[Show abstract][Hide abstract] ABSTRACT: Obesity-associated inflammation contributes to the development of metabolic diseases. While brite adipocytes have been shown to ameliorate metabolic parameters in rodents, their origin and differentiation remain to be characterized in humans. Native CD45-/CD34+/CD31- cells have been previously described as human adipocyte progenitors. Using two additional cell surface markers, MSCA1 (tissue non-specific alkaline phosphatase), and CD271 (nerve growth factor receptor), we are able to partition the CD45-/CD34+/CD31- cell population into three subsets. We establish serum-free culture conditions without cell expansion to promote either white/brite adipogenesis using rosiglitazone or Bone Morphogenetic Protein 7 (BMP7), or specifically brite adipogenesis using 3-Isobuthyl-1-Methylxanthine. We demonstrate that adipogenesis leads to an increase of MSCA1 activity, expression of white/brite adipocyte-related genes and mitochondriogenesis. Using pharmacological inhibition and gene silencing approaches, we show that MSCA1 activity is required for triglyceride accumulation and for the expression of white/brite-related genes in human cells. Moreover, native immunoselected MSCA1+ cells exhibit brite precursor characteristics and the highest adipogenic potential of the three progenitor subsets. Finally, we provided evidence that MSCA1+ white/brite precursors accumulate with obesity in subcutaneous adipose tissue (sAT) and that local BMP7 and inflammation regulate brite adipogenesis by modulating MSCA1 in human sAT. The accumulation of MSCA1+ white/brite precursors in sAT with obesity may reveal a blockade of their differentiation by immune cells, suggesting that local inflammation contributes to metabolic disorders through impairment of white/brite adipogenesis. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Rhabdomyolysis can be life threatening if complicated by AKI. Macrophage infiltration has been observed in rat kidneys after glycerol-induced rhabdomyolysis, but the role of macrophages in rhabdomyolysis-induced AKI remains unknown. Here, in a patient diagnosed with rhabdomyolysis, we detected substantial macrophage infiltration in the kidney. In a mouse model of rhabdomyolysis-induced AKI, diverse renal macrophage phenotypes were observed depending on the stage of the disease. Two days after rhabdomyolysis, F4/80(low)CD11b(high)Ly6b(high)CD206(low) kidney macrophages were dominant, whereas by day 8, F4/80(high)CD11b(+)Ly6b(low)CD206(high) cells became the most abundant. Single-cell gene expression analyses of FACS-sorted macrophages revealed that these subpopulations were heterogeneous and that individual cells simultaneously expressed both M1 and M2 markers. Liposomal clodronate-mediated macrophage depletion significantly reduced the early infiltration of F4/80(low)CD11b(high)Ly6b(high)CD206(low) macrophages. Furthermore, transcriptionally regulated targets potentially involved in disease progression, including fibronectin, collagen III, and chemoattractants that were identified via single-cell analysis, were verified as macrophage-dependent in situ. In vitro, myoglobin treatment induced proximal tubular cells to secrete chemoattractants and macrophages to express proinflammatory markers. At day 30, liposomal clodronate-mediated macrophage depletion reduced fibrosis and improved both kidney repair and mouse survival. Seven months after rhabdomyolysis, histologic lesions were still present but were substantially reduced with prior depletion of macrophages. These results suggest an important role for macrophages in rhabdomyolysis-induced AKI progression and advocate the utility of long-term follow-up for patients with this disease.
No preview · Article · Sep 2014 · Journal of the American Society of Nephrology
[Show abstract][Hide abstract] ABSTRACT: Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.
No preview · Article · Mar 2014 · Nature Structural & Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Weight control diets favorably affect parameters of the metabolic syndrome and delay the onset of diabetic complications. The adaptations occurring in adipose tissue (AT) are likely to have a profound impact on the whole body response as AT is a key target of dietary intervention. Identification of environmental and individual factors controlling AT adaptation is therefore essential. Here, expression of 271 transcripts, selected for regulation according to obesity and weight changes, was determined in 515 individuals before, after 8-week low-calorie diet-induced weight loss, and after 26-week ad libitum weight maintenance diets. For 175 genes, opposite regulation was observed during calorie restriction and weight maintenance phases, independently of variations in body weight. Metabolism and immunity genes showed inverse profiles. During the dietary intervention, network-based analyses revealed strong interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently controlled AT gene expression. These analyses help understanding the relative importance of environmental and individual factors that control the expression of human AT genes and therefore may foster strategies aimed at improving AT function in metabolic diseases.
[Show abstract][Hide abstract] ABSTRACT: Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of gammaH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls gammaH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes gammaH2AX spreading. Remarkably, depletion of cohesin leads to an increase of gammaH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome.
[Show abstract][Hide abstract] ABSTRACT: Genomic DNA (gDNA) contamination is an inherent problem during RNA purification that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT-qPCR). Currently, there is no alternative to RT(-) controls to evaluate the impact of the gDNA background on RT-PCR data. We propose a novel method (ValidPrime) that is more accurate than traditional RT(-) controls to test qPCR assays with respect to their sensitivity toward gDNA. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA) and gDNA reference sample(s). The VPA, targeting a non-transcribed locus, is used to measure the gDNA contents in RT(+) samples and the gDNA reference is used to normalize for GOI-specific differences in gDNA sensitivity. We demonstrate that the RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for both exogenous (spiked) and endogenous gDNA, contributing ∼60% of the total signal, whereas substantially reducing the number of required qPCR control reactions. In conclusion, ValidPrime offers a cost-efficient alternative to RT(-) controls and accurately corrects for signals derived from gDNA in RT-qPCR.
Full-text · Article · Jan 2012 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Aims/hypothesis
Evidence suggests that bacterial components in blood could play an early role in events leading to diabetes. To test this hypothesis, we studied the capacity of a broadly specific bacterial marker (16S rDNA) to predict the onset of diabetes and obesity in a general population.
Data from an Epidemiological Study on the Insulin Resistance Syndrome (D.E.S.I.R.) is a longitudinal study with the primary aim of describing the history of the metabolic syndrome. The 16S rDNA concentration was measured in blood at baseline and its relationship with incident diabetes and obesity over 9 years of follow-up was assessed. In addition, in a nested case–control study in which participants later developed diabetes, bacterial phylotypes present in blood were identified by pyrosequencing of the overall 16S rDNA gene content.
We analysed 3,280 participants without diabetes or obesity at baseline. The 16S rDNA concentration was higher in those destined to have diabetes. No difference was observed regarding obesity. However, the 16S rDNA concentration was higher in those who had abdominal adiposity at the end of follow-up. The adjusted OR (95% CIs) for incident diabetes and for abdominal adiposity were 1.35 (1.11, 1.60), p = 0.002 and 1.18 (1.03, 1.34), p = 0.01, respectively. Moreover, pyrosequencing analyses showed that participants destined to have diabetes and the controls shared a core blood microbiota, mostly composed of the Proteobacteria phylum (85–90%).
16S rDNA was shown to be an independent marker of the risk of diabetes. These findings are evidence for the concept that tissue bacteria are involved in the onset of diabetes in humans.
[Show abstract][Hide abstract] ABSTRACT: Glucocorticoids are frequently prescribed drugs with important side-effects such as glucose intolerance and tissue remodeling. The goal was to explore the molecular basis of the response of skeletal muscle and adipose tissue during a short-term dexamethasone treatment to better understand the induction of side-effects of glucocorticoids on these metabolic tissues. Fifteen healthy male subjects were assigned to a 4-day treatment with dexamethasone at 4 mg/day. The primary outcome measures were changes in gene expression profiling of subcutaneous skeletal muscle and adipose tissue. Urinary cortisol, plasma, and metabolic biochemistry were also assessed. In both tissues the prominent observation was a response to stress and increased inflammatory responses. An upregulation of the serum amyloid A was detected in skeletal muscle, adipose tissue, and plasma, whereas circulating levels of C reactive protein, another acute phase protein, decreased along with a worsened insulin sensitivity index. As tissue-specific features, tissue remodeling was shown in skeletal muscle while the adipose tissue exhibited a decreased energy metabolism. Several limitations might be raised due to the small number of subjects investigated: a possible cross talk with the mineralocorticoid receptor, and a single time point may not identify regulations occurring during longitudinal treatment. In line with the known physiological effect of glucocorticoids the early modulation of stress response genes was observed. An unexpected feature was the upregulation of the inflammatory and immune pathways. The identification of novel impact on two glucocorticoid target tissues provides a molecular basis for the design of more specific glucocorticoids devoid of adverse effects.
No preview · Article · Nov 2011 · Physiological Genomics
[Show abstract][Hide abstract] ABSTRACT: The gut microbiota, which is considered a causal factor in metabolic diseases as shown best in animals, is under the dual influence of the host genome and nutritional environment. This study investigated whether the gut microbiota per se, aside from changes in genetic background and diet, could sign different metabolic phenotypes in mice.
The unique animal model of metabolic adaptation was used, whereby C57Bl/6 male mice fed a high-fat carbohydrate-free diet (HFD) became either diabetic (HFD diabetic, HFD-D) or resisted diabetes (HFD diabetes-resistant, HFD-DR). Pyrosequencing of the gut microbiota was carried out to profile the gut microbial community of different metabolic phenotypes. Inflammation, gut permeability, features of white adipose tissue, liver and skeletal muscle were studied. Furthermore, to modify the gut microbiota directly, an additional group of mice was given a gluco-oligosaccharide (GOS)-supplemented HFD (HFD+GOS).
Despite the mice having the same genetic background and nutritional status, a gut microbial profile specific to each metabolic phenotype was identified. The HFD-D gut microbial profile was associated with increased gut permeability linked to increased endotoxaemia and to a dramatic increase in cell number in the stroma vascular fraction from visceral white adipose tissue. Most of the physiological characteristics of the HFD-fed mice were modulated when gut microbiota was intentionally modified by GOS dietary fibres.
The gut microbiota is a signature of the metabolic phenotypes independent of differences in host genetic background and diet.
[Show abstract][Hide abstract] ABSTRACT: The preclinical stage of systolic heart failure (HF), known as asymptomatic left ventricular dysfunction (ALVD), is diagnosed only by echocardiography, frequent in the general population and leads to a high risk of developing severe HF. Large scale screening for ALVD is a difficult task and represents a major unmet clinical challenge that requires the determination of ALVD biomarkers.
294 individuals were screened by echocardiography. We identified 9 ALVD cases out of 128 subjects with cardiovascular risk factors. White blood cell gene expression profiling was performed using pangenomic microarrays. Data were analyzed using principal component analysis (PCA) and Significant Analysis of Microarrays (SAM). To build an ALVD classifier model, we used the nearest centroid classification method (NCCM) with the ClaNC software package. Classification performance was determined using the leave-one-out cross-validation method. Blood transcriptome analysis provided a specific molecular signature for ALVD which defined a model based on 7 genes capable of discriminating ALVD cases. Analysis of an ALVD patients validation group demonstrated that these genes are accurate diagnostic predictors for ALVD with 87% accuracy and 100% precision. Furthermore, Receiver Operating Characteristic curves of expression levels confirmed that 6 out of 7 genes discriminate for left ventricular dysfunction classification.
These targets could serve to enhance the ability to efficiently detect ALVD by general care practitioners to facilitate preemptive initiation of medical treatment preventing the development of HF.
[Show abstract][Hide abstract] ABSTRACT: Objectif
Des données expérimentales suggèrent que la présence de composants bactériens dans le sang et les tissus est une des étapes initiales conduisant au diabète de type 2. L’objectif de l’étude est d’analyser la relation entre la concentration sanguine d’un gène hautement conservé au sein des espèces bactériennes : le gène 16S ARNr et la survenue du diabète dans une population générale.
Patients et méthodes
L’étude DESIR est une étude de cohorte dont l’objectif était de décrire l’histoire naturelle du syndrome métabolique. Les participants ont été évalués à l’inclusion et à 3, 6 et 9 ans. La concentration sanguine du gène 16S ARNr a été mesurée à l’inclusion. De plus nous avons réalisé dans un sous groupe de la population une étude cas témoin pour identifier par technique de pyroséquençage, l’ADN bactérien associé à la survenue du diabète.
3 650 participants indemnes de diabète à l’inclusion ont été analysés. En référence au quartile de concentration sanguine d’ADN bactérien le plus faible et après ajustement sur les facteurs confondants, l’odds ratio de développer un diabète pour la dernière période de suivi était de 1,92 (0,76–4,81) dans le quartile 2, 3.50 (1,42–8,62) dans le quartile 3 and 3,63 (1,52–8,70) dans le quartile 4. L’analyse par pyroséquençage de l’ADN bactérien a montré que les sujets destinés à devenir diabétiques et les témoins partageaient le même ensemble de gènes bactériens appartenant pour l’essentiel au phylum des protéobactéries avec des différences au niveau des genres bactériens.
La concentration sanguine en gènes bactériens présents dans le sang est un marqueur de risque de développer un diabète. Le microbiome tissulaire pourrait être une cible thérapeutique pour prévenir les maladies métaboliques.
No preview · Article · Mar 2011 · Diabetes & Metabolism
[Show abstract][Hide abstract] ABSTRACT: DNA double strand breaks (DSBs) are among the most deleterious forms of lesions and deciphering the details of the chromatin landscape induced around DSBs represents a great challenge for molecular biologists. Chromatin Immunoprecipitation, followed by microarray hybridisation (ChIP-chip) or high-throughput sequencing (ChIP-seq), are powerful techniques that provide high-resolution maps of protein-genome interactions. However, applying these techniques to study chromatin changes induced around DSBs was previously hindered due to a lack of suitable DSB induction techniques. We have recently developed an experimental system utilizing a restriction enzyme fused to a modified oestrogen receptor ligand binding domain (AsiSI-ER), which generates multiple, sequence-specific and unambiguously positioned DSBs across the genome upon induction with 4-hydroxytamoxifen (4OHT).(1) Cell lines expressing this construct represent a powerful tool to study specific chromatin changes during DSB repair, enabling high-resolution profiling of DNA repair complexes and chromatin modifications induced around DSBs. Using this system, we have recently produced the first map of gammaH2AX, a DSB-induced chromatin modification, on two human chromosomes and have investigated its spreading properties.(1) Here we provide additional data characterizing the cell lines, present a genome-wide profile of gammaH2AX obtained by ChIP-seq, and discuss the potential of our system towards investigations of previously uncharacterized aspects of DSB repair.
[Show abstract][Hide abstract] ABSTRACT: Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein–genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of γH2AX around DSBs. We found that all DSBs trigger large γH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of γH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that γH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within γH2AX domains, indicating that mechanisms may exist to protect gene transcription from γH2AX spreading and from the chromatin rearrangements induced by DSBs.
[Show abstract][Hide abstract] ABSTRACT: Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3' end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3' end formation. We identify PTB as the protein factor whose binding to the human beta-globin (HBB) 3' UTR is abrogated by a 3' end processing-inactivating mutation. We show that PTB promotes both in vitro 3' end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.
Full-text · Article · Jul 2009 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Uncoupling protein (UCP) 3 is an inner mitochondrial membrane transporter mainly produced in skeletal muscle in humans. UCP3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. In humans, UCP3 content is higher in fast-twitch glycolytic muscle than in slow-twitch oxidative muscle and is dysregulated in type 2 diabetes. Here, we studied the molecular mechanisms determining human UCP3 levels in skeletal muscle and their regulation by fasting in transgenic mice.
We produced a series of transgenic lines with constructs bearing different putative regulatory regions of the human UCP3 gene, including promoter and intron sequences. UCP3 mRNA and reporter gene expression and activity were measured in different skeletal muscles and tissues.
The profile of expression and the response to fasting and thyroid hormone of human UCP3 mRNA in transgenic mice with 16 kb of the human UCP3 gene were similar to that of the endogenous human gene. Various parts of the UCP3 promoter did not confer expression in transgenic lines. Inclusion of intron 1 resulted in an expression profile in skeletal muscle that was identical to that of human UCP3 mRNA. Further dissection of intron 1 revealed that distinct regions were involved in skeletal muscle expression, distribution among fibre types and response to fasting.
The control of human UCP3 transcription in skeletal muscle is not solely conferred by the promoter, but depends on several cis-acting elements in intron 1, suggesting a complex interplay between the promoter and intronic sequences.