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1.
Cell ; 165(4): 882-95, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-27133169

RESUMO

High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not in lean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associated macrophage activation a homeostatic role to restore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity.


Assuntos
Encéfalo/metabolismo , Dieta Hiperlipídica , Glucose/metabolismo , Obesidade/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Cognição , Células Endoteliais/metabolismo , Ácidos Graxos/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Camundongos , Células Mieloides/metabolismo
2.
Cell ; 165(1): 125-138, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-27015310

RESUMO

Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis.


Assuntos
Tecido Adiposo Marrom/metabolismo , Regulação do Apetite , Glucose/metabolismo , Resistência à Insulina , Neurônios/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Comportamento Alimentar , Camundongos , Miostatina/genética , Optogenética , Transcriptoma
3.
Cell ; 156(3): 495-509, 2014 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-24462248

RESUMO

Maternal metabolic homeostasis exerts long-term effects on the offspring's health outcomes. Here, we demonstrate that maternal high-fat diet (HFD) feeding during lactation predisposes the offspring for obesity and impaired glucose homeostasis in mice, which is associated with an impairment of the hypothalamic melanocortin circuitry. Whereas the number and neuropeptide expression of anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP) neurons, electrophysiological properties of POMC neurons, and posttranslational processing of POMC remain unaffected in response to maternal HFD feeding during lactation, the formation of POMC and AgRP projections to hypothalamic target sites is severely impaired. Abrogating insulin action in POMC neurons of the offspring prevents altered POMC projections to the preautonomic paraventricular nucleus of the hypothalamus (PVH), pancreatic parasympathetic innervation, and impaired glucose-stimulated insulin secretion in response to maternal overnutrition. These experiments reveal a critical timing, when altered maternal metabolism disrupts metabolic homeostasis in the offspring via impairing neuronal projections, and show that abnormal insulin signaling contributes to this effect.


Assuntos
Dieta Hiperlipídica , Hiperglicemia/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Lactação , Obesidade/metabolismo , Animais , Axônios/metabolismo , Feminino , Masculino , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Gravidez , Pró-Opiomelanocortina/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais
4.
Nat Immunol ; 15(5): 423-30, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24681566

RESUMO

Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6Rα chain of the receptor for IL-6 in myeloid cells (Il6ra(Δmyel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(Δmyel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.


Assuntos
Endotoxemia/imunologia , Resistência à Insulina , Interleucina-6/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Obesidade/imunologia , Animais , Células Cultivadas , Humanos , Resistência à Insulina/genética , Resistência à Insulina/imunologia , Interleucina-4/imunologia , Interleucina-6/genética , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Receptores de Interleucina-6/genética , Transdução de Sinais/genética
6.
Nature ; 541(7637): 371-375, 2017 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28002401

RESUMO

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2'-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.


Assuntos
Adenosina/análogos & derivados , Capuzes de RNA/química , Capuzes de RNA/metabolismo , Estabilidade de RNA , Adenosina/química , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Endorribonucleases/metabolismo , Epigênese Genética , Guanosina/análogos & derivados , Guanosina/metabolismo , Células HEK293 , Meia-Vida , Humanos , Masculino , Metilação , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Especificidade por Substrato , Sítio de Iniciação de Transcrição , Transcriptoma
7.
Nat Chem Biol ; 15(4): 340-347, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30778204

RESUMO

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.


Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/fisiologia , RNA Nuclear Pequeno/biossíntese , Adenosina/biossíntese , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Processamento Alternativo , Animais , Células HEK293 , Humanos , Masculino , Metilação , Camundongos , Camundongos Knockout , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Nuclear Pequeno/metabolismo
8.
J Immunol ; 198(7): 2927-2934, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28193830

RESUMO

Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that focal sites of inflammation around dying adipocytes, so-called crown-like structures, exhibit a unique microenvironment for macrophage proliferation. Interestingly, locally proliferating macrophages were not classically activated (M1), but they exhibited a rather alternatively activated (M2) immune phenotype. In this study, we established organotypic cell cultures of AT explants to study the impact of cytokine treatment on local ATM proliferation, without the bias of early monocyte recruitment. We show that exposure of AT to Th2 cytokines, such as IL-4, IL-13, and GM-CSF, stimulates ATM proliferation, whereas Th1 cytokines, such as TNF-α, inhibit local ATM proliferation. Furthermore, AT from obese mice exhibits an increased sensitivity to IL-4 stimulation, indicated by an increased phosphorylation of STAT6. In line with this, gene expression of the IL-4 receptor (Il4ra) and its ligand IL-13 are elevated in AT of obese C57BL/6 mice. Most importantly, Il4ra expression and susceptibility to IL-4 or IL-13 treatment depend on IL-6 signaling, which seems to be the underlying mechanism of local ATM proliferation in obesity. We conclude that IL-6 acts as a Th2 cytokine in obesity by stimulating M2 polarization and local ATM proliferation, presumably due to upregulation of the IL-4 receptor α.


Assuntos
Tecido Adiposo/imunologia , Proliferação de Células , Interleucina-6/imunologia , Macrófagos/imunologia , Obesidade/imunologia , Animais , Western Blotting , Proliferação de Células/fisiologia , Diabetes Mellitus Tipo 2/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transcriptoma
9.
Trends Immunol ; 36(2): 92-101, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25616716

RESUMO

Owing to its abundance in inflammatory settings, interleukin IL-6 is frequently viewed as a proinflammatory cytokine, with functions that parallel those of tumor necrosis factor (TNF) and IL-1ß in the context of inflammation. However, accumulating evidence points to a broader role for IL-6 in a variety of (patho)physiological conditions, including functions related to the resolution of inflammation. We review recent findings on the complex biological functions governed by IL-6 signaling, focusing on its role in inflammation-associated cancer and metabolic disorders such as obesity and type 2 diabetes mellitus (T2DM). We propose that the anti-inflammatory functions of IL-6 may extend to multiple settings and cell types, and suggest that these dimensions should be incorporated in therapeutic approaches to these diseases. Finally, we outline important areas of inquiry towards understanding this pleiotropic cytokine.


Assuntos
Interleucina-6/metabolismo , Neoplasias/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Fígado/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Músculo Esquelético/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Obesidade/genética , Obesidade/imunologia , Obesidade/metabolismo , Transdução de Sinais
10.
PLoS Genet ; 10(6): e1004385, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24945157

RESUMO

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.


Assuntos
Metabolismo Energético/genética , Ácidos Graxos/metabolismo , Canais Iônicos/genética , Mitocôndrias Cardíacas/metabolismo , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Acidose Láctica/metabolismo , Animais , Cardiomiopatias/patologia , Ingestão de Alimentos/genética , Expectativa de Vida , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Doenças Mitocondriais/metabolismo , Miocárdio/metabolismo , Oxirredução , Estresse Oxidativo , Bombas de Próton/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2
11.
J Neurosci ; 35(36): 12584-92, 2015 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-26354923

RESUMO

Variations in the fat mass and obesity-associated (FTO) gene are linked to obesity. However, the underlying neurobiological mechanisms by which these genetic variants influence obesity, behavior, and brain are unknown. Given that Fto regulates D2/3R signaling in mice, we tested in humans whether variants in FTO would interact with a variant in the ANKK1 gene, which alters D2R signaling and is also associated with obesity. In a behavioral and fMRI study, we demonstrate that gene variants of FTO affect dopamine (D2)-dependent midbrain brain responses to reward learning and behavioral responses associated with learning from negative outcome in humans. Furthermore, dynamic causal modeling confirmed that FTO variants modulate the connectivity in a basic reward circuit of meso-striato-prefrontal regions, suggesting a mechanism by which genetic predisposition alters reward processing not only in obesity, but also in other disorders with altered D2R-dependent impulse control, such as addiction. Significance statement: Variations in the fat mass and obesity-associated (FTO) gene are associated with obesity. Here we demonstrate that variants of FTO affect dopamine-dependent midbrain brain responses and learning from negative outcomes in humans during a reward learning task. Furthermore, FTO variants modulate the connectivity in a basic reward circuit of meso-striato-prefrontal regions, suggesting a mechanism by which genetic vulnerability in reward processing can increase predisposition to obesity.


Assuntos
Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Receptores de Dopamina D2/metabolismo , Recompensa , Adulto , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Conectoma , Feminino , Humanos , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/fisiologia
12.
Neuroimage ; 128: 21-31, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26767945

RESUMO

Variations in the fat mass and obesity associated (FTO) gene are currently the strongest known genetic factor predisposing humans to non-monogenic obesity. Recent experiments have linked these variants to a broad spectrum of behavioural alterations, including food choice and substance abuse. Yet, the underlying neurobiological mechanisms by which these genetic variations influence body weight remain elusive. Here, we explore the brain structural substrate of the obesity-predisposing rs9939609 T/A variant of the FTO gene in non-obese subjects by means of multivariate classification and use fMRI to investigate genotype-specific differences in neural food-cue reactivity by analysing correlates of a visual food perception task. Our findings demonstrate that MRI-derived measures of morphology along middle and posterior fusiform gyrus (FFG) are highly predictive for FTO at-risk allele carriers, who also show enhanced neural responses elicited by food cues in the same posterior FFG area. In brief, these findings provide first-time evidence for FTO-specific differences in both brain structure and function already in non-obese individuals, thereby contributing to a mechanistic understanding of why FTO is a predisposing factor for obesity.


Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Obesidade/genética , Lobo Temporal/fisiologia , Percepção Visual , Adulto , Feminino , Alimentos , Predisposição Genética para Doença/genética , Genótipo , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Máquina de Vetores de Suporte
13.
PLoS Genet ; 6(5): e1000938, 2010 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-20463885

RESUMO

A major component of obesity-related insulin resistance is the establishment of a chronic inflammatory state with invasion of white adipose tissue by mononuclear cells. This results in the release of pro-inflammatory cytokines, which in turn leads to insulin resistance in target tissues such as skeletal muscle and liver. To determine the role of insulin action in macrophages and monocytes in obesity-associated insulin resistance, we conditionally inactivated the insulin receptor (IR) gene in myeloid lineage cells in mice (IR(Deltamyel)-mice). While these animals exhibit unaltered glucose metabolism on a normal diet, they are protected from the development of obesity-associated insulin resistance upon high fat feeding. Euglycemic, hyperinsulinemic clamp studies demonstrate that this results from decreased basal hepatic glucose production and from increased insulin-stimulated glucose disposal in skeletal muscle. Furthermore, IR(Deltamyel)-mice exhibit decreased concentrations of circulating tumor necrosis factor (TNF) alpha and thus reduced c-Jun N-terminal kinase (JNK) activity in skeletal muscle upon high fat feeding, reflecting a dramatic reduction of the chronic and systemic low-grade inflammatory state associated with obesity. This is paralleled by a reduced accumulation of macrophages in white adipose tissue due to a pronounced impairment of matrix metalloproteinase (MMP) 9 expression and activity in these cells. These data indicate that insulin action in myeloid cells plays an unexpected, critical role in the regulation of macrophage invasion into white adipose tissue and in the development of obesity-associated insulin resistance.


Assuntos
Resistência à Insulina , Macrófagos/imunologia , Monócitos/imunologia , Obesidade/imunologia , Receptor de Insulina/imunologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/imunologia , Obesidade/genética , Obesidade/metabolismo , Receptor de Insulina/genética
14.
Proc Natl Acad Sci U S A ; 107(13): 6028-33, 2010 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-20231445

RESUMO

c-Jun N-terminal kinase (JNK) 1-dependent signaling plays a crucial role in the development of obesity-associated insulin resistance. Here we demonstrate that JNK activation not only occurs in peripheral tissues, but also in the hypothalamus and pituitary of obese mice. To resolve the importance of JNK1 signaling in the hypothalamic/pituitary circuitry, we have generated mice with a conditional inactivation of JNK1 in nestin-expressing cells (JNK1(DeltaNES) mice). JNK1(DeltaNES) mice exhibit improved insulin sensitivity both in the CNS and in peripheral tissues, improved glucose metabolism, as well as protection from hepatic steatosis and adipose tissue dysfunction upon high-fat feeding. Moreover, JNK1(DeltaNES) mice also show reduced somatic growth in the presence of reduced circulating growth hormone (GH) and insulin-like growth factor 1 (IGF1) concentrations, as well as increased thyroid axis activity. Collectively, these experiments reveal an unexpected, critical role for hypothalamic/pituitary JNK1 signaling in the coordination of metabolic/endocrine homeostasis.


Assuntos
Glucose/metabolismo , Hipotálamo/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Hipófise/metabolismo , Adiposidade/fisiologia , Animais , Peso Corporal/fisiologia , Gorduras na Dieta/administração & dosagem , Hormônio do Crescimento/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Insulina/metabolismo , Resistência à Insulina/fisiologia , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Proteína Quinase 8 Ativada por Mitógeno/deficiência , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Transdução de Sinais , Glândula Tireoide/metabolismo
15.
J Cell Biol ; 222(5)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36880935

RESUMO

Talin-1 is the core mechanosensitive adapter protein linking integrins to the cytoskeleton. The TLN1 gene is comprised of 57 exons that encode the 2,541 amino acid TLN1 protein. TLN1 was previously considered to be expressed as a single isoform. However, through differential pre-mRNA splicing analysis, we discovered a cancer-enriched, non-annotated 51-nucleotide exon in TLN1 between exons 17 and 18, which we refer to as exon 17b. TLN1 is comprised of an N-terminal FERM domain, linked to 13 force-dependent switch domains, R1-R13. Inclusion of exon 17b introduces an in-frame insertion of 17 amino acids immediately after Gln665 in the region between R1 and R2 which lowers the force required to open the R1-R2 switches potentially altering downstream mechanotransduction. Biochemical analysis of this isoform revealed enhanced vinculin binding, and cells expressing this variant show altered adhesion dynamics and motility. Finally, we showed that the TGF-ß/SMAD3 signaling pathway regulates this isoform switch. Future studies will need to consider the balance of these two TLN1 isoforms.


Assuntos
Neoplasias , Talina , Humanos , Talina/genética , Mecanotransdução Celular , Éxons/genética , Proteínas Adaptadoras de Transdução de Sinal
16.
Front Bioinform ; 2: 786898, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36304260

RESUMO

RNA-seq analysis of alternative pre-mRNA splicing has facilitated an unprecedented understanding of transcriptome complexity in health and disease. However, despite the availability of countless bioinformatic pipelines for transcriptome-wide splicing analysis, the use of these tools is often limited to expert bioinformaticians. The need for high computational power, combined with computational outputs that are complicated to visualize and interpret present obstacles to the broader research community. Here we introduce DJExpress, an R package for differential expression analysis of transcriptomic features and expression-trait associations. To determine gene-level differential junction usage as well as associations between junction expression and molecular/clinical features, DJExpress uses raw splice junction counts as input data. Importantly, DJExpress runs on an average laptop computer and provides a set of interactive and intuitive visualization formats. In contrast to most existing pipelines, DJExpress can handle both annotated and de novo identified splice junctions, thereby allowing the quantification of novel splice events. Moreover, DJExpress offers a web-compatible graphical interface allowing the analysis of user-provided data as well as the visualization of splice events within our custom database of differential junction expression in cancer (DJEC DB). DJEC DB includes not only healthy and tumor tissue junction expression data from TCGA and GTEx repositories but also cancer cell line data from the DepMap project. The integration of DepMap functional genomics data sets allows association of junction expression with molecular features such as gene dependencies and drug response profiles. This facilitates identification of cancer cell models for specific splicing alterations that can then be used for functional characterization in the lab. Thus, DJExpress represents a powerful and user-friendly tool for exploration of alternative splicing alterations in RNA-seq data, including multi-level data integration of alternative splicing signatures in healthy tissue, tumors and cancer cell lines.

17.
J Biol Chem ; 285(9): 6198-207, 2010 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-19996103

RESUMO

The unfolded protein response (UPR) or endoplasmic reticulum (ER) stress response is a physiological process enabling cells to cope with altered protein synthesis demands. However, under conditions of obesity, prolonged activation of the UPR has been shown to have deteriorating effects on different metabolic pathways. Here we identify Bax inhibitor-1 (BI-1), an evolutionary conserved ER-membrane protein, as a novel modulator of the obesity-associated alteration of the UPR. BI-1 partially inhibits the UPR by interacting with IRE1alpha and inhibiting IRE1alpha endonuclease activity as seen on the splicing of the transcription factor Xbp-1. Because we observed a down-regulation of BI-1 expression in liver and muscle of genetically obese ob/ob and db/db mice as well as in mice with diet-induced obesity in vivo, we investigated the effect of restoring BI-1 expression on metabolic processes in these mice. Importantly, BI-1 overexpression by adenoviral gene transfer dramatically improved glucose metabolism in both standard diet-fed mice as well as in mice with diet-induced obesity and, critically, reversed hyperglycemia in db/db mice. This improvement in whole body glucose metabolism and insulin sensitivity was due to dramatically reduced gluconeogenesis as shown by reduction of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase expression. Taken together, these results identify BI-1 as a critical regulator of ER stress responses in the development of obesity-associated insulin resistance and provide proof of concept evidence that gene transfer-mediated elevations in hepatic BI-1 may represent a promising approach for the treatment of type 2 diabetes.


Assuntos
Endorribonucleases/antagonistas & inibidores , Intolerância à Glucose/terapia , Resistência à Insulina , Proteínas de Membrana/farmacologia , Obesidade/complicações , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Retículo Endoplasmático/patologia , Terapia Genética/métodos , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Hiperglicemia/terapia , Fígado/metabolismo , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/uso terapêutico , Camundongos , Camundongos Obesos , Resposta a Proteínas não Dobradas
18.
Nat Commun ; 11(1): 4077, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796846

RESUMO

Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ciclo Celular , Sobrevivência Celular , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Avaliação Pré-Clínica de Medicamentos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerase-1/genética
19.
FEBS Lett ; 592(12): 2012-2022, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29754392

RESUMO

The fate of mRNA is regulated by epitranscriptomic nucleotide modifications, the most abundant of which is N6 -methyladenosine (m6 A). Although the pattern and distribution of m6 A in mRNA is mediated by specific methyltransferases, a recent hypothesis is that specific demethylases or 'erasers' allow m6 A to be dynamically reversed by signaling pathways. In this Review, we discuss the data in support and against this model. New insights into the function of fat mass and obesity-associated protein (FTO), the original enzyme thought to be an m6 A eraser, reveal that its physiologic target is not m6 A, but instead is N6 ,2'-O-dimethyladenosine (m6 Am ). Another m6 A demethylase, ALKBH5, appears to have functions limited to sperm development in normal mice. Overall, the majority of the data suggest that m6 A is generally not reversible, although m6 A may be susceptible to demethylation in pathophysiological states such as cancer.


Assuntos
Adenosina/análogos & derivados , Epigênese Genética , RNA Mensageiro/química , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Humanos , RNA Mensageiro/metabolismo
20.
Nat Commun ; 9(1): 1646, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29695802

RESUMO

Colorectal cancer (CRC) is one of the most lethal cancers worldwide in which the vast majority of cases exhibit little genetic risk but are associated with a sedentary lifestyle and obesity. Although the mechanisms underlying CRC and colitis-associated colorectal cancer (CAC) remain unclear, we hypothesised that obesity-induced inflammation predisposes to CAC development. Here, we show that diet-induced obesity accelerates chemically-induced CAC in mice via increased inflammation and immune cell recruitment. Obesity-induced interleukin-6 (IL-6) shifts macrophage polarisation towards tumour-promoting macrophages that produce the chemokine CC-chemokine-ligand-20 (CCL-20) in the CAC microenvironment. CCL-20 promotes CAC progression by recruiting CC-chemokine-receptor-6 (CCR-6)-expressing B cells and γδ T cells via chemotaxis. Compromised cell recruitment as well as inhibition of B and γδ T cells protects against CAC progression. Collectively, our data reveal a function for IL-6 in the CAC microenvironment via lymphocyte recruitment through the CCL-20/CCR-6 axis, thereby implicating a potential therapeutic intervention for human patients.


Assuntos
Quimiocina CCL20/metabolismo , Colite Ulcerativa/patologia , Neoplasias Colorretais/imunologia , Interleucina-6/metabolismo , Obesidade/imunologia , Receptores CCR6/metabolismo , Animais , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Quimiocina CCL20/imunologia , Quimiotaxia/imunologia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Neoplasias Colorretais/patologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-6/genética , Subunidade alfa de Receptor de Interleucina-6/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Obesidade/etiologia , Receptores CCR6/genética , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia
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