi-stable disease phenotype. Adipose tissue transcriptome analyses in children indicate that humans too cluster into distinct sub-populations, stratifying according to Trim28 expression, transcriptome organization, and obesity-associated imprinted gene dysregulation. These data provide evidence of discrete polyphenism in mouse and man and thus carry important implications for complex trait genetics, evolution, and medicine.
Asunto(s)
Epigénesis Genética , Haploinsuficiencia , Proteínas Nucleares/genética , Obesidad/genética , Proteínas Represoras/genética , Delgadez/genética , Adolescente , Animales , Índice de Masa Corporal , Niño , Preescolar , Humanos , Ratones , Encuestas Nutricionales , Polimorfismo Genético , Proteína 28 que Contiene Motivos TripartitoRESUMEN
The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Imprinted genes, which are expressed in a parent-of-origin-specific manner, are known to have important roles in development, growth and metabolism. However, our knowledge regarding their roles in the control of pancreatic growth and function remains limited. Here we show that many imprinted genes are highly expressed in pancreatic mesenchyme-derived cells and explore the role of the paternally-expressed insulin-like growth factor 2 (Igf2) gene in mesenchymal and epithelial pancreatic lineages using a newly developed conditional Igf2 mouse model. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of any discernible growth or functional phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Additionally, increased IGF2 levels specifically in the mesenchyme, through conditional Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Furthermore, ex-vivo exposure of primary acinar cells to exogenous IGF2 activates AKT, a key signalling node, and increases their number and amylase production. Based on these findings, we propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/genética , Mesodermo/crecimiento & desarrollo , Páncreas/crecimiento & desarrollo , Comunicación Paracrina/genética , Células Acinares/metabolismo , Células Acinares/patología , Aminoácidos/genética , Animales , Linaje de la Célula/genética , Cromo , Metilación de ADN/genética , Femenino , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica/genética , Impresión Genómica/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones , Ácidos Nicotínicos/genética , Páncreas/citología , Páncreas/metabolismo , Embarazo , ARN Largo no Codificante/genéticaRESUMEN
The Slc26 gene family encodes several conserved anion transporters implicated in human genetic disorders, including Pendred syndrome, diastrophic dysplasia and congenital chloride diarrhea. We previously characterized the TAT1 (testis anion transporter 1; SLC26A8) protein specifically expressed in male germ cells and mature sperm and showed that in the mouse, deletion of Tat1 caused male sterility due to a lack of sperm motility, impaired sperm capacitation and structural defects of the flagella. Ca(2+), Cl(-) and HCO(3)(-) influxes trigger sperm capacitation events required for oocyte fertilization; these events include the intracellular rise of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA)-dependent protein phosphorylation. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in mature sperm and has been shown to contribute to Cl(-) and HCO(3)(-) movements during capacitation. Furthermore, several members of the SLC26 family have been described to form complexes with CFTR, resulting in the reciprocal regulation of their activities. We show here that TAT1 and CFTR physically interact and that in Xenopus laevis oocytes and in CHO-K1 cells, TAT1 expression strongly stimulates CFTR activity. Consistent with this, we show that Tat1 inactivation in mouse sperm results in deregulation of the intracellular cAMP content, preventing the activation of PKA-dependent downstream phosphorylation cascades essential for sperm activation. These various results suggest that TAT1 and CFTR may form a molecular complex involved in the regulation of Cl(-) and HCO(3)(-) fluxes during sperm capacitation. In humans, mutations in CFTR and/or TAT1 may therefore be causes of asthenozoospermia and low fertilizing capacity of sperm.
Asunto(s)
Proteínas de Transporte de Anión/fisiología , Antiportadores/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Capacitación Espermática/fisiología , Testículo/metabolismo , Animales , Bicarbonatos/metabolismo , Células COS , Células Cultivadas , Cloruros/metabolismo , Chlorocebus aethiops , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Electrofisiología , Humanos , Immunoblotting , Inmunoprecipitación , Masculino , Ratones , Ratones Transgénicos , Oocitos/citología , Oocitos/metabolismo , Fosforilación , Motilidad Espermática , Transportadores de Sulfato , Testículo/citología , Xenopus laevisRESUMEN
Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.
Asunto(s)
Metilación de ADN , Embrión de Mamíferos/citología , Genoma , Células Madre/fisiología , Cromosoma X/genética , Animales , Inestabilidad Cromosómica , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Impresión Genómica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , ADN Metiltransferasa 3BRESUMEN
The hormone GDF15 is secreted in response to cellular stressors. Metformin elevates circulating levels of GDF15, an action important for the drug's beneficial effects on body weight. Metformin can also inhibit mammalian respiratory complex I, leading to decreases in ATP:AMP ratio, activation of AMP Kinase (AMPK), and increased GDF15 production. We undertook studies using a range of mice with tissue-specific loss of Gdf15 (namely gut, liver and global deletion) to determine the relative contributions of two classical metformin target tissues, the gut and liver, to the elevation of GDF15 seen with metformin. In addition, we performed comparative studies with another pharmacological agent, the AMP kinase pan-activator, MK-8722. Deletion of Gdf15 from the intestinal epithelium significantly reduced the circulating GDF15 response to oral metformin, whereas deletion of Gdf15 from the liver had no effect. In contrast, deletion of Gdf15 from the liver, but not the gut, markedly reduced circulating GDF15 responses to MK-8722. Further, our data show that, while GDF15 restricts high-fat diet-induced weight gain, the intestinal production of GDF15 is not necessary for this effect. These findings add to the body of evidence implicating the intestinal epithelium in key aspects of the pharmacology of metformin action.
Asunto(s)
Factor 15 de Diferenciación de Crecimiento , Metformina , Animales , Ratones , Adenilato Quinasa/metabolismo , Transporte Biológico , Mucosa Intestinal , Hígado , Mamíferos , Metformina/farmacología , Factor 15 de Diferenciación de Crecimiento/metabolismoRESUMEN
OBJECTIVES: Obesity in humans and mice is associated with elevated levels of two hormones responsive to cellular stress, namely GDF15 and FGF21. Over-expression of each of these is associated with weight loss and beneficial metabolic changes but where they are secreted from and what they are required for physiologically in the context of overfeeding remains unclear. METHODS: Here we used tissue selective knockout mouse models and human transcriptomics to determine the source of circulating GDF15 in obesity. We then generated and characterized the metabolic phenotypes of GDF15/FGF21 double knockout mice. RESULTS: Circulating GDF15 and FGF21 are both largely derived from the liver, rather than adipose tissue or skeletal muscle, in obese states. Combined whole body deletion of FGF21 and GDF15 does not result in any additional weight gain in response to high fat feeding but it does result in significantly greater hepatic steatosis and insulin resistance than that seen in GDF15 single knockout mice. CONCLUSIONS: Collectively the data suggest that overfeeding activates a stress response in the liver which is the major source of systemic rises in GDF15 and FGF21. These hormones then activate pathways which reduce this metabolic stress.
Asunto(s)
Hígado Graso , Resistencia a la Insulina , Animales , Peso Corporal , Hígado Graso/genética , Hígado Graso/metabolismo , Factores de Crecimiento de Fibroblastos , Factor 15 de Diferenciación de Crecimiento/genética , Hormonas , Humanos , Resistencia a la Insulina/genética , Ratones , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismoRESUMEN
Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/-p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/-p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/-p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/-p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/-p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/-p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.
Asunto(s)
Ingestión de Alimentos/fisiología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Obesidad/metabolismo , Animales , Biomarcadores/metabolismo , Peso Corporal , Dieta Alta en Grasa , Metabolismo Energético , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/patologíaRESUMEN
OBJECTIVE: Insulin signalling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, as well as lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidaemia. We sought to investigate this discordance. METHODS: The human pathogenic Pik3r1 Y657∗ mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high-fat diet (HFD). We examined adipose and liver histology, and assessed liver responses to fasting and refeeding transcriptomically. RESULTS: Like humans with SHORT syndrome, Pik3r1WT/Y657∗ mice were small with severe IR, and adipose expansion on HFD was markedly reduced. Also as in humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycaemia nor intestinal malabsorption, provided an alternative explanation for the uncoupling of IR from dyslipidaemia. CONCLUSIONS: Pik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather, increased energy expenditure, and we suggest that further study of brown adipose tissue in both humans and mice is warranted.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/genética , Trastornos del Crecimiento/metabolismo , Hipercalcemia/metabolismo , Resistencia a la Insulina/genética , Enfermedades Metabólicas/metabolismo , Nefrocalcinosis/metabolismo , Tejido Adiposo Pardo/metabolismo , Adiposidad , Animales , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Dieta Alta en Grasa , Dislipidemias/genética , Metabolismo Energético/genética , Hígado Graso/metabolismo , Trastornos del Crecimiento/genética , Hipercalcemia/genética , Inflamación/metabolismo , Insulina/metabolismo , Lipogénesis , Hígado/metabolismo , Masculino , Enfermedades Metabólicas/genética , Ratones , Ratones Endogámicos C57BL , Nefrocalcinosis/genética , Obesidad/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Previous studies have implicated the Eed-Enx1 Polycomb group complex in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Here we show that recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure.
Asunto(s)
Compensación de Dosificación (Genética) , Embrión de Mamíferos/embriología , N-Metiltransferasa de Histona-Lisina , Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Células Madre Totipotentes/metabolismo , Cromosoma X/genética , Secuencia de Aminoácidos/genética , Animales , Diferenciación Celular/genética , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Metilación de ADN , Femenino , Feto , Regulación del Desarrollo de la Expresión Génica/genética , Histona Metiltransferasas , Histonas/genética , Histonas/metabolismo , Lisina/genética , Lisina/metabolismo , Masculino , Metiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteína Metiltransferasas , ARN Largo no Codificante , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteínas Represoras/genética , Células Madre Totipotentes/citologíaRESUMEN
Ambiguity regarding the role of glucose-dependent insulinotropic polypeptide (GIP) in obesity arises from conflicting reports asserting that both GIP receptor (GIPR) agonism and antagonism are effective strategies for inhibiting weight gain. To enable identification and manipulation of Gipr-expressing (Gipr) cells, we created Gipr-Cre knockin mice. As GIPR-agonists have recently been reported to suppress food intake, we aimed to identify central mediators of this effect. Gipr cells were identified in the arcuate, dorsomedial, and paraventricular nuclei of the hypothalamus, as confirmed by RNAscope in mouse and human. Single-cell RNA-seq identified clusters of hypothalamic Gipr cells exhibiting transcriptomic signatures for vascular, glial, and neuronal cells, the latter expressing somatostatin but little pro-opiomelanocortin or agouti-related peptide. Activation of Gq-DREADDs in hypothalamic Gipr cells suppressed food intake in vivo, which was not obviously additive with concomitant GLP1R activation. These data identify hypothalamic GIPR as a target for the regulation of energy balance.
Asunto(s)
Ingestión de Alimentos/fisiología , Hipotálamo/citología , Neuronas/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Anciano de 80 o más Años , Animales , Ingestión de Alimentos/efectos de los fármacos , Femenino , Polipéptido Inhibidor Gástrico/metabolismo , Técnicas de Sustitución del Gen , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/tratamiento farmacológico , Receptores de la Hormona Gastrointestinal/agonistas , Receptores de la Hormona Gastrointestinal/genéticaRESUMEN
Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear.
Asunto(s)
Antiportadores/genética , Hipotiroidismo Congénito/genética , Bocio/genética , Transportadores de Sulfato/genética , Adulto , Animales , Niño , Preescolar , Codón sin Sentido , Hipotiroidismo Congénito/diagnóstico , Análisis Mutacional de ADN , Femenino , Bocio/congénito , Bocio/diagnóstico , Células HEK293 , Homocigoto , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Linaje , Glándula Tiroides/patología , Secuenciación del ExomaRESUMEN
The co-stimulatory molecule CD28 is essential for activation of helper T cells. Despite this critical role, it is not known whether CD28 has functions in maintaining T cell responses following activation. To determine the role for CD28 after T cell priming, we generated a strain of mice where CD28 is removed from CD4(+) T cells after priming. We show that continued CD28 expression is important for effector CD4(+) T cells following infection; maintained CD28 is required for the expansion of T helper type 1 cells, and for the differentiation and maintenance of T follicular helper cells during viral infection. Persistent CD28 is also required for clearance of the bacterium Citrobacter rodentium from the gastrointestinal tract. Together, this study demonstrates that CD28 persistence is required for helper T cell polarization in response to infection, describing a novel function for CD28 that is distinct from its role in T cell priming.
Asunto(s)
Antígenos CD28/metabolismo , Citrobacter rodentium/fisiología , Reactividad Cruzada/inmunología , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/microbiología , Inmunidad , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Diferenciación Celular , Proliferación Celular , Factores de Transcripción Forkhead/metabolismo , Inmunidad Celular , Virus de la Influenza A/fisiología , Integrasas/metabolismo , Ligandos , Ratones , Infecciones por Orthomyxoviridae/inmunología , Receptores OX40/metabolismo , Transducción de Señal/inmunologíaRESUMEN
X chromosome inactivation involves multiple levels of chromatin modification, established progressively and in a stepwise manner during early development. The chromosomal protein Smchd1 was recently shown to play an important role in DNA methylation of CpG islands (CGIs), a late step in the X inactivation pathway that is required for long-term maintenance of gene silencing. Here we show that inactive X chromosome (Xi) CGI methylation can occur via either Smchd1-dependent or -independent pathways. Smchd1-dependent CGI methylation, the primary pathway, is acquired gradually over an extended period, whereas Smchd1-independent CGI methylation occurs rapidly after the onset of X inactivation. The de novo methyltransferase Dnmt3b is required for methylation of both classes of CGI, whereas Dnmt3a and Dnmt3L are dispensable. Xi CGIs methylated by these distinct pathways differ with respect to their sequence characteristics and immediate chromosomal environment. We discuss the implications of these results for understanding CGI methylation during development.