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1.
Cell Mol Gastroenterol Hepatol ; 12(2): 599-632, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33813036

RESUMO

BACKGROUND: Inflammatory bowel disease (IBD) is a complex disease with variable presentation, progression, and response to therapies. Current disease classification is based on subjective clinical phenotypes. The peripheral blood immunophenome can reflect local inflammation, and thus we measured 39 circulating immune cell types in a large cohort of IBD and control subjects and performed immunotype:phenotype associations. METHODS: We performed fluorescence-activated cell sorting or CyTOF analysis on blood from 728 Crohn's disease, 464 ulcerative colitis, and 334 non-IBD patients, with available demographics, endoscopic and clinical examinations and medication use. RESULTS: We observed few immune cell types commonly affected in IBD (lowered natural killer cells, B cells, and CD45RA- CD8 T cells). Generally, the immunophenome was distinct between ulcerative colitis and Crohn's disease. Within disease subtype, there were further distinctions, with specific immune cell types associating with disease duration, behavior, and location. Thiopurine monotherapy altered abundance of many cell types, often in the same direction as disease association, while anti-tumor necrosis factor (anti-TNF) monotherapy demonstrated an opposing pattern. Concomitant use of an anti-TNF and thiopurine was not synergistic, but rather was additive. For example, thiopurine monotherapy use alone or in combination with anti-TNF was associated with a dramatic reduction in major subclasses of B cells. CONCLUSIONS: We present a peripheral map of immune cell changes in IBD related to disease entity and therapies as a resource for hypothesis generation. We propose the changes in B cell subsets could affect antibody formation and potentially explain the mechanism behind the superiority of combination therapy through the impact of thiopurines on pharmacokinetics of anti-TNFs.

2.
Nat Commun ; 12(1): 547, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483510

RESUMO

Elevated plasma cholesterol and type 2 diabetes (T2D) are associated with coronary artery disease (CAD). Individuals treated with cholesterol-lowering statins have increased T2D risk, while individuals with hypercholesterolemia have reduced T2D risk. We explore the relationship between lipid and glucose control by constructing network models from the STARNET study with sequencing data from seven cardiometabolic tissues obtained from CAD patients during coronary artery by-pass grafting surgery. By integrating gene expression, genotype, metabolomic, and clinical data, we identify a glucose and lipid determining (GLD) regulatory network showing inverse relationships with lipid and glucose traits. Master regulators of the GLD network also impact lipid and glucose levels in inverse directions. Experimental inhibition of one of the GLD network master regulators, lanosterol synthase (LSS), in mice confirms the inverse relationships to glucose and lipid levels as predicted by our model and provides mechanistic insights.


Assuntos
Glicemia/metabolismo , Doença da Artéria Coronariana/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Metabolismo dos Lipídeos , Modelos Biológicos , Animais , Colesterol/sangue , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/genética , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único
3.
Mol Genet Metab ; 126(4): 388-396, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709776

RESUMO

Inbred mouse strains are a cornerstone of translational research but paradoxically many strains carry mild inborn errors of metabolism. For example, α-aminoadipic acidemia and branched-chain ketoacid dehydrogenase deficiency are known in C57BL/6J mice. Using RNA sequencing, we now reveal the causal variants in Dhtkd1 and Bckdhb, and the molecular mechanism underlying these metabolic defects. C57BL/6J mice have decreased Dhtkd1 mRNA expression due to a solitary long terminal repeat (LTR) in intron 4 of Dhtkd1. This LTR harbors an alternate splice donor site leading to a partial splicing defect and as a consequence decreased total and functional Dhtkd1 mRNA, decreased DHTKD1 protein and α-aminoadipic acidemia. Similarly, C57BL/6J mice have decreased Bckdhb mRNA expression due to an LTR retrotransposon in intron 1 of Bckdhb. This transposable element encodes an alternative exon 1 causing aberrant splicing, decreased total and functional Bckdhb mRNA and decreased BCKDHB protein. Using a targeted metabolomics screen, we also reveal elevated plasma C5-carnitine in 129 substrains. This biochemical phenotype resembles isovaleric acidemia and is caused by an exonic splice mutation in Ivd leading to partial skipping of exon 10 and IVD protein deficiency. In summary, this study identifies three causal variants underlying mild inborn errors of metabolism in commonly used inbred mouse strains.


Assuntos
Erros Inatos do Metabolismo/genética , Camundongos Endogâmicos/genética , Animais , Elementos de DNA Transponíveis/genética , Cetona Oxirredutases/genética , Masculino , Erros Inatos do Metabolismo/diagnóstico , Metabolômica , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fenótipo , Análise de Sequência de RNA
4.
FASEB J ; 33(3): 4355-4364, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30540494

RESUMO

Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid ß-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Ácidos Graxos/metabolismo , Proteína Multifuncional do Peroxissomo-2/fisiologia , Peroxissomos/enzimologia , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Sistemas CRISPR-Cas , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/fisiologia , Células HEK293 , Humanos , Ácidos Láuricos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Oxirredução , Ácido Palmítico/metabolismo , Enzima Bifuncional do Peroxissomo/deficiência , Proteína Multifuncional do Peroxissomo-2/deficiência , Proteína Multifuncional do Peroxissomo-2/genética , Proteínas Recombinantes/metabolismo
5.
Cardiovasc Res ; 114(10): 1324-1334, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29635338

RESUMO

Aims: Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism. Methods and results: We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1bE3A/E3A hearts compared with Cpt1bWT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1bE3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis. Conclusions: Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions.


Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Malonil Coenzima A/metabolismo , Mitocôndrias Cardíacas/enzimologia , Miocárdio/enzimologia , Animais , Carnitina O-Palmitoiltransferase/genética , Genótipo , Glucose/metabolismo , Glicólise , Preparação de Coração Isolado , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Oxirredução , Fenótipo , Função Ventricular Esquerda
6.
Autophagy ; 14(3): 437-449, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29455584

RESUMO

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in cell culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members-TFEB, TFE3 and MITF-from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of MTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in cell culture media due to their potentially confounding effects on experimental results.


Assuntos
Autofagia/fisiologia , Redes Reguladoras de Genes/genética , HEPES/metabolismo , Lisossomos/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Humanos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
7.
BMC Genomics ; 18(1): 987, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29273013

RESUMO

BACKGROUND: Exosomes and other extracellular vesicles (EVs) have emerged as an important mechanism of cell-to-cell communication. However, previous studies either did not fully resolve what genetic materials were shuttled by exosomes or only focused on a specific set of miRNAs and mRNAs. A more systematic method is required to identify the genetic materials that are potentially transferred during cell-to-cell communication through EVs in an unbiased manner. RESULTS: In this work, we present a novel next generation of sequencing (NGS) based approach to identify EV mediated mRNA exchanges between co-cultured adipocyte and macrophage cells. We performed molecular and genomic profiling and jointly considered data from RNA sequencing (RNA-seq) and genotyping to track the "sequence varying mRNAs" transferred between cells. We identified 8 mRNAs being transferred from macrophages to adipocytes and 21 mRNAs being transferred in the opposite direction. These mRNAs represented biological functions including extracellular matrix, cell adhesion, glycoprotein, and signal peptides. CONCLUSIONS: Our study sheds new light on EV mediated RNA communications between adipocyte and macrophage cells, which may play a significant role in developing insulin resistance in diabetic patients. This work establishes a new method that is applicable to examining genetic material exchanges in many cellular systems and has the potential to be extended to in vivo studies as well.


Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , RNA Mensageiro/metabolismo , Adipócitos/metabolismo , Linhagem Celular , Técnicas de Cocultura , Expressão Gênica , Técnicas de Genotipagem , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Macrófagos/metabolismo , Transporte de RNA , Análise de Sequência de RNA
8.
Biochim Biophys Acta Mol Basis Dis ; 1863(12): 3277-3285, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28962896

RESUMO

OBJECTIVE: The transcription factor Krüppel-like factor 14 (KLF14) has been associated with type 2 diabetes and high-density lipoprotein-cholesterol (HDL-C) through genome-wide association studies. The mechanistic underpinnings of KLF14's control of metabolic processes remain largely unknown. We studied the physiological roles of KLF14 in a knockout (KO) mouse model. METHODS: Male whole body Klf14 KO mice were fed a chow or high fat diet (HFD) and diet induced phenotypes were analyzed. Additionally, tissue-specific expression of Klf14 was determined using RT-PCR, RNA sequencing, immunoblotting and whole mount lacZ staining. Finally, the consequences of KLF14 loss-of-function were studied using RNA sequencing in tissues with relatively high Klf14 expression levels. RESULTS: KLF14 loss-of-function did not affect HFD-induced weight gain or insulin resistance. Fasting plasma concentrations of glucose, insulin, cholesterol, HDL-C and ApoA-I were also comparable between Klf14+/+ and Klf14-/- mice on chow and HFD. We found that in mice expression of Klf14 was the highest in the anterior pituitary (adenohypophysis), lower but detectable in white adipose tissue and undetectable in liver. Loss of KLF14 function impacted on the pituitary transcriptome with extracellular matrix organization as the primary affected pathway and a predicted link to glucocorticoid receptor signaling. CONCLUSIONS: Whole body loss of KLF14 function in male mice does not result in metabolic abnormalities as assessed under chow and HFD conditions. Mostly likely there is redundancy for the role of KLF14 in the mouse and a diverging function in humans.


Assuntos
Fatores de Transcrição Kruppel-Like/deficiência , Síndrome Metabólica/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , HDL-Colesterol/metabolismo , Dieta Hiperlipídica , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Síndrome Metabólica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glucocorticoides/metabolismo , Análise de Sequência de RNA
9.
Science ; 352(6291): aad0189, 2016 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-27284200

RESUMO

Recent improvements in quantitative proteomics approaches, including Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), permit reproducible large-scale protein measurements across diverse cohorts. Together with genomics, transcriptomics, and other technologies, transomic data sets can be generated that permit detailed analyses across broad molecular interaction networks. Here, we examine mitochondrial links to liver metabolism through the genome, transcriptome, proteome, and metabolome of 386 individuals in the BXD mouse reference population. Several links were validated between genetic variants toward transcripts, proteins, metabolites, and phenotypes. Among these, sequence variants in Cox7a2l alter its protein's activity, which in turn leads to downstream differences in mitochondrial supercomplex formation. This data set demonstrates that the proteome can now be quantified comprehensively, serving as a key complement to transcriptomics, genomics, and metabolomics--a combination moving us forward in complex trait analysis.


Assuntos
Colesterol/metabolismo , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteômica , Animais , Dieta , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Variação Genética , Células Hep G2 , Humanos , Redes e Vias Metabólicas/genética , Metaboloma , Metabolômica , Camundongos , Camundongos Endogâmicos , Mitocôndrias Hepáticas/genética , Dados de Sequência Molecular , Proteoma , Locos de Características Quantitativas , Transcriptoma
10.
Diabetes ; 65(9): 2591-605, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27325287

RESUMO

Aberrant mitochondrial fission plays a pivotal role in the pathogenesis of skeletal muscle insulin resistance. However, fusion-fission dynamics are physiologically regulated by inherent tissue-specific and nutrient-sensitive processes that may have distinct or even opposing effects with respect to insulin sensitivity. Based on a combination of mouse population genetics and functional in vitro assays, we describe here a regulatory circuit in which peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte master regulator and receptor for the thiazolidinedione class of antidiabetic drugs, controls mitochondrial network fragmentation through transcriptional induction of Bnip3. Short hairpin RNA-mediated knockdown of Bnip3 in cultured adipocytes shifts the balance toward mitochondrial elongation, leading to compromised respiratory capacity, heightened fatty acid ß-oxidation-associated mitochondrial reactive oxygen species generation, insulin resistance, and reduced triacylglycerol storage. Notably, the selective fission/Drp1 inhibitor Mdivi-1 mimics the effects of Bnip3 knockdown on adipose mitochondrial bioenergetics and glucose disposal. We further show that Bnip3 is reciprocally regulated in white and brown fat depots of diet-induced obesity and leptin-deficient ob/ob mouse models. Finally, Bnip3(-/-) mice trade reduced adiposity for increased liver steatosis and develop aggravated systemic insulin resistance in response to high-fat feeding. Together, our data outline Bnip3 as a key effector of PPARγ-mediated adipose mitochondrial network fragmentation, improving insulin sensitivity and limiting oxidative stress.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , PPAR gama/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Feminino , Glucose/metabolismo , Immunoblotting , Imuno-Histoquímica , Insulina/metabolismo , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/genética , Obesidade/genética , Obesidade/metabolismo , PPAR gama/genética , Ensaio de Radioimunoprecipitação , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Cell Metab ; 23(1): 13-26, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26712461

RESUMO

Inborn errors of metabolism (IEM) are not unlike common diseases. They often present as a spectrum of disease phenotypes that correlates poorly with the severity of the disease-causing mutations. This greatly impacts patient care and reveals fundamental gaps in our knowledge of disease modifying biology. Systems biology approaches that integrate multi-omics data into molecular networks have significantly improved our understanding of complex diseases. Similar approaches to study IEM are rare despite their complex nature. We highlight that existing common disease-derived datasets and networks can be repurposed to generate novel mechanistic insight in IEM and potentially identify candidate modifiers. While understanding disease pathophysiology will advance the IEM field, the ultimate goal should be to understand per individual how their phenotype emerges given their primary mutation on the background of their whole genome, not unlike personalized medicine. We foresee that panomics and network strategies combined with recent experimental innovations will facilitate this.


Assuntos
Erros Inatos do Metabolismo/genética , Animais , Redes Reguladoras de Genes , Humanos , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/metabolismo , Mutação , Fenótipo , Transcriptoma
12.
Cell ; 158(6): 1415-1430, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25215496

RESUMO

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.


Assuntos
Perfilação da Expressão Gênica , Fígado/química , Camundongos/metabolismo , Mitocôndrias/química , Proteoma/análise , Soro/química , Animais , Glucose/metabolismo , Humanos , Cetona Oxirredutases/metabolismo , Fígado/citologia , Fígado/metabolismo , Camundongos/classificação , Camundongos/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mitocôndrias/metabolismo , Locos de Características Quantitativas , Soro/metabolismo , Resposta a Proteínas não Dobradas
13.
PLoS One ; 9(9): e108046, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25265318

RESUMO

PURPOSE: To identify proteins and (molecular/biological) pathways associated with differences between benign and malignant epithelial ovarian tumors. EXPERIMENTAL PROCEDURES: Serum of six patients with a serous adenocarcinoma of the ovary was collected before treatment, with a control group consisting of six matched patients with a serous cystadenoma. In addition to the serum, homogeneous regions of cells exhibiting uniform histology were isolated from benign and cancerous tissue by laser microdissection. We subsequently employed label-free liquid chromatography tandem mass spectrometry (LC-MSe) to identify proteins in these serum and tissues samples. Analyses of differential expression between samples were performed using Bioconductor packages and in-house scripts in the statistical software package R. Hierarchical clustering and pathway enrichment analyses were performed, as well as network enrichment and interactome analysis using MetaCore. RESULTS: In total, we identified 20 and 71 proteins that were significantly differentially expressed between benign and malignant serum and tissue samples, respectively. The differentially expressed protein sets in serum and tissue largely differed with only 2 proteins in common. MetaCore network analysis, however inferred GCR-alpha and Sp1 as common transcriptional regulators. Interactome analysis highlighted 14-3-3 zeta/delta, 14-3-3 beta/alpha, Alpha-actinin 4, HSP60, and PCBP1 as critical proteins in the tumor proteome signature based on their relative overconnectivity. The data have been deposited to the ProteomeXchange with identifier PXD001084. DISCUSSION: Our analysis identified proteins with both novel and previously known associations to ovarian cancer biology. Despite the small overlap between differentially expressed protein sets in serum and tissue, APOA1 and Serotransferrin were significantly lower expressed in both serum and cancer tissue samples, suggesting a tissue-derived effect in serum. Pathway and subsequent interactome analysis also highlighted common regulators in serum and tissue samples, suggesting a yet unknown role for PCBP1 in ovarian cancer pathophysiology.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Adulto , Idoso , Análise por Conglomerados , Feminino , Redes Reguladoras de Genes , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/patologia , Proteoma
14.
Biochim Biophys Acta ; 1841(7): 987-94, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24747043

RESUMO

Acylcarnitines have been linked to obesity-induced insulin resistance. However the majority of these studies have focused on acylcarnitines in plasma. It is currently unclear to what extent plasma levels of acylcarnitines reflect tissue acylcarnitine metabolism. We investigated the correlation of plasma acylcarnitine levels with selected tissue acylcarnitines as measured with tandem mass spectrometry, in both fed and fasted BALB/cJ (BALB) and C57BL/6N (Bl6) mice. Fasting affected acylcarnitine levels in all tissues. These changes varied substantially between the different tissue compartments. No significant correlations were found between plasma acylcarnitine species and their tissue counterparts in both mouse strains, with the exception of plasma C4OH-carnitine in BALB mice. We suggest that this lack of correlation is due to differences in acylcarnitine turnover rates between plasma and tissue compartments and the fact that the plasma acylcarnitine profile is a composition of acylcarnitines derived from different compartments. Therefore, plasma acylcarnitine levels do not reflect tissue levels and should be interpreted with caution. A focus on tissue acylcarnitine levels is warranted in metabolic studies.


Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Carnitina/análogos & derivados , Carnitina/sangue , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Algoritmos , Animais , Análise por Conglomerados , Jejum , Ácidos Graxos/metabolismo , Resistência à Insulina , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Valor Preditivo dos Testes , Espectrometria de Massas em Tandem
15.
FASEB J ; 28(3): 1365-74, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24344334

RESUMO

Oxidation of unsaturated fatty acids requires the action of auxiliary enzymes, such as Δ(3),Δ(2)-enoyl-CoA isomerases. Here we describe a detailed biochemical, molecular, histological, and evolutionary characterization of Eci3, the fourth member of the mammalian enoyl-CoA isomerase family. Eci3 specifically evolved in rodents after gene duplication of Eci2. Eci3 is with 79% identity homologous to Eci2 and contains a peroxisomal targeting signal type 1. Subcellular fractionation of mouse kidney and immunofluorescence studies revealed a specific peroxisomal localization for Eci3. Expression studies showed that mouse Eci3 is almost exclusively expressed in kidney. By using immunohistochemistry, we found that Eci3 is not only expressed in cells of the proximal tubule, but also in a subset of cells in the tubulointerstitium and the glomerulus. In vitro, Eci3 catalyzed the isomerization of trans-3-nonenoyl-CoA to trans-2-nonenoyl-CoA equally efficient as Eci2, suggesting a role in oxidation of unsaturated fatty acids. However, in contrast to Eci2, in silico gene coexpression and enrichment analysis for Eci3 in kidney did not yield carboxylic acid metabolism, but diverse biological functions, such as ion transport (P=7.1E-3) and tissue morphogenesis (P=1.0E-3). Thus, Eci3 picked up a novel and unexpected role in kidney function during rodent evolution.


Assuntos
Dodecenoil-CoA Isomerase/metabolismo , Rim/enzimologia , Animais , Sequência de Bases , Primers do DNA , Imunofluorescência , Humanos , Camundongos
16.
Hum Mol Genet ; 22(25): 5249-61, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23933733

RESUMO

The importance of mitochondrial fatty acid ß-oxidation (FAO) as a glucose-sparing process is illustrated by patients with inherited defects in FAO, who may present with life-threatening fasting-induced hypoketotic hypoglycemia. It is unknown why peripheral glucose demand outpaces hepatic gluconeogenesis in these patients. In this study, we have systematically addressed the fasting response in long-chain acyl-CoA dehydrogenase-deficient (LCAD KO) mice. We demonstrate that the fasting-induced hypoglycemia in LCAD KO mice was initiated by an increased glucose requirement in peripheral tissues, leading to rapid hepatic glycogen depletion. Gluconeogenesis did not compensate for the increased glucose demand, which was not due to insufficient hepatic glucogenic capacity but rather caused by a shortage in the supply of glucogenic precursors. This shortage in supply was explained by a suppressed glucose-alanine cycle, decreased branched-chain amino acid metabolism and ultimately impaired protein mobilization. We conclude that during fasting, FAO not only serves to spare glucose but is also indispensable for amino acid metabolism, which is essential for the maintenance of adequate glucose production.


Assuntos
Gluconeogênese/genética , Glucose/metabolismo , Hipoglicemia/metabolismo , Oxirredução , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Aminoácidos/metabolismo , Animais , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Humanos , Hipoglicemia/genética , Hipoglicemia/patologia , Erros Inatos do Metabolismo Lipídico/metabolismo , Glicogênio Hepático/genética , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia
17.
J Lipid Res ; 53(7): 1296-303, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22534643

RESUMO

L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid ß-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor α (PPARα) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadh's expression by PPARα, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ω-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Ácidos Dicarboxílicos/metabolismo , Enoil-CoA Hidratase/metabolismo , Isomerases/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/deficiência , 3-Hidroxiacil-CoA Desidrogenases/genética , Animais , Biologia Computacional , Enoil-CoA Hidratase/deficiência , Enoil-CoA Hidratase/genética , Jejum , Isomerases/deficiência , Isomerases/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Enzima Bifuncional do Peroxissomo , Peroxissomos/metabolismo
18.
Cell ; 147(4): 719-21, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-22078872

RESUMO

Cholesterol regulates activation of sterol regulatory element-binding protein (SREBP) through a classic feedback loop. Walker et al. (2011) extend the regulatory inputs governing SREBP activity to include an independent loop modulated by phosphatidylcholine (PC) and cellular methylation capacity. These findings suggest a link between lipid synthesis and cellular pathways involved in methylation.

19.
Biochim Biophys Acta ; 1771(8): 999-1013, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17475546

RESUMO

The peroxisome proliferator activated receptor gamma (PPARgamma) is a member in the nuclear receptor superfamily which mediates part of the regulatory effects of dietary fatty acids on gene expression. As PPARgamma also coordinates adipocyte differentiation, it is an important component in storing the excess nutritional energy as fat. Our genes have evolved into maximizing energy storage, and PPARgamma has a central role in the mismatch between our genes and our affluent western society which results in a broad range of metabolic disturbances, collectively known as the metabolic syndrome. A flurry of human and mouse studies has shed new light on the mechanisms how the commonly used insulin sensitizer drugs and PPARgamma activators, thiazolidinediones, act, and which of their physiological effects are dependent of PPARgamma. It is now evident that the full activation of PPARgamma is less advantageous than targeted modulation of its activity. Furthermore, new roles for PPARgamma signaling have been discovered in inflammation, bone morphogenesis, endothelial function, cancer, longevity, and atherosclerosis, to mention a few. Here we draw together and discuss these recent advances in the research into PPARgamma biology.


Assuntos
PPAR gama/fisiologia , Tecido Adiposo/fisiologia , Substituição de Aminoácidos , Animais , Fenômenos Fisiológicos Cardiovasculares , Trato Gastrointestinal/fisiologia , Regulação da Expressão Gênica , Variação Genética , Humanos , Rim/fisiologia , Pulmão/fisiologia , Camundongos , Músculo Esquelético/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , PPAR gama/genética , Pâncreas/fisiologia , Polimorfismo de Nucleotídeo Único
20.
Curr Protoc Mol Biol ; Chapter 29: Unit 29B.4, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18265402

RESUMO

Due to the small size of the mouse, evaluating its clinical phenotype is sometimes problematic. In contrast, mouse models are readily accessible to post-mortem analyses at any time during the course of a disease and prior to its clinical onset. RNA, protein, and histological analyses following sacrifice represent a powerful means to identify affected cell types and molecular events underlying the altered phenotype, and therefore to understanding the signaling or metabolic pathways involved. In this unit, an overview of post-mortem analyses is provided with a strong emphasis on the principles of routine histology, including tissue fixation, processing, embedding, and staining with hematoxylin and eosin. There are also several protocols for staining with specialized histological stains used in the metabolic field to detect intracellular lipids, intracellular lipid "ghosts", cholesterol esters, polysaccharides, mitochondria, pathological collagen deposits, and atherosclerotic plaques.


Assuntos
Técnicas Histológicas/métodos , Envelhecimento , Animais , Feminino , Masculino , Camundongos , Camundongos Mutantes , Fenótipo , Coloração e Rotulagem , Fixação de Tecidos
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