RESUMEN
The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.
Asunto(s)
Adipocitos , Diferenciación Celular , Oxígeno , Oxígeno/metabolismo , Adipocitos/metabolismo , Adipocitos/citología , Humanos , Técnicas de Cultivo de Célula/métodos , Animales , Glucólisis , Hepatocitos/metabolismo , Hipoxia de la Célula , Mitocondrias/metabolismo , Ratones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células Cultivadas , Glucosa/metabolismo , Macrófagos/metabolismoRESUMEN
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
RATIONALE: Atherosclerosis is a chronic inflammatory disease. Recent studies have shown that dysfunctional autophagy in endothelial cells, smooth muscle cells, and macrophages, plays a detrimental role during atherogenesis, leading to the suggestion that autophagy-stimulating approaches may provide benefit. OBJECTIVE: Dendritic cells (DCs) are at the crossroad of innate and adaptive immune responses and profoundly modulate the development of atherosclerosis. Intriguingly, the role of autophagy in DC function during atherosclerosis and how the autophagy process would impact disease development has not been addressed. METHODS AND RESULTS: Here, we show that the autophagic flux in atherosclerosis-susceptible Ldlr-/- (low-density lipoprotein receptor-deficient) mice is substantially higher in splenic and aortic DCs compared with macrophages and is further activated under hypercholesterolemic conditions. RNA sequencing and functional studies on selective cell populations reveal that disruption of autophagy through deletion of Atg16l1 differentially affects the biology and functions of DC subsets in Ldlr-/- mice under high-fat diet. Atg16l1 deficient CD11b+ DCs develop a TGF (transforming growth factor)-ß-dependent tolerogenic phenotype and promote the expansion of regulatory T cells, whereas no such effects are seen with Atg16l1 deficient CD8α+ DCs. Atg16l1 deletion in DCs (all CD11c-expressing cells) expands aortic regulatory T cells in vivo, limits the accumulation of T helper cells type 1, and reduces the development of atherosclerosis in Ldlr-/- mice. In contrast, no such effects are seen when Atg16l1 is deleted selectively in conventional CD8α+ DCs and CD103+ DCs. Total T-cell or selective regulatory T-cell depletion abrogates the atheroprotective effect of Atg16l1 deficient DCs. CONCLUSIONS: In contrast to its proatherogenic role in macrophages, autophagy disruption in DCs induces a counter-regulatory response that maintains immune homeostasis in Ldlr-/- mice under high-fat diet and limits atherogenesis. Selective modulation of autophagy in DCs could constitute an interesting therapeutic target in atherosclerosis.
Asunto(s)
Aorta/inmunología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Autofagia , Antígeno CD11b/inmunología , Comunicación Celular , Proliferación Celular , Células Dendríticas/inmunología , Activación de Linfocitos , Linfocitos T Reguladores/inmunología , Animales , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Trasplante de Médula Ósea , Antígenos CD11/genética , Antígenos CD11/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Femenino , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transducción de Señal , Linfocitos T Reguladores/metabolismoRESUMEN
OBJECTIVE: Myelosuppression is a life-threatening complication of thiopurine therapy, and the incidence of thiopurine-induced myelosuppression is higher in East Asians than in Europeans. We investigated genetic factors associated with thiopurine-induced leukopenia in patients with IBD. DESIGN: A genome-wide association study (GWAS) was conducted in thiopurine-treated patients with IBD, followed by high-throughput sequencing of genes identified as significant in the GWAS or those involved in thiopurine metabolism (n=331). Significant loci associated with thiopurine-induced leukopenia were validated in two additional replication cohorts (n=437 and n=330). Functional consequences of FTO (fat mass and obesity-associated) variant were examined both in vitro and in vivo. RESULTS: The GWAS identified two loci associated with thiopurine-induced leukopenia (rs16957920, FTO intron; rs2834826, RUNX1 intergenic). High-throughput targeted sequencing indicated that an FTO coding variant (rs79206939, p.A134T) linked to rs16957920 is associated with thiopurine-induced leukopenia. This result was further validated in two replication cohorts (combined p=1.3×10-8, OR=4.3). The frequency of FTO p.A134T is 5.1% in Koreans but less than 0.1% in Western populations. The p.A134T variation reduced FTO activity by 65% in the nucleotide demethylase assay. In vivo experiments revealed that Fto-/- and Fto+/- mice were more susceptible to thiopurine-induced myelosuppression than wild-type mice. CONCLUSIONS: The results suggest that the hypomorphic FTO p.A134T variant is associated with thiopurine-induced leukopenia. These results shed light on the novel physiological role of FTO and provide a potential pharmacogenetic biomarker for thiopurine therapy.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Azatioprina/efectos adversos , Inmunosupresores/efectos adversos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Leucopenia/inducido químicamente , Mercaptopurina/efectos adversos , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Anciano , Animales , Azatioprina/uso terapéutico , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Marcadores Genéticos , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunosupresores/uso terapéutico , Enfermedades Inflamatorias del Intestino/genética , Leucopenia/genética , Masculino , Mercaptopurina/uso terapéutico , Ratones , Ratones Noqueados , Persona de Mediana Edad , República de Corea , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
SNPs in the first intron of FTO (fat mass and obesity associated) are strongly associated with human obesity. While it is not yet formally established that this effect is mediated through the actions of the FTO protein itself, loss of function mutations in FTO or its murine homologue Fto result in severe growth retardation, and mice globally overexpressing FTO are obese. The mechanisms through which FTO influences growth and body composition are unknown. We describe a role for FTO in the coupling of amino acid levels to mammalian target of rapamycin complex 1 signaling. These findings suggest that FTO may influence body composition through playing a role in cellular nutrient sensing.
Asunto(s)
Aminoácidos/metabolismo , Composición Corporal/genética , Obesidad/genética , Proteínas/genética , Proteínas/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Fraccionamiento Celular , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Fibroblastos , Células HEK293 , Humanos , Inmunoprecipitación , Ratones , Polimorfismo de Nucleótido Simple/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en TándemRESUMEN
AIMS: The adaptive immune response plays an important role in atherosclerosis. In response to a high-fat/high-cholesterol (HF/HC) diet, marginal zone B (MZB) cells activate an atheroprotective programme by regulating the differentiation and accumulation of 'poorly differentiated' T follicular helper (Tfh) cells. On the other hand, Tfh cells activate the germinal centre response, which promotes atherosclerosis through the production of class-switched high-affinity antibodies. We therefore investigated the direct role of Tfh cells and the role of IL18 in Tfh differentiation in atherosclerosis. METHODS AND RESULTS: We generated atherosclerotic mouse models with selective genetic deletion of Tfh cells, MZB cells, or IL18 signalling in Tfh cells. Surprisingly, mice lacking Tfh cells had increased atherosclerosis. Lack of Tfh not only reduced class-switched IgG antibodies against oxidation-specific epitopes (OSEs) but also reduced atheroprotective natural IgM-type anti-phosphorylcholine (PC) antibodies, despite no alteration of natural B1 cells. Moreover, the absence of Tfh cells was associated with an accumulation of MZB cells with substantially reduced ability to secrete antibodies. In the same manner, MZB cell deficiency in Ldlr-/- mice was associated with a significant decrease in atheroprotective IgM antibodies, including natural anti-PC IgM antibodies. In humans, we found a positive correlation between circulating MZB-like cells and anti-OSE IgM antibodies. Finally, we identified an important role for IL18 signalling in HF/HC diet-induced Tfh. CONCLUSION: Our findings reveal a previously unsuspected role of MZB cells in regulating atheroprotective 'natural' IgM antibody production in a Tfh-dependent manner, which could have important pathophysiological and therapeutic implications.
Asunto(s)
Aterosclerosis , Interleucina-18 , Humanos , Ratones , Animales , Inmunoglobulina M , Linfocitos B , Aterosclerosis/genética , Aterosclerosis/prevención & control , Colesterol , Linfocitos T Colaboradores-InductoresRESUMEN
Genomewide-association studies have revealed that SNPs (single nucleotide polymorphisms) in FTO (fat mass and obesity-associated) are robustly associated with BMI (body mass index) and obesity. FTO is an Fe(II) 2-OG (2-oxoglutarate)-dependent dioxygenase that can demethylate 3-meT (3-methylthymine) in single-stranded DNA, as well as 3-meU (3-methyluracil) and N6-methyl adenosine in RNA. In the present paper we describe the development of an RNase-cleavage assay measuring the demethylation activity of FTO on 3-meU. RNase A cleaves at the 3'-end of pyrimidines, including uracil, and a methyl group at position three of uracil inhibits cleavage. An oligonucleotide probe was designed consisting of a DNA stem, an RNA loop containing a single 3-meU as the only RNase A-cleavage site, a fluorescent reporter on one end and a quencher at the other end. FTO demethylation of the unique 3-meU enables RNase A cleavage, releasing the quencher and enabling a fluorescent signal. In the presence of excess RNase A, FTO activity is limiting to the development of fluorescent signal, which can be read continuously and is able to discriminate between wild-type and the catalytically dead R316Q FTO. 2-OG is a co-substrate of FTO and, as a metabolite in the citric acid cycle, is a marker of intracellular nutritional status. The assay described in the present paper was used to measure, for the first time, the K(m) of FTO for 2-OG. The K(m) of 2.88 µM is up to 10-fold lower than the estimated intracellular concentrations of 2-OG, rendering it unlikely that FTO functions as a sensor for 2-OG levels.
Asunto(s)
Técnicas Biosensibles/métodos , Índice de Masa Corporal , Ácidos Cetoglutáricos/metabolismo , Proteínas/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Técnicas Biosensibles/normas , Metilación de ADN/genética , Humanos , Cinética , Obesidad , Polimorfismo de Nucleótido Simple/genética , Proteínas/genética , Proteínas/fisiología , Reproducibilidad de los Resultados , Especificidad por Sustrato/genéticaRESUMEN
At the moment of union in fertilization, sperm and oocyte are transcriptionally silent. The ensuing onset of embryonic transcription (embryonic genome activation [EGA]) is critical for development, yet its timing and profile remain elusive in any vertebrate species. We here dissect transcription during EGA by high-resolution single-cell RNA sequencing of precisely synchronized mouse one-cell embryos. This reveals a program of embryonic gene expression (immediate EGA [iEGA]) initiating within 4 h of fertilization. Expression during iEGA produces canonically spliced transcripts, occurs substantially from the maternal genome, and is mostly downregulated at the two-cell stage. Transcribed genes predict regulation by transcription factors (TFs) associated with cancer, including c-Myc. Blocking c-Myc or other predicted regulatory TF activities disrupts iEGA and induces acute developmental arrest. These findings illuminate intracellular mechanisms that regulate the onset of mammalian development and hold promise for the study of cancer.
Asunto(s)
Embrión de Mamíferos , Perfilación de la Expresión Génica , Masculino , Animales , Ratones , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Semen , Expresión Génica , Desarrollo Embrionario/genética , Mamíferos/genéticaRESUMEN
In human embryos, the initiation of transcription (embryonic genome activation [EGA]) occurs by the eight-cell stage, but its exact timing and profile are unclear. To address this, we profiled gene expression at depth in human metaphase II oocytes and bipronuclear (2PN) one-cell embryos. High-resolution single-cell RNA sequencing revealed previously inaccessible oocyte-to-embryo gene expression changes. This confirmed transcript depletion following fertilization (maternal RNA degradation) but also uncovered low-magnitude upregulation of hundreds of spliced transcripts. Gene expression analysis predicted embryonic processes including cell-cycle progression and chromosome maintenance as well as transcriptional activators that included cancer-associated gene regulators. Transcription was disrupted in abnormal monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos. These findings indicate that human embryonic transcription initiates at the one-cell stage, sooner than previously thought. The pattern of gene upregulation promises to illuminate processes involved at the onset of human development, with implications for epigenetic inheritance, stem-cell-derived embryos, and cancer.
Asunto(s)
Embrión de Mamíferos , Genoma Humano , Blastocisto , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , OocitosRESUMEN
Shexiang Baoxin Pill (SBP) is an oral formulation of Chinese materia medica for the treatment of angina pectoris. It displays pleiotropic roles in protecting the cardiovascular system. However, the mode of action of SBP in promoting angiogenesis, and in particular the synergy between its constituents is currently not fully understood. The combination of ginsenosides Rb2 and Rg3 were studied in human umbilical vein endothelial cells (HUVECs) for their proangiogenic effects. To understand the mode of action of the combination in more mechanistic detail, RNA-Seq analysis was conducted, and differentially expressed genes (DEGs), pathway analysis and Weighted Gene Correlation Network Analysis (WGCNA) were applied to further identify important genes that a play pivotal role in the combination treatment. The effects of pathway-specific inhibitors were observed to provide further support for the hypothesized mode of action of the combination. Ginsenosides Rb2 and Rg3 synergistically promoted HUVEC proliferation and tube formation under defined culture conditions. Also, the combination of Rb2/Rg3 rescued cells from homocysteine-induced damage. mRNA expression of CXCL8, CYR61, FGF16 and FGFRL1 was significantly elevated by the Rb2/Rg3 treatment, and representative signaling pathways induced by these genes were found. The increase of protein levels of phosphorylated-Akt and ERK42/44 by the Rb2/Rg3 combination supports the notion that it promotes endothelial cell proliferation via the PI3K/Akt and MAPK/ERK signaling pathways. The present study provides the hypothesis that SBP, via ginsenosides Rb2 and Rg3, involves the CXCR1/2 CXCL8 (IL8)-mediated PI3K/Akt and MAPK/ERK signaling pathways in achieving its proangiogenic effects.
RESUMEN
Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig-/-) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig-/- mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion.
Asunto(s)
Tejido Adiposo/metabolismo , Leptina/metabolismo , Proteínas Nucleares/genética , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis , Adiponectina/genética , Adiponectina/metabolismo , Animales , Peso Corporal , Dieta Alta en Grasa , Femenino , Prueba de Tolerancia a la Glucosa , Leptina/sangre , Leptina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Proteínas Nucleares/deficiencia , Fenotipo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
OBJECTIVES: Combinatorial therapies are under intense investigation to develop more efficient anti-obesity drugs; however, little is known about how they act in the brain to produce enhanced anorexia and weight loss. The goal of this study was to identify the brain sites and neuronal populations engaged during the co-administration of GLP-1R and CCK1R agonists, an efficient combination therapy in obese rodents. METHODS: We measured acute and long-term feeding and body weight responses and neuronal activation patterns throughout the neuraxis and in specific neuronal subsets in response to GLP-1R and CCK1R agonists administered alone or in combination in lean and high-fat diet fed mice. We used PhosphoTRAP to obtain unbiased molecular markers for neuronal populations selectively activated by the combination of the two agonists. RESULTS: The initial anorectic response to GLP-1R and CCK1R co-agonism was mediated by a reduction in meal size, but over a few hours, a reduction in meal number accounted for the sustained feeding suppressive effects. The nucleus of the solitary tract (NTS) is one of the few brain sites where GLP-1R and CCK1R signalling interact to produce enhanced neuronal activation. None of the previously categorised NTS neuronal subpopulations relevant to feeding behaviour were implicated in this increased activation. However, we identified NTS/AP Calcrl+ neurons as treatment targets. CONCLUSIONS: Collectively, these studies indicated that circuit-level integration of GLP-1R and CCK1R co-agonism in discrete brain nuclei including the NTS produces enhanced rapid and sustained appetite suppression and weight loss.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/metabolismo , Obesidad/tratamiento farmacológico , Receptores de Colecistoquinina/metabolismo , Animales , Fármacos Antiobesidad/farmacología , Regulación del Apetito , Encéfalo/metabolismo , Dieta Alta en Grasa , Ingestión de Alimentos/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Obesidad/metabolismo , Núcleo Solitario/metabolismo , Pérdida de Peso/efectos de los fármacosRESUMEN
Macrophages exhibit a spectrum of activation states ranging from classical to alternative activation1. Alternatively, activated macrophages are involved in diverse pathophysiological processes such as confining tissue parasites2, improving insulin sensitivity3 or promoting an immune-tolerant microenvironment that facilitates tumour growth and metastasis4. Recently, the metabolic regulation of macrophage function has come into focus as both the classical and alternative activation programmes require specific regulated metabolic reprogramming5. While most of the studies regarding immunometabolism have focussed on the catabolic pathways activated to provide energy, little is known about the anabolic pathways mediating macrophage alternative activation. In this study, we show that the anabolic transcription factor sterol regulatory element binding protein 1 (SREBP1) is activated in response to the canonical T helper 2 cell cytokine interleukin-4 to trigger the de novo lipogenesis (DNL) programme, as a necessary step for macrophage alternative activation. Mechanistically, DNL consumes NADPH, partitioning it away from cellular antioxidant defences and raising reactive oxygen species levels. Reactive oxygen species serves as a second messenger, signalling sufficient DNL, and promoting macrophage alternative activation. The pathophysiological relevance of this mechanism is validated by showing that SREBP1/DNL is essential for macrophage alternative activation in vivo in a helminth infection model.
Asunto(s)
Antioxidantes/metabolismo , Ácidos Grasos/biosíntesis , Macrófagos/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Dexametasona/farmacología , Humanos , Interleucina-4/farmacología , Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Nippostrongylus/aislamiento & purificación , Nippostrongylus/patogenicidad , Células RAW 264.7 , Análisis de Secuencia de ARN/métodos , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitología , Regulación hacia ArribaRESUMEN
Even though metformin is widely used to treat type2 diabetes, reducing glycaemia and body weight, the mechanisms of action are still elusive. Recent studies have identified the gastrointestinal tract as an important site of action. Here we used intestinal organoids to explore the effects of metformin on intestinal cell physiology. Bulk RNA-sequencing analysis identified changes in hexose metabolism pathways, particularly glycolytic genes. Metformin increased expression of Slc2a1 (GLUT1), decreased expression of Slc2a2 (GLUT2) and Slc5a1 (SGLT1) whilst increasing GLUT-dependent glucose uptake and glycolytic rate as observed by live cell imaging of genetically encoded metabolite sensors and measurement of oxygen consumption and extracellular acidification rates. Metformin caused mitochondrial dysfunction and metformin's effects on 2D-cultures were phenocopied by treatment with rotenone and antimycin-A, including upregulation of GDF15 expression, previously linked to metformin dependent weight loss. Gene expression changes elicited by metformin were replicated in 3D apical-out organoids and distal small intestines of metformin treated mice. We conclude that metformin affects glucose uptake, glycolysis and GDF-15 secretion, likely downstream of the observed mitochondrial dysfunction. This may explain the effects of metformin on intestinal glucose utilisation and food balance.
Asunto(s)
Glucosa/metabolismo , Factor 15 de Diferenciación de Crecimiento/biosíntesis , Metformina/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Animales , Transporte Biológico , Respiración de la Célula/efectos de los fármacos , Células Cultivadas , Biología Computacional/métodos , Perfilación de la Expresión Génica , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Factor 15 de Diferenciación de Crecimiento/genética , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Masculino , Ratones , Mitocondrias/genética , Fosforilación Oxidativa/efectos de los fármacos , TranscriptomaRESUMEN
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.
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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
BACKGROUND: Innate lymphoid cells type 2 (ILC2s) play critical homeostatic functions in peripheral tissues. ILC2s reside in perivascular niches and limit atherosclerosis development. OBJECTIVES: ILC2s also reside in the pericardium but their role in postischemic injury is unknown. METHODS: We examined the role of ILC2 in a mouse model of myocardial infarction (MI), and compared mice with or without genetic deletion of ILC2. We determined infarct size using histology and heart function using echocardiography. We assessed cardiac ILC2 using flow cytometry and RNA sequencing. Based on these data, we devised a therapeutic strategy to activate ILC2 in mice with acute MI, using exogenous interleukin (IL)-2. We also assessed the ability of low-dose IL-2 to activate ILC2 in a double-blind randomized clinical trial of patients with acute coronary syndromes (ACS). RESULTS: We found that ILC2 levels were increased in pericardial adipose tissue after experimental MI, and genetic ablation of ILC2 impeded the recovery of heart function. RNA sequencing revealed distinct transcript signatures in ILC2, and pointed to IL-2 axis as a major upstream regulator. Treatment of T-cell-deficient mice with IL-2 (to activate ILC2) significantly improved the recovery of heart function post-MI. Administration of low-dose IL-2 to patients with ACS led to activation of circulating ILC2, with significant increase in circulating IL-5, a prototypic ILC2-derived cytokine. CONCLUSIONS: ILC2s promote cardiac healing and improve the recovery of heart function after MI in mice. Activation of ILC2 using low-dose IL-2 could be a novel therapeutic strategy to promote a reparative response after MI.
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Síndrome Coronario Agudo , Interleucina-2 , Linfocitos , Infarto del Miocardio , Recuperación de la Función , Animales , Femenino , Síndrome Coronario Agudo/tratamiento farmacológico , Tejido Adiposo/inmunología , Interleucina-2/metabolismo , Interleucina-2/uso terapéutico , Linfocitos/fisiología , Ratones Endogámicos C57BL , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Recuperación de la Función/inmunología , Función VentricularRESUMEN
Background: Development of adipose tissue before birth is essential for energy storage and thermoregulation in the neonate and for cardiometabolic health in later life. Thyroid hormones are important regulators of growth and maturation in fetal tissues. Offspring hypothyroid in utero are poorly adapted to regulate body temperature at birth and are at risk of becoming obese and insulin resistant in childhood. The mechanisms by which thyroid hormones regulate the growth and development of adipose tissue in the fetus, however, are unclear. Methods: This study examined the structure, transcriptome, and protein expression of perirenal adipose tissue (PAT) in a fetal sheep model of thyroid hormone deficiency during late gestation. Proportions of unilocular (UL) (white) and multilocular (ML) (brown) adipocytes, and UL adipocyte size, were assessed by histological and stereological techniques. Changes to the adipose transcriptome were investigated by RNA sequencing and bioinformatic analysis, and proteins of interest were quantified by Western blotting. Results: Hypothyroidism in utero resulted in elevated plasma insulin and leptin concentrations and overgrowth of PAT in the fetus, specifically due to hyperplasia and hypertrophy of UL adipocytes with no change in ML adipocyte mass. RNA sequencing and genomic analyses showed that thyroid deficiency affected 34% of the genes identified in fetal adipose tissue. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathways were associated with adipogenic, metabolic, and thermoregulatory processes, insulin resistance, and a range of endocrine and adipocytokine signaling pathways. Adipose protein levels of signaling molecules, including phosphorylated S6-kinase (pS6K), glucose transporter isoform 4 (GLUT4), and peroxisome proliferator-activated receptor γ (PPARγ), were increased by fetal hypothyroidism. Fetal thyroid deficiency decreased uncoupling protein 1 (UCP1) protein and mRNA content, and UCP1 thermogenic capacity without any change in ML adipocyte mass. Conclusions: Growth and development of adipose tissue before birth is sensitive to thyroid hormone status in utero. Changes to the adipose transcriptome and phenotype observed in the hypothyroid fetus may have consequences for neonatal survival and the risk of obesity and metabolic dysfunction in later life.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Hipotiroidismo Congénito/metabolismo , Termogénesis/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Insulina/sangre , Leptina/sangre , PPAR gamma/metabolismo , Ovinos , Transducción de Señal/fisiología , Transcriptoma , Proteína Desacopladora 1/metabolismoRESUMEN
Leptin plays a major role in coordinating the integrated response of the CNS to changes in nutritional state. Neurons within the paraventricular nucleus (PVN) of the hypothalamus express leptin receptors and receive dense innervation from leptin receptor-expressing neurons in the arcuate nucleus. To obtain new insights into the effects of circulating leptin on PVN function, we compared global transcriptional profiles of laser-captured PVN from ad libitum fed mice versus 48 h fasted mice receiving either sham or leptin treatment intraperitoneally. Five hundred twenty-seven PVN-expressed genes were altered by fasting in a manner that was at least partially reversible by leptin. Consistent with previous reports, thyrotrophin releasing hormone mRNA levels were decreased by fasting but restored to fed levels with leptin treatment. mRNA levels of oxytocin, vasopressin, and somatostatin were also reduced by fasting and restored by leptin. Given the known effects of leptin on synaptic remodeling, it is notable that, among the top 15 genes that were positively regulated by leptin, five have been implicated in synaptic function and/or plasticity (basigin, apolipoprotein E, Gap43, GABA(A) receptor-associated protein, and synuclein-gamma). Pathway analysis identified oxidative phosphorylation, in particular, genes encoding complex 1 proteins that play a role in ubiquinone biosynthesis, to be the predominant gene set that was significantly regulated in a leptin-dependent manner. Thus, in addition to its effects on the expression of a broad range of neuropeptides, leptin may also exert more general influences on synaptic function in, and the bioenergetic state of, the PVN.
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Regulación de la Expresión Génica/efectos de los fármacos , Leptina/administración & dosificación , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Algoritmos , Animales , Privación de Alimentos/fisiología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Masculino , Redes y Vías Metabólicas/genética , Ratones , Microdisección/métodos , Plasticidad Neuronal/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fosforilación Oxidativa , ARN Mensajero/metabolismo , Sinapsis/genética , Hormona Liberadora de Tirotropina/genética , Hormona Liberadora de Tirotropina/metabolismoRESUMEN
Enteroendocrine cells (EECs) produce hormones such as glucagon-like peptide 1 and peptide YY that regulate food absorption, insulin secretion, and appetite. Based on the success of glucagon-like peptide 1-based therapies for type 2 diabetes and obesity, EECs are themselves the focus of drug discovery programs to enhance gut hormone secretion. The aim of this study was to identify the transcriptome and peptidome of human EECs and to provide a cross-species comparison between humans and mice. By RNA sequencing of human EECs purified by flow cytometry after cell fixation and staining, we present a first transcriptomic analysis of human EEC populations and demonstrate a strong correlation with murine counterparts. RNA sequencing was deep enough to enable identification of low-abundance transcripts such as G-protein-coupled receptors and ion channels, revealing expression in human EECs of G-protein-coupled receptors previously found to play roles in postprandial nutrient detection. With liquid chromatography-tandem mass spectrometry, we profiled the gradients of peptide hormones along the human and mouse gut, including their sequences and posttranslational modifications. The transcriptomic and peptidomic profiles of human and mouse EECs and cross-species comparison will be valuable tools for drug discovery programs and for understanding human metabolism and the endocrine impacts of bariatric surgery.
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Diabetes Mellitus Tipo 2 , Transcriptoma , Animales , Células Enteroendocrinas , Péptido 1 Similar al Glucagón , Humanos , Ratones , Receptores Acoplados a Proteínas GRESUMEN
Context: Self-limited delayed puberty (DP) is often associated with a delay in physical maturation, but although highly heritable the causal genetic factors remain elusive. Genome-wide association studies of the timing of puberty have identified multiple loci for age at menarche in females and voice break in males, particularly in pathways controlling energy balance. Objective/Main Outcome Measures: We sought to assess the contribution of rare variants in such genes to the phenotype of familial DP. Design/Patients: We performed whole-exome sequencing in 67 pedigrees (125 individuals with DP and 35 unaffected controls) from our unique cohort of familial self-limited DP. Using a whole-exome sequencing filtering pipeline one candidate gene [fat mass and obesity-associated gene (FTO)] was identified. In silico, in vitro, and mouse model studies were performed to investigate the pathogenicity of FTO variants and timing of puberty in FTO+/- mice. Results: We identified potentially pathogenic, rare variants in genes in linkage disequilibrium with genome-wide association studies of age at menarche loci in 283 genes. Of these, five genes were implicated in the control of body mass. After filtering for segregation with trait, one candidate, FTO, was retained. Two FTO variants, found in 14 affected individuals from three families, were also associated with leanness in these patients with DP. One variant (p.Leu44Val) demonstrated altered demethylation activity of the mutant protein in vitro. Fto+/- mice displayed a significantly delayed timing of pubertal onset (P < 0.05). Conclusions: Mutations in genes implicated in body mass and timing of puberty in the general population may contribute to the pathogenesis of self-limited DP.