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1.
Int J Hyg Environ Health ; 260: 114391, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38781750

RESUMO

The hygienic quality of urban surfaces can be impaired by multiple sources of microbiological contaminants. These surfaces can trigger the development of multiple bacterial taxa and favor their spread during rain events through the circulation of runoff waters. These runoff waters are commonly directed toward sewer networks, stormwater infiltration systems or detention tanks prior a release into natural water ways. With water scarcity becoming a major worldwide issue, these runoffs are representing an alternative supply for some usage like street cleaning and plant watering. Microbiological hazards associated with these urban runoffs, and surveillance guidelines must be defined to favor these uses. Runoff microbiological quality from a recently implemented city center rainwater harvesting zone was evaluated through classical fecal indicator bacteria (FIB) assays, quantitative PCR and DNA meta-barcoding analyses. The incidence of socio-urbanistic patterns on the organization of these urban microbiomes were investigated. FIB and DNA from Human-specific Bacteroidales and pathogens such as Staphylococcus aureus were detected from most runoffs and showed broad distribution patterns. 16S rRNA DNA meta-barcoding profilings further identified core recurrent taxa of health concerns like Acinetobacter, Mycobacterium, Aeromonas and Pseudomonas, and divided these communities according to two main groups of socio-urbanistic patterns. One of these was highly impacted by heavy traffic, and showed recurrent correlation networks involving bacterial hydrocarbon degraders harboring significant virulence properties. The tpm-based meta-barcoding approach identified some of these taxa at the species level for more than 30 genera. Among these, recurrent pathogens were recorded such as P. aeruginosa, P. paraeruginosa, and Aeromonas caviae. P. aeruginosa and A. caviae tpm reads were found evenly distributed over the study site but those of P. paraeruginosa were higher among sub-catchments impacted by heavy traffic. Health risks associated with these runoff P. paraeruginosa emerging pathogens were high and associated with strong cytotoxicity on A549 lung cells. Recurrent detections of pathogens in runoff waters highlight the need of a microbiological surveillance prior allowing their use. Good microbiological quality can be obtained for certain typologies of sub-catchments with good hygienic practices but not all. A reorganization of Human mobility and behaviors would likely trigger changes in these bacterial diversity patterns and reduce the occurrences of the most hazardous groups.


Assuntos
Bactérias , Cidades , Monitoramento Ambiental , Microbiota , Chuva , Microbiologia da Água , Humanos , Bactérias/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Monitoramento Ambiental/métodos , RNA Ribossômico 16S/genética , Fezes/microbiologia
2.
Int J Mol Sci ; 24(2)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36674544

RESUMO

Adipose tissue hypertrophy during obesity plays pleiotropic effects on health. Adipose tissue expandability depends on adipocyte size and number. In mature adipocytes, lipid accumulation as triglycerides into droplets is imbalanced by lipid uptake and lipolysis. In previous studies, we showed that adipogenesis induced by oleic acid is signed by size increase and reduction of FAT/CD36 (SR-B2) activity. The present study aims to decipher the mechanisms involved in fat mass regulation by fatty acid/FAT-CD36 signalling. Human adipose stem cells, 3T3-L1, and its 3T3-MBX subclone cell lines were used in 2D cell cultures or co-cultures to monitor in real-time experiments proliferation, differentiation, lipolysis, and/or lipid uptake and activation of FAT/CD36 signalling pathways regulated by oleic acid, during adipogenesis and/or regulation of adipocyte size. Both FABP4 uptake and its induction by fatty acid-mediated FAT/CD36-PPARG gene transcription induce accumulation of intracellular FABP4, which in turn reduces FAT/CD36, and consequently exerts a negative feedback loop on FAT/CD36 signalling in both adipocytes and their progenitors. Both adipocyte size and recruitment of new adipocytes are under the control of FABP4 stores. This study suggests that FABP4 controls fat mass homeostasis.


Assuntos
Adipócitos , Ácido Oleico , Humanos , Camundongos , Animais , Ácido Oleico/farmacologia , Ácido Oleico/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Lipólise , Adipogenia , Diferenciação Celular , Ácidos Graxos/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células 3T3-L1 , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo
3.
Adipocyte ; 11(1): 510-528, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35946137

RESUMO

Metabolic disorders related to obesity are largely dependent on adipose tissue hypertrophy, which involves adipocyte hypertrophy and increased adipogenesis. Adiposize is regulated by lipid accumulation as a result of increased lipogenesis (mainly lipid uptake in mature adipocytes) and reduced lipolysis. Using realtime 2D cell culture analyses of lipid uptake, we show (1) that high glucose concentration (4.5 g/L) was required to accumulate oleic acid increasing lipid droplet size until unilocularization similar to mature adipocytes in few days, (2) oleic acid reduced Peroxisome-Proliferator Activated Receptor Gamma (PPARG) gene transcription and (3) insulin counteracted oleic acid-induced increase of lipid droplet size. Although the lipolytic activity observed in high versus low glucose (1 g/L) conditions was not altered, insulin was found to inhibit oleic acid induced gene transcription required for lipid storage such as Cell Death Inducing DFFA Like Effectors (CIDEC) and G0S2 (G0 switch gene S2), possibly through PPARA activity. Although this signalling pathway requires more detailed investigation, the results point out the differential mechanisms involved in the pro-adipogenic effect of insulin in absence versus its protective effect on adiposity in presence of oleic acid uptake.Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide-1-D-ribofuranoside; AMPK, AMP-Activated protein kinase, ASCs, adipose stem cell; ATGL, adipose triglyceride lipase; BSA, Bovine serum albumin; CEBPA, CCAAT enhancer binding protein alpha; CIDEs, Cell Death Inducing DFFA Like Effectors; dA, differentiated adipocyte; DMEM, Dulbecco's Modified Eagle's Medium; FABPs, Fatty Acid Binding Proteins; FAT/CD36, Fatty acid translocase; FCS, Foetal calf serum; FN1, fibronectin 1; FFA, free fatty acid; G0S2, G0 switch gene S2; GLUTs, Glucose transporters; GPR120, G protein-coupled receptor 120; HG, high glucose; HSL, hormone sensitive lipase; INSR, insulin receptor; LG, low glucose; OA, oleic acid; PBS, Phosphate buffer saline; PPARs, Peroxisome-Proliferator Activated Receptors; PKA, Protein kinase cyclic AMP-dependent; PKG, Protein kinase cyclic GMP dependent; PTGS2, cytochrome oxidase 2; RTCA, realtime cell analysis; TG, triglyceride.


Assuntos
Ácidos Graxos , Insulina , Adipócitos/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Humanos , Hipertrofia/metabolismo , Insulina/metabolismo , Lipólise , Obesidade/metabolismo , Ácido Oleico/metabolismo , Ácido Oleico/farmacologia , Proteínas Quinases/metabolismo
4.
Cancer Gene Ther ; 29(10): 1429-1438, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35379907

RESUMO

Cell migration depends on the dynamic organisation of the actin cytoskeleton and assembly and disassembly of focal adhesions (FAs). However, the precise mechanisms coordinating these processes remain poorly understood. We previously identified the oestrogen-related receptor α (ERRα) as a major regulator of cell migration. Here, we show that loss of ERRα leads to abnormal accumulation of actin filaments that is associated with an increased level of inactive form of the actin-depolymerising factor cofilin. We further show that ERRα depletion decreases cell adhesion and results in defective FA formation and turnover. Interestingly, specific inhibition of the RhoA-ROCK-LIMK-cofilin pathway rescues the actin polymerisation defects resulting from ERRα silencing, but not cell adhesion. Instead, we found that MAP4K4 is a direct target of ERRα and down-regulation of its activity rescues cell adhesion and FA formation in the ERRα-depleted cells. Altogether, our results highlight a crucial role of ERRα in coordinating the dynamic of actin network and FAs through the independent regulation of the RhoA and MAP4K4 pathways.


Assuntos
Actinas , Adesões Focais , Fatores de Despolimerização de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Movimento Celular/fisiologia , Adesões Focais/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptor ERRalfa Relacionado ao Estrogênio
5.
Sci Rep ; 11(1): 21626, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34732797

RESUMO

We have determined the lipid, protein and miRNA composition of skeletal muscle (SkM)-released extracellular vesicles (ELVs) from Ob/ob (OB) vs wild-type (WT) mice. The results showed that atrophic insulin-resistant OB-SkM released less ELVs than WT-SkM, highlighted by a RAB35 decrease and an increase in intramuscular cholesterol content. Proteomic analyses of OB-ELVs revealed a group of 37 proteins functionally connected, involved in lipid oxidation and with catalytic activities. OB-ELVs had modified contents for phosphatidylcholine (PC 34-4, PC 40-3 and PC 34-0), sphingomyelin (Sm d18:1/18:1) and ceramides (Cer d18:1/18:0) and were enriched in cholesterol, likely to alleviated intracellular accumulation. Surprisingly many ELV miRNAs had a nuclear addressing sequence, and targeted genes encoding proteins with nuclear activities. Interestingly, SkM-ELV miRNA did not target mitochondria. The most significant function targeted by the 7 miRNAs altered in OB-ELVs was lipid metabolism. In agreement, OB-ELVs induced lipid storage in recipient adipocytes and increased lipid up-take and fatty acid oxidation in recipient muscle cells. In addition, OB-ELVs altered insulin-sensitivity and induced atrophy in muscle cells, reproducing the phenotype of the releasing OB muscles. These data suggest for the first time, a cross-talk between muscle cells and adipocytes, through the SkM-ELV route, in favor of adipose tissue expansion.


Assuntos
Homeostase , Resistência à Insulina , Lipídeos/análise , MicroRNAs/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/patologia , Tecido Adiposo , Animais , Exossomos/genética , Exossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas Musculares/genética , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Proteoma/análise , Proteoma/metabolismo
6.
Sensors (Basel) ; 21(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34372238

RESUMO

The measuring of nanoparticle toxicity faces an important limitation since it is based on metrics exposure, the concentration at which cells are exposed instead the true concentration inside the cells. In vitro studies of nanomaterials would benefit from the direct measuring of the true intracellular dose of nanoparticles. The objective of the present study was to state whether the intracellular detection of nanodiamonds is possible by measuring the refractive index. Based on optical diffraction tomography of treated live cells, the results show that unlabeled nanoparticles can be detected and localized inside cells. The results were confirmed by fluorescence measurements. Optical diffraction tomography paves the way to measuring the true intracellular concentrations and the localization of nanoparticles which will improve the dose-response paradigm of pharmacology and toxicology in the field of nanomaterials.


Assuntos
Nanodiamantes , Nanopartículas , Nanopartículas/toxicidade , Refratometria
7.
Life (Basel) ; 11(4)2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917485

RESUMO

In 2018, seven million people died prematurely due to exposure to pollution. Polycyclic aromatic hydrocarbons (PAHs) are a significant source of secondary organic aerosol (SOA) in urban areas. We investigated the toxic effects of by-products of naphthalene SOA on lung cells. These by-products were 1,4-naphthoquinone (1,4-NQ), 2-hydroxy-1,4-naphthoquinone (2-OH-NQ), phthalic acid (PA) and phthaldialdehyde (OPA). Two different assessment methodologies were used to monitor the toxic effects: real-time cell analysis (RTCA) and the Holomonitor, a quantitative phase contrast microscope. The chemicals were tested in concentrations of 12.5 to 100 µM for 1,4-NQ and 1 to 10 mM for 2-OH-NQ, PA and OPA. We found that 1,4-NQ is toxic to cells from 25 to 100 µM (EC50: 38.7 µM ± 5.2); 2-OH-NQ is toxic from 1 to 10mM (EC50: 5.3 mM ± 0.6); PA is toxic from 5 to 10 mM (EC50: 5.2 mM ± 0.3) and OPA is toxic from 2.5 to 10 mM (EC50: 4.2 mM ± 0.5). Only 1,4-NQ and OPA affected cell parameters (migration, motility, motility speed and optical volume). Furthermore, 1,4-NQ is the most toxic by-product of naphthalene, with an EC50 value that was one hundred times higher than those of the other compounds. RTCA and Holomonitor analysis showed a complementarity when studying the toxicity induced by chemicals.

8.
Mol Ther Methods Clin Dev ; 18: 880-892, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32953937

RESUMO

We have determined whether orange juice-derived nanovesicles (ONVs) could be used for the treatment of obesity-associated intestinal complications. ONVs were characterized by lipidomic, metabolomic, electron microscopy. In vitro, intestinal barriers (IBs = Caco-2+HT-29-MTX) were treated with ONVs and co-cultured with adipocytes to monitor IB fat release. In vivo, obesity was induced with a high-fat, high-sucrose diet (HFHSD mice) for 12 weeks. Then, half of HFHSD mice were gavaged with ONVs. One-month ONV treatment did not modify HFHSD-induced insulin resistance but reversed diet-induced gut modifications. In the jejunum, ONVs increased villi size, reduced triglyceride content, and modulated mRNA levels of genes involved in immune response (tumor necrosis factor [TNF]-α and interleukin [IL]-1ß), barrier permeability (CLDN1, OCLN, ZO1), fat absorption, and chylomicron release. ONVs targeted microsomal triglyceride transfer protein (MTP) and angiopoietin-like protein-4 (ANGPTL4), two therapeutic targets to reduce plasma lipids and inflammation in gastrointestinal diseases. Interestingly, ONV treatment did not aggravate liver steatosis, as MTP mRNA was increased in the liver. Therefore, ONVs protected both intestine and the liver from fat overload associated with the HFHSD. As ONVs concentrated amino acids and bioactive lipids versus orange juice, which are deficient in obese patients, the use of ONVs as a dietary supplement could bring physiological relevant compounds in the jejunum to accelerate the restoration of intestinal functions during weight loss in obese patients.

9.
Adipocyte ; 8(1): 83-97, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30905315

RESUMO

Adipose tissue function in the regulation of lipemia is highly dependent on intestinal absorption of nutrients. Therefore the aim of the present study was the development and validation of an in vitro multiculture model allowing to measure intestinal absorption using adipocytes as lipid sensors. We previously described (1) novel methods to study oleic acid induction of adipogenesis and lipogenesis and (2) a functional reconstituted intestinal barrier using human cell lines Caco-2/HT29-MTX (9:1). In the present study we develop a co-culture model with either adipocytes or hepatocytes as sensors for intestinal lipid absorption. This model was validated using oleic acid (OA) pre-absorbed onto the intestinal barrier. Optimized experimental conditions were obtained with partially differentiated 3T3L1-MBX adipocytes sensing up to 5 µM OA in solution or 40 µM OA pre-absorbed by Caco2/HT29-MTX intestinal barriers. Metabolism including glycemia and insulinemia greatly influenced the ability to  TG accumulation in adipocytes. By comparison AML12 hepatocytes found less sensitive to OA (up to 1 µM). The present study demonstrates a much better functionality for fatty acid uptake and release in Caco2/HT29-MTX versus Caco-2 intestinal barriers. Taken together these results open new opportunities to study in vitro lipid transfer between intestinal barriers and either adipocytes or hepatocytes. Abbreviations: BSA: Bovine serum albumin; CIDEs: Cell Death Inducing DFFA Like Effectors; DMEM, Dulbecco's Modified Eagle's Medium; FABPs: Fatty Acid Binding Proteins; FAT/CD36: Fatty acid translocase; FCS: Fetal calf serum; GLP2: Glucagon-like peptide-2; NAFLD: Nonalcoholic fatty liver disease; OA: oleic acid; PBS: Phosphate buffer saline; PPARs: Peroxisome-Proliferator Activated Receptors; RTCA: realtime cell analysis; TG: triglyceride.


Assuntos
Adipócitos/fisiologia , Absorção Intestinal/fisiologia , Ácido Oleico/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Transporte Biológico/fisiologia , Células CACO-2 , Diferenciação Celular , Técnicas de Cocultura , Proteínas de Ligação a Ácido Graxo/metabolismo , Células HT29 , Hepatócitos/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Intestinos/fisiologia , Lipídeos/fisiologia , Lipogênese/fisiologia , Camundongos , Hepatopatia Gordurosa não Alcoólica , Transporte Proteico/fisiologia , Triglicerídeos/metabolismo
10.
Int J Mol Sci ; 18(7)2017 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-28726765

RESUMO

Gastrointestinal epithelium is the unique route for nutrients and for many pharmaceuticals to enter the body. The present study aimed to analyze precisely whether co-culture of two colon cancer cell lines, mucus-producing cells HT29-MTX and enterocyte-like Caco-2 cells, ameliorate differentiation into an in vitro intestinal barrier model and the signaling pathways involved. Differentiated Caco-2 cells gene datasets were compared first to intestinal or cancer phenotypes and second to signaling pathway gene datasets. Experimental validations were performed in real-time experiments, immunochemistry, and gene expression analyses on Caco-2 versus co-cultures of Caco-2 and HT29-MTX (10%) cells. Partial maintenance of cancer-cell phenotype in differentiated Caco-2 cells was confirmed and fatty acids merged as potential regulators of cancer signaling pathways. HT29-MTX cells induced morphological changes in Caco-2 cells, slightly increased their proliferation rate and profoundly modified gene transcription of phenotype markers, fatty acid receptors, intracellular transporters, and lipid droplet components as well as functional responses to oleic acid. In vitro, enterocyte phenotype was rescued partially by co-culture of cancer cells with goblet cells and completed through oleic acid interaction with signaling pathways dysregulated in cancer cells.


Assuntos
Neoplasias do Colo/metabolismo , Enterócitos/metabolismo , Ácido Oleico/metabolismo , Fenótipo , Células CACO-2 , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Transcrição Gênica
11.
Oncotarget ; 8(10): 17140-17155, 2017 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-28188308

RESUMO

Melanoma is well known for its propensity for lethal metastasis and resistance to most current therapies. Tumor progression and drug resistance depend to a large extent on the interplay between tumor cells and the surrounding matrix. We previously identified Tetraspanin 8 (Tspan8) as a critical mediator of melanoma invasion, whose expression is absent in healthy skin. The present study investigated whether Tspan8 may influence cell-matrix anchorage and regulate downstream molecular pathways leading to an aggressive behavior. Using silencing and ectopic expression strategies, we showed that Tspan8-mediated invasion of melanoma cells resulted from defects in cell-matrix anchorage by interacting with ß1 integrins and by interfering with their clustering, without affecting their surface or global expression levels. These effects were associated with impaired phosphorylation of integrin-linked kinase (ILK) and its downstream target Akt-S473, but not FAK. Specific blockade of Akt or ILK activity strongly affected cell-matrix adhesion. Moreover, expression of a dominant-negative form of ILK reduced ß1 integrin clustering and cell-matrix adhesion. Finally, we observed a tumor-promoting effect of Tspan8 in vivo and a mutually exclusive expression pattern between Tspan8 and phosphorylated ILK in melanoma xenografts and human melanocytic lesions. Altogether, the in vitro, in vivo and in situ data highlight a novel regulatory role for Tspan8 in melanoma progression by modulating cell-matrix interactions through ß1 integrin-ILK axis and establish Tspan8 as a negative regulator of ILK activity. These findings emphasize the importance of targeting Tspan8 as a means of switching from low- to firm-adhesive states, mandatory to prevent tumor dissemination.


Assuntos
Integrina beta1/genética , Melanoma/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Tetraspaninas/genética , Animais , Western Blotting , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina beta1/metabolismo , Masculino , Melanoma/metabolismo , Melanoma/patologia , Camundongos Nus , Microscopia Confocal , Mutação , Invasividade Neoplásica , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Tetraspaninas/metabolismo , Transplante Heterólogo
12.
Diabetologia ; 59(5): 1049-58, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26852333

RESUMO

AIMS/HYPOTHESIS: The crosstalk between skeletal muscle (SkM) and beta cells plays a role in diabetes aetiology. In this study, we have investigated whether SkM-released exosome-like vesicles (ELVs) can be taken up by pancreatic beta cells and can deliver functional cargoes. METHODS: Mice were fed for 16 weeks with standard chow diet (SCD) or with standard diet enriched with 20% palmitate (HPD) and ELVs were purified from quadriceps muscle. Fluorescent ELVs from HPD or SCD quadriceps were injected i.v. or intramuscularly (i.m.) into mice to determine their biodistributions. Micro (mi)RNA quantification in ELVs was determined using quantitative real-time RT-PCR (qRT-PCR)-based TaqMan low-density arrays. Microarray analyses were performed to determine whether standard diet ELVs (SD-ELVs) and high palmitate diet ELVs (HPD-ELVs) induced specific transcriptional signatures in MIN6B1 cells. RESULTS: In vivo, muscle ELVs were taken up by pancreas, 24 h post-injection. In vitro, both SD-ELVs and HPD-ELVs transferred proteins and miRNAs to MIN6B1 cells and modulated gene expressions whereas only HPD-ELVs induced proliferation of MIN6B1 cells and isolated islets. Bioinformatic analyses suggested that transferred HPD-ELV miRNAs may participate in these effects. To validate this, we demonstrated that miR-16, which is overexpressed in HPD-ELVs, was transferred to MIN6B1 cells and regulated Ptch1, involved in pancreas development. In vivo, islets from HPD mice showed increased size and altered expression of genes involved in development, including Ptch1, suggesting that the effect of palm oil on islet size in vivo was reproduced in vitro by treating beta cells with HPD-ELVs. CONCLUSIONS/INTERPRETATION: Our data suggest that muscle ELVs might have an endocrine effect and could participate in adaptations in beta cell mass during insulin resistance.


Assuntos
Exossomos/metabolismo , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Músculo Esquelético/metabolismo , Animais , Linhagem Celular , Masculino , Camundongos , MicroRNAs/metabolismo , Fibras Musculares Esqueléticas/metabolismo
13.
Biomed Res Int ; 2015: 821761, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26380295

RESUMO

Cancer progression may be affected by metabolism. In this study, we aimed to analyze the effect of glucose on the proliferation and/or survival of human hepatocellular carcinoma (HCC) cells. Human gene datasets regulated by glucose were compared to gene datasets either dysregulated in HCC or regulated by other signaling pathways. Significant numbers of common genes suggested putative involvement in transcriptional regulations by glucose. Real-time proliferation assays using high (4.5 g/L) versus low (1 g/L) glucose on two human HCC cell lines and specific inhibitors of selected pathways were used for experimental validations. High glucose promoted HuH7 cell proliferation but not that of HepG2 cell line. Gene network analyses suggest that gene transcription by glucose could be mediated at 92% through ChREBP in HepG2 cells, compared to 40% in either other human cells or rodent healthy liver, with alteration of LKB1 (serine/threonine kinase 11) and NOX (NADPH oxidases) signaling pathways and loss of transcriptional regulation of PPARGC1A (peroxisome-proliferator activated receptors gamma coactivator 1) target genes by high glucose. Both PPARA and PPARGC1A regulate transcription of genes commonly regulated by glycolysis, by the antidiabetic agent metformin and by NOX, suggesting their major interplay in the control of HCC progression.


Assuntos
Carcinoma Hepatocelular/genética , Glucose/metabolismo , Neoplasias Hepáticas/genética , PPAR alfa/genética , Fatores de Transcrição/genética , Quinases Proteína-Quinases Ativadas por AMP , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Glicólise/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metformina/administração & dosagem , PPAR alfa/biossíntese , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/biossíntese
14.
PLoS One ; 9(8): e106214, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25170835

RESUMO

INTRODUCTION: Adipocyte size and body fat distribution are strongly linked to the metabolic complications of obesity. The aim of the present study was to test the plasticity of white adipose tissue in response to insulin deprivation and replacement. We have characterized the changes of adipose cell size repartition and gene expressions in type 1 diabetes Sprague-Dawley rats and type 1 diabetic supplemented with insulin. METHODS: Using streptozotocin (STZ)-induced diabetes, we induced rapid changes in rat adipose tissue weights to study the changes in the distribution of adipose cell sizes in retroperitoneal (rWAT), epididymal (eWAT) and subcutaneous adipose tissues (scWAT). Adipose tissue weights of type 1 diabetic rats were then rapidly restored by insulin supplementation. Cell size distributions were analyzed using multisizer IV (Beckman Coulter). Cell size changes were correlated to transcriptional regulation of genes coding for proteins involved in lipid and glucose metabolisms and adipocytokines. RESULTS: The initial body weight of the rats was 465±5.2 g. Insulin privation was stopped when rats lost 100 g which induced reductions in fat mass of 68% for rWAT, 42% for eWAT and 59% for scWAT corresponding to decreased mode cell diameters by 31.1%, 20%, 25.3%, respectively. The most affected size distribution by insulin deprivation was observed in rWAT. The bimodal distribution of adipose cell sizes disappeared in response to insulin deprivation in rWAT and scWAT. The most important observation is that cell size distribution returned close to control values in response to insulin treatment. mRNAs coding for adiponectin, leptin and apelin were more stimulated in scWAT compared to other depots in diabetic plus insulin group. CONCLUSION: Fat depots have specific responses to insulin deprivation and supplementation. The results show that insulin is a major determinant of bimodal cell repartition in adipose tissues.


Assuntos
Adipócitos/metabolismo , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina , Transcrição Gênica/efeitos dos fármacos , Adipócitos/patologia , Tecido Adiposo Branco , Animais , Tamanho Celular , Diabetes Mellitus Experimental/patologia , Insulina/deficiência , Insulina/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley
15.
Diabetologia ; 57(10): 2155-64, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25073444

RESUMO

AIMS/HYPOTHESIS: Exosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route. METHODS: Exosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA). RESULTS: HP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues. CONCLUSIONS/INTERPRETATION: Exosomes act as 'paracrine-like' signals and modify muscle homeostasis during high-fat diets.


Assuntos
Exossomos/metabolismo , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Palmitatos/farmacologia , Animais , Western Blotting , Linhagem Celular , Homeostase/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Oleico/farmacologia , Reação em Cadeia da Polimerase em Tempo Real
16.
Nutr Cancer ; 66(4): 645-55, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24738610

RESUMO

Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, a hormone excessively produced during obesity, is suggested to be involved in breast cancer. The aim of the study was to investigate procarcinogenic potential of leptin by evaluating influence of leptin on cell proliferation, cell cycle, apoptosis, and signaling on numerous breast cells lines, including 184B5 normal cells, MCF10A fibrocystic cells and MCF-7, MDA-MB-231, and T47D cancer cells. Expressions of leptin and Ob-R were analyzed using qRT-PCR and immunohistochemistry, proliferation using fluorimetric resazurin reduction test and xCELLigence system, apoptosis and cell cycle by flow cytometry, and effect of leptin on different signalling pathways using qRT-PCR and Western blot. Cells were exposed to increasing concentrations of leptin. All cell lines expressed mRNA and protein of leptin and Ob-R. Leptin stimulated proliferation of all cell lines except for 184B5 and MDA-MB-231 cells. Leptin inhibited apoptosis but didn't alter proportion of cells within cell cycle in MCF7 cells. Leptin induced overexpression of leptin, Ob-R, estrogen receptor, and aromatase mRNA in MCF-7 and T47D cells. Autoregulation induced by leptin, relationship with estrogen pathway, and proliferative and antiapoptic activity in breast cancer cells may explain that obesity-associated hyperleptinemia may be a breast cancer risk factor.


Assuntos
Neoplasias da Mama/sangue , Proliferação de Células/efeitos dos fármacos , Leptina/sangue , Obesidade/sangue , Apoptose/efeitos dos fármacos , Neoplasias da Mama/etiologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Doença da Mama Fibrocística/sangue , Doença da Mama Fibrocística/etiologia , Humanos , Imuno-Histoquímica , Leptina/genética , Células MCF-7 , Obesidade/complicações , Receptores para Leptina/sangue , Receptores para Leptina/genética , Transdução de Sinais/efeitos dos fármacos
17.
PLoS One ; 9(1): e84153, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24392111

RESUMO

Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.


Assuntos
Exossomos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Proteoma , Proteômica , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Exossomos/ultraestrutura , Camundongos , Mioblastos/citologia , Transporte Proteico , Proteômica/métodos
18.
Biotechnol J ; 7(11): 1395-404, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22930530

RESUMO

Hepatocellular carcinoma (HCC) represents one of the most frequently diagnosed human cancers; however, there are currently few treatment alternatives to surgical resection. In this study we performed bioinformatic analysis of previously published transcriptomic data in order to characterize liver specific networks, including biological functions, signaling pathways and transcription factors, potentially dysregulated in HCC. By incorporating specific signaling inhibitors into real-time proliferation assays using HepG2 cells, we then validated these in silico results. We found that G protein subunits Gi/G0, protein kinase C, Mek1/2, and Erk1/2 (P42/44), JAK1, PPARA and NFκB p65 subunit were the major signaling molecules required for survival and proliferation of human HCC cell lines. We also found that these pathways regulate the expression of key hepatic transcription factors involved in cell differentiation, such as CEBPA, EGR1, FOXM1 and PPARs. By combining bioinformatic and functional analyses, major signaling pathways related to tumorigenicity in HCC are revealed, thereby elucidating potential targets for drug therapies.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Redes Reguladoras de Genes , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/metabolismo , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Análise por Conglomerados , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
19.
Physiol Genomics ; 42A(1): 61-70, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20571111

RESUMO

The adipocyte-derived hormone adiponectin exerts protective actions in several disorders, including some cancers. However, while growing data suggest that adiponectin could be an effective anticancer agent, its mechanism of action in cancer cells is still poorly known. Here, using microarrays, we identified a set of 1,301 genes commonly modulated in three cancer-derived cell lines in response to short-term stimulation with full-length recombinant human adiponectin. Most of these genes are involved in translation regulation, immune or stress responses, and cell proliferation. Furthermore, among genes linked to disease that were retrieved by functional enrichment tests using text mining based on PubMed analysis, we found that 66% are involved in malignant neoplasms, further supporting the link between adiponectin and cancer mechanisms. Bioinformatic analysis demonstrated the diversity of signaling pathways and transcription factors potentially mediating adiponectin effects on gene expression, illustrating the complexity of adiponectin mechanisms of action in cancer cells.


Assuntos
Adiponectina/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Células Hep G2 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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