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1.
Eur J Nutr ; 61(4): 2201-2215, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35092460

RESUMO

PURPOSE: Excessive fat mass accumulation in obesity leads to diverse metabolic disorders, increased risks of cardiovascular diseases and in some cases, mortality. The aim of this study was to screen the actions of botanical extracts intended for oral use on human adipose tissue, using an in vitro screening model combining human intestinal cells with human adipose cells. This was to find the most effective extracts on lipid accumulation, UCP1 expression and ATP production in pre-adipocytes and on adipocyte lipolysis. METHODS: In this study, 25 individual plant extracts were screened for their effects on human adipose cells. Consequently, an original in vitro model was set up using the Caco-2 cell line, to mimic the intestinal passage of the extracts and then exposing human adipose cells to them. The biological actions of extracts were thus characterized, and compared with a coffee extract standard. The most effective extracts, and their combinations, were retained for their actions on lipid accumulation, the expression of the thermogenic effector UCP1 and ATP production in pre-adipocytes as well as on lipolysis activity of mature adipocytes. RESULTS: The biphasic culture system combining human Caco-2 cells with human adipose cells was verified as functional using the green coffee extract standard. Out of the 25 plant extracts studied, only 7 and their combinations were retained due to their potent effects on adipose cells biology. The data showed that compared to the coffee extract standard, Immortelle, Catechu, Carrot and Rose hip extracts were the most effective in reducing lipid accumulation and increased UCP1 expression in human pre-adipocytes. CONCLUSION: This study reveals the potential inhibitory effects on lipid accumulation and thermogenic activity of Immortelle, Catechu, Carrot and Rose hip extracts, and for the first time synergies in their combinations, using an in vitro model mimicking as closely as possible, human intestinal passage linked to adipose cells. These findings need to be confirmed by in vivo trials.


Assuntos
Café , Lipólise , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Adipócitos , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom , Células CACO-2 , Café/metabolismo , Humanos , Lipídeos , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia
2.
J Clin Endocrinol Metab ; 101(6): 2578-87, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27049236

RESUMO

CONTEXT: Collagen accumulation around adipocytes and vessels (ie, pericellular fibrosis) is a hallmark of obese adipose tissue associated with altered metabolism. OBJECTIVE: Our objective was to evaluate components of basement membrane (BM) in adipose tissue, including collagen IV, a major BM component, and its relationships with metabolic parameters and TGFß isoforms. DESIGN AND SETTING: We used immuno-techniques and gene expression approaches to detect BM components in subcutaneous and visceral adipose tissue samples. Adipocytes and endothelial cells were isolated from lean and obese adipose tissue. We also focused on the expression of COL4A1 correlated to metabolic variables in moderate obesity and, in severe obesity before and after bariatric surgery. Using in vitro analysis, we explored the impact of TGFß isoforms on the expression of inflammatory and extracellular matrix genes in adipocytes and endothelial cells. RESULTS: BM components were detected around adipocytes and endothelial cells, and were increased in obese adipocytes. COL4A1 expression was positively correlated with insulin-resistance indices in obese subjects and showed less reduction in severely obese subjects with poorer insulin-resistance outcomes 6 months after gastric bypass. COL4A1 expression also correlated with TGFß1 and TGFß3 gene expressions in subcutaneous adipose tissue. Stimulating isolated adipocytes and endothelial cells in vitro with these TGFß isoforms showed an inflammatory and pro-fibrotic phenotype. However, TGFß1 and TGFß3 exposure only provoked COL4A1 overexpression in endothelial cells and not in adipocytes. CONCLUSION: The disorganization of several BM components, including collagen IV, could contribute to pathological alterations of obese adipose tissue and cells.


Assuntos
Tecido Adiposo/metabolismo , Membrana Basal/metabolismo , Obesidade/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Adipócitos/metabolismo , Células Cultivadas , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta3/genética
3.
Diabetes ; 64(9): 3121-34, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25695947

RESUMO

Inflammation and lipid accumulation are hallmarks of muscular pathologies resulting from metabolic diseases such as obesity and type 2 diabetes. During obesity, the hypertrophy of visceral adipose tissue (VAT) contributes to muscle dysfunction, particularly through the dysregulated production of adipokines. We have investigated the cross talk between human adipocytes and skeletal muscle cells to identify mechanisms linking adiposity and muscular dysfunctions. First, we demonstrated that the secretome of obese adipocytes decreased the expression of contractile proteins in myotubes, consequently inducing atrophy. Using a three-dimensional coculture of human myotubes and VAT adipocytes, we showed the decreased expression of genes corresponding to skeletal muscle contractility complex and myogenesis. We demonstrated an increased secretion by cocultured cells of cytokines and chemokines with interleukin (IL)-6 and IL-1ß as key contributors. Moreover, we gathered evidence showing that obese subcutaneous adipocytes were less potent than VAT adipocytes in inducing these myotube dysfunctions. Interestingly, the atrophy induced by visceral adipocytes was corrected by IGF-II/insulin growth factor binding protein-5. Finally, we observed that the skeletal muscle of obese mice displayed decreased expression of muscular markers in correlation with VAT hypertrophy and abnormal distribution of the muscle fiber size. In summary, we show the negative impact of obese adipocytes on muscle phenotype, which could contribute to muscle wasting associated with metabolic disorders.


Assuntos
Adipócitos/metabolismo , Proteínas Contráteis/metabolismo , Gordura Intra-Abdominal/citologia , Fibras Musculares Esqueléticas/metabolismo , Obesidade Mórbida/metabolismo , Adipócitos/imunologia , Adulto , Animais , Atrofia/imunologia , Atrofia/metabolismo , Técnicas de Cocultura , Citocinas/imunologia , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Gordura Intra-Abdominal/imunologia , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos , Camundongos Obesos , Fibras Musculares Esqueléticas/imunologia , Fibras Musculares Esqueléticas/patologia , Obesidade Mórbida/imunologia , Gordura Subcutânea/citologia , Gordura Subcutânea/imunologia , Gordura Subcutânea/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
4.
Diabetes ; 63(2): 535-49, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24130331

RESUMO

During obesity, chronic inflammation of human white adipose tissue (WAT) is associated with metabolic and vascular alterations. Endothelial cells from visceral WAT (VAT-ECs) exhibit a proinflammatory and senescent phenotype and could alter adipocyte functions. We aimed to determine the contribution of VAT-ECs to adipocyte dysfunction related to inflammation and to rescue these alterations by anti-inflammatory strategies. We developed an original three-dimensional setting allowing maintenance of unilocular adipocyte functions. Coculture experiments demonstrated that VAT-ECs provoked a decrease in the lipolytic activity, adipokine secretion, and insulin sensitivity of adipocytes from obese subjects, as well as an increased production of several inflammatory molecules. Interleukin (IL)-6 and IL-1ß were identified as potential actors in these adipocyte alterations. The inflammatory burst was not observed in cocultured cells from lean subjects. Interestingly, pericytes, in functional interactions with ECs, exhibited a proinflammatory phenotype with diminished angiopoietin-1 (Ang-1) secretion in WAT from obese subjects. Using the anti-inflammatory Ang-1, we corrected some deleterious effects of WAT-ECs on adipocytes, improving lipolytic activity and insulin sensitivity and reducing the secretion of proinflammatory molecules. In conclusion, we identified a negative impact of VAT-ECs on adipocyte functions during human obesity. Therapeutic options targeting EC inflammation could prevent adipocyte alterations that contribute to obesity comorbidities.


Assuntos
Adipócitos/fisiologia , Tecido Adiposo/citologia , Angiopoietina-1/farmacologia , Células Endoteliais/fisiologia , Animais , Técnicas de Cultura de Células , Humanos , Inflamação , Insulina , Lipólise/fisiologia , Obesidade/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Endocrinology ; 154(3): 1069-79, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23372021

RESUMO

The hypertrophied white adipose tissue (WAT) during human obesity produces inflammatory mediators, including cytokines (IL-6 and TNFα) and chemokines ([C-C motif] chemokine ligand 2 and IL-8). These inflammatory factors are preferentially produced by the nonadipose cells, particularly the adipose tissue infiltrating macrophages. We identified the chemokine (C-X-C motif) ligand 2 (CXCL2) by a transcriptomic approach. Because CXCL2 could represent a WAT-produced chemokine, we explored its role in obesity-associated inflammation. CXCL2 levels in serum and mRNA in WAT were higher in obese subjects compared with lean ones. CXCL2 secretions were higher in sc and visceral (vis) WAT from obese compared with lean subjects. In vis WAT, CXCL2 mRNA expression was higher in macrophages compared with other WAT cells and positively correlated with the inflammatory macrophage markers TNFα and IL-6. CXCL2 triggered the in vitro adhesion of the neutrophils, its selective cell targets, to endothelial cells (ECs) of vis WAT (vis WAT-ECs). Immunohistological analysis indicated that activated neutrophils were adherent to the endothelium of vis WAT from human obese subjects. Blood neutrophils from obese subjects released high levels of proinflammatory mediators (IL-8, chemokine motif ligand 2 [CCL2], matrix metalloproteinase [MMP] 9, and myeloperoxidase [MPO]). Visceral WAT-ECs, treated by neutrophil-conditioned media prepared from obese subjects, displayed an increase of the expression of inflammatory molecules associated with senescence and angiogenic capacities. To conclude, CXCL2, a WAT-produced chemokine being up-regulated in obesity, stimulates neutrophil adhesion to vis WAT-ECs. Activated neutrophils in obesity may influence vis WAT-ECs functions and contribute to WAT inflammation.


Assuntos
Tecido Adiposo Branco/fisiopatologia , Quimiocina CXCL2/fisiologia , Neutrófilos/fisiologia , Obesidade Mórbida/fisiopatologia , Tecido Adiposo Branco/patologia , Adulto , Estudos de Casos e Controles , Adesão Celular/fisiologia , Quimiocina CXCL2/genética , Quimiocinas/fisiologia , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Feminino , Humanos , Inflamação/etiologia , Inflamação/patologia , Inflamação/fisiopatologia , Mediadores da Inflamação/fisiologia , Ativação de Neutrófilo/fisiologia , Neutrófilos/patologia , Obesidade Mórbida/genética , Obesidade Mórbida/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
J Clin Endocrinol Metab ; 97(9): E1677-85, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22745246

RESUMO

AIMS AND HYPOTHESIS: Mast cells are immune cells known for their role in several inflammatory and fibrotic diseases. Recent works in mice suggest that mast cells could be cellular actors involved in the pathophysiology of obesity, a disease characterized by white adipose tissue (WAT) and systemic inflammation. The aim of the study was to better characterize mast cells in WAT of obese with or without type 2 diabetes and lean subjects as well as to explore the relationship with WAT inflammation and fibrosis. METHODS: Subcutaneous and omental adipose tissue from six lean subjects, 10 obese nondiabetic, and 10 diabetic patients was analyzed by immunohistochemistry and real-time PCR for inflammatory and fibrosis markers. Cytokines secretion of mast cells isolated from WAT and cultured in different conditions was estimated by cytokine array kit. RESULTS: We found that mast cells are activated in human adipose tissue and localized preferentially in fibrosis depots, a local condition that stimulates their inflammatory state. Mast cells with tryptase(+) chymase(+) staining tended to be higher in obese omental adipose tissue. We found positive links between mast cell number and several characteristics of obese WAT including fibrosis, macrophage accumulation, and endothelial cell inflammation. Mast cell number and their inflammatory phenotype are associated with diabetes parameters. CONCLUSION AND INTERPRETATION: Mast cells are cellular actors of WAT inflammation and possibly fibrotic state found in obesity and diabetes. Whether mast cells could be involved in the pathophysiology of diabetes needs additional study as well as the positioning of these cells in driving pathological alterations of WAT in these chronic metabolic diseases.


Assuntos
Tecido Adiposo/patologia , Diabetes Mellitus Tipo 2/patologia , Inflamação/patologia , Mastócitos/patologia , Obesidade Mórbida/patologia , Tecido Adiposo Branco/patologia , Adulto , Biomarcadores/análise , Glicemia/metabolismo , Contagem de Células , Separação Celular , Quimases/química , Células Endoteliais/patologia , Feminino , Fibrose/patologia , Homeostase/fisiologia , Humanos , Imuno-Histoquímica , Lipídeos/sangue , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Obesidade Mórbida/metabolismo , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Triptases/química
7.
Diabetes ; 59(11): 2817-25, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20713683

RESUMO

OBJECTIVE: Fibrosis is a newly appreciated hallmark of the pathological alteration of human white adipose tissue (WAT). We investigated the composition of subcutaneous (scWAT) and omental WAT (oWAT) fibrosis in obesity and its relationship with metabolic alterations and surgery-induced weight loss. RESEARCH DESIGN AND METHODS: Surgical biopsies for scWAT and oWAT were obtained in 65 obese (BMI 48.2 ± 0.8 kg/m(2)) and 9 lean subjects (BMI 22.8 ± 0.7 kg/m(2)). Obese subjects who were candidates for bariatric surgery were clinically characterized before, 3, 6, and 12 months after surgery, including fat mass evaluation by dual energy X-ray absorptiometry. WAT fibrosis was quantified and characterized using quantitative PCR, microscopic observation, and immunohistochemistry. RESULTS: Fibrosis amount, distribution and collagen types (I, III, and VI) present distinct characteristics in lean and obese subjects and with WAT depots localization (subcutaneous or omental). Obese subjects had more total fibrosis in oWAT and had more pericellular fibrosis around adipocytes than lean subjects in both depots. Macrophages and mastocytes were highly represented in fibrotic bundles in oWAT, whereas scWAT was more frequently characterized by hypocellular fibrosis. The oWAT fibrosis negatively correlated with omental adipocyte diameters (R = -0.30, P = 0.02), and with triglyceride levels (R = -0.42, P < 0.01), and positively with apoA1 (R = 0.25, P = 0.05). Importantly, scWAT fibrosis correlated negatively with fat mass loss measured at the three time points after surgery. CONCLUSIONS: Our data suggest differential clinical consequences of fibrosis in human WAT. In oWAT, fibrosis could contribute to limit adipocyte hypertrophy and is associated with a better lipid profile, whereas scWAT fibrosis may hamper fat mass loss induced by surgery.


Assuntos
Tecido Adiposo Branco/patologia , Tecido Adiposo/patologia , Fibrose/patologia , Obesidade Mórbida/patologia , Obesidade/metabolismo , Omento/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/metabolismo , Compostos Azo/metabolismo , Biópsia , Composição Corporal , Colágeno/metabolismo , Colágeno Tipo VI/metabolismo , Feminino , Fibrose/metabolismo , Humanos , Metabolismo dos Lipídeos , Masculino , Obesidade/patologia , Obesidade Mórbida/metabolismo , Omento/metabolismo , Reação em Cadeia da Polimerase/métodos , Procedimentos Cirúrgicos Operatórios/métodos , Magreza/genética , Magreza/metabolismo
8.
Diabetes ; 59(10): 2513-21, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20530742

RESUMO

OBJECTIVE: Growth of white adipose tissue takes place in normal development and in obesity. A pool of adipose progenitors is responsible for the formation of new adipocytes and for the potential of this tissue to expand in response to chronic energy overload. However, factors controlling self-renewal of human adipose progenitors are largely unknown. We investigated the expression profile and the role of activin A in this process. RESEARCH DESIGN AND METHODS: Expression of INHBA/activin A was investigated in three types of human adipose progenitors. We then analyzed at the molecular level the function of activin A during human adipogenesis. We finally investigated the status of activin A in adipose tissues of lean and obese subjects and analyzed macrophage-induced regulation of its expression. RESULTS: INHBA/activin A is expressed by adipose progenitors from various fat depots, and its expression dramatically decreases as progenitors differentiate into adipocytes. Activin A regulates the number of undifferentiated progenitors. Sustained activation or inhibition of the activin A pathway impairs or promotes, respectively, adipocyte differentiation via the C/EBPß-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared with the expression levels in lean subjects. Indeed, activin A levels in adipose progenitors are dramatically increased by factors secreted by macrophages derived from obese adipose tissue. CONCLUSIONS: Altogether, our data show that activin A plays a significant role in human adipogenesis. We propose a model in which macrophages that are located in adipose tissue regulate adipose progenitor self-renewal through activin A.


Assuntos
Ativinas/fisiologia , Tecido Adiposo/citologia , Glucosefosfato Desidrogenase/genética , Obesidade Mórbida/patologia , Células-Tronco/citologia , Magreza/patologia , Ativinas/genética , Ativinas/farmacologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Adulto , Diferenciação Celular , Divisão Celular , RNA Polimerases Dirigidas por DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/genética , Dexametasona/farmacologia , Regulação da Expressão Gênica , Glucosefosfato Desidrogenase/efeitos dos fármacos , Humanos , Obesidade Mórbida/genética , Obesidade Mórbida/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Proteína de Ligação a TATA-Box/efeitos dos fármacos , Proteína de Ligação a TATA-Box/genética
9.
Arterioscler Thromb Vasc Biol ; 30(1): 39-45, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19893003

RESUMO

OBJECTIVE: To examine the role of adipose-produced chemokine, chemokine ligand (CCL) 5, on the recruitment and survival of macrophages in human white adipose tissue (WAT). METHODS AND RESULTS: CCL5 levels measured by enzyme immunoassay in serum and by real-time polymerase chain reaction in WAT were higher in obese compared to lean subjects. CCL5, but not CCL2, secretion was higher in visceral compared to subcutaneous WAT. CCL5 mRNA expression was positively correlated with the inflammatory macrophage markers as CD11b, tumor necrosis factor-alpha, and IL-6 in visceral WAT (n=24 obese subjects), and was higher in macrophages than other WAT cells. We found that CCL5 triggered adhesion and transmigration of blood monocytes to/through endothelial cells of human WAT. Whereas in obese WAT apoptotic macrophages were located around necrotic adipocytes, we demonstrated that CCL5, but not CCL2, protected macrophages from free cholesterol-induced apoptosis via activation of the Akt/Erk pathways. CONCLUSIONS: CCL5 could participate in the inflammation of obese WAT by recruiting blood monocytes and exerting antiapoptotic properties on WAT macrophages. This specific role of CCL5 on macrophage survival with maintenance of their lipid scavenging function should be taken into account for future therapeutic strategies in obesity-related diseases.


Assuntos
Tecido Adiposo Branco/imunologia , Quimiocina CCL5/sangue , Quimiocina CCL5/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Obesidade Mórbida/imunologia , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Apoptose/imunologia , Biópsia , Peso Corporal/imunologia , Antígeno CD11b/metabolismo , Adesão Celular/imunologia , Movimento Celular/imunologia , Sobrevivência Celular/imunologia , Quimiocina CCL5/genética , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Macrófagos/citologia , Obesidade Mórbida/metabolismo , Obesidade Mórbida/patologia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
J Nutr ; 139(11): 2055-60, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19759245

RESUMO

Obesity is a state of chronic low-grade inflammation. Limiting white adipose tissue (WAT) expansion and therefore reducing inflammation could be effective in preventing the progression of obesity and the development of associated complications. We investigated the effects of 1,2-vinyldithiin (1,2-DT), a garlic-derived organosulfur, on the differentiation and inflammatory state of human preadipocytes. Preadipocytes were prepared from subcutaneous adipose tissue of nonobese young women and differentiated in the presence of 1,2-DT. Inflammatory preadipocytes were obtained following treatment with human macrophage-secreted factors. 1,2-DT (100 micromol/L) significantly reduced gene expression of PPARgamma2 (-40%), CCAAT/enhancer binding protein-alpha (-25%), lipoprotein lipase (-22%), leptin (-30%), and adiponectin (-15%). Lipid accumulation was also significantly diminished in preadipocytes differentiated in the presence of 100 micromol/L 1,2-DT (-37%) compared with controls. Furthermore, 100 micromol/L 1,2-DT treatment for 10 d significantly reduced PPARgamma activity (-27%). The protein expression of perilipin and the secretion levels for 2 adipokines, leptin and adiponectin, were significantly diminished in 1,2-DT-cultured preadipocytes (-37, -51, and -43%, respectively). Moreover, the secretion of inflammatory molecules (interleukin-6 and monocyte chemoattractant protein-1) induced by macrophage-secreted factors was partially abolished in 100 micromol/L 1,2-DT-treated preadipocytes (-28 and -25%, respectively). In conclusion, we demonstrated that 1,2-DT, a garlic-derived organosulfur, has antiadipogenic and antiinflammatory actions on human preadipocytes and may be a novel, antiobesity nutraceutical.


Assuntos
Adipócitos/citologia , Fármacos Antiobesidade/farmacologia , Diferenciação Celular/efeitos dos fármacos , Alho , Inflamação/prevenção & controle , Extratos Vegetais/farmacologia , Adipócitos/efeitos dos fármacos , Adiponectina/genética , Anti-Inflamatórios/farmacologia , Proteínas de Transporte , Feminino , Humanos , Leptina/genética , PPAR gama/efeitos dos fármacos , PPAR gama/metabolismo , Perilipina-1 , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/genética , Adulto Jovem
11.
Mol Endocrinol ; 23(1): 11-24, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18945811

RESUMO

White adipose tissue (WAT) in obese humans is characterized by macrophage accumulation the effects of which on WAT biology are not fully understood. We previously demonstrated that macrophage-secreted factors impair preadipocyte differentiation and induce inflammation, and we described the excessive fibrotic deposition in WAT from obese individuals. Microarray analysis revealed significant overexpression of extracellular matrix (ECM) genes in inflammatory preadipocytes. We show here an organized deposition of fibronectin, collagen I, and tenascin-C and clustering of the ECM receptor alpha5 integrin, characterizing inflammatory preadipocytes. Anti-alpha5 integrin-neutralizing antibody decreased proliferation of these cells, underlining the importance of the fibronectin/integrin partnership. Fibronectin-cultured preadipocytes exhibited increased proliferation and expression of both nuclear factor-kappaB and cyclin D1. Small interfering RNA deletion of nuclear factor-kappaB and cyclin D1 showed that these factors link preadipocyte proliferation with inflammation and ECM remodeling. Macrophage-secreted molecules increased preadipocyte migration through an increase in active/phosphorylated focal adhesion kinase. Gene expression and neutralizing antibody experiments suggest that inhibin beta A, a TGF-beta family member, is a major fibrotic factor. Interactions between preadipocytes and macrophages were favored in a three-dimensional collagen I matrix mimicking the fibrotic context of WAT. Cell-rich regions were immunostained for preadipocytes, proliferation, and macrophages in the vicinity of fibrotic WAT from obese individuals. In conclusion, an inflammatory environment leads to profound modifications of the human preadipocyte phenotype, producing fibrotic components with increased migration and proliferation. This phenomenon might play a role in facilitating the constitution of quiescent preadipocyte pools and eventually in the maintenance and aggravation of increased fat mass in obesity.


Assuntos
Adipócitos Brancos/citologia , Adipócitos Brancos/fisiologia , Macrófagos/metabolismo , Sequência de Bases , Adesão Celular , Comunicação Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados , Proteínas da Matriz Extracelular/genética , Fibrose , Expressão Gênica , Genes bcl-1 , Humanos , Inflamação/genética , Inflamação/patologia , Inflamação/fisiopatologia , Modelos Biológicos , Obesidade/patologia , Obesidade/fisiopatologia , Fenótipo , RNA Interferente Pequeno/genética , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética
12.
Genome Biol ; 9(1): R14, 2008 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-18208606

RESUMO

BACKGROUND: Investigations performed in mice and humans have acknowledged obesity as a low-grade inflammatory disease. Several molecular mechanisms have been convincingly shown to be involved in activating inflammatory processes and altering cell composition in white adipose tissue (WAT). However, the overall importance of these alterations, and their long-term impact on the metabolic functions of the WAT and on its morphology, remain unclear. RESULTS: Here, we analyzed the transcriptomic signature of the subcutaneous WAT in obese human subjects, in stable weight conditions and after weight loss following bariatric surgery. An original integrative functional genomics approach was applied to quantify relations between relevant structural and functional themes annotating differentially expressed genes in order to construct a comprehensive map of transcriptional interactions defining the obese WAT. These analyses highlighted a significant up-regulation of genes and biological themes related to extracellular matrix (ECM) constituents, including members of the integrin family, and suggested that these elements could play a major mediating role in a chain of interactions that connect local inflammatory phenomena to the alteration of WAT metabolic functions in obese subjects. Tissue and cellular investigations, driven by the analysis of transcriptional interactions, revealed an increased amount of interstitial fibrosis in obese WAT, associated with an infiltration of different types of inflammatory cells, and suggest that phenotypic alterations of human pre-adipocytes, induced by a pro-inflammatory environment, may lead to an excessive synthesis of ECM components. CONCLUSION: This study opens new perspectives in understanding the biology of human WAT and its pathologic changes indicative of tissue deterioration associated with the development of obesity.


Assuntos
Tecido Adiposo/metabolismo , Matriz Extracelular/patologia , Perfilação da Expressão Gênica , Obesidade/genética , Tecido Adiposo/patologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Cirurgia Bariátrica , Fibrose , Humanos , Inflamação , Obesidade/patologia , Redução de Peso
13.
Endocrinology ; 148(5): 2444-52, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17303666

RESUMO

Estrogens exert multiple genomic effects on adipose tissue through binding to nuclear estrogen receptors. However, there is evidence for additional nongenomic mechanisms whereby estrogens may exert their control on adipose tissue metabolism through rapid activation of various membrane-initiated kinase cascades. Here, we tested rapid effects of estrogens on nitric oxide production in white adipose tissue using 17-beta estradiol (E2) and its membrane impermeant albumin conjugated form (17-beta estradiol hemisuccinate BSA, E2-BSA). We found that both E2 and E2-BSA stimulate nitric oxide synthase (NOS) activity in adipocytes. These effects were abolished by 1) ICI 182-780, a selective estrogen receptor antagonist; 2) wortmannin, an inhibitor of phosphatidylinositol 3-kinase; and 3) N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89) an inhibitor of protein kinase A. In contrast to NOS activation by E2, E2-BSA-induced NOS activity was abolished by UO126, an inhibitor of MAPK kinase/ERK (p42/p44 MAPKs). Immunoblotting studies have shown that both estrogens phosphorylate endothelial NOS (NOS III) on Ser(1179), an effect that is prevented by wortmannin and H89, suggesting that NOS III is the target for estrogen-induced NOS activity. Furthermore, only the E2-BSA-induced NOS III phosphorylation on Ser(1179) was totally abolished by UO126. These results indicate that the signaling cascades involved in adipocyte NOS stimulation by estrogens are different depending on whether estrogens are free or conjugated to albumin and therefore underline the importance of estrogen receptor locations in the nongenomic actions of estrogens in these cells.


Assuntos
Adipócitos Brancos/enzimologia , Estradiol/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Genômica , Masculino , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismo , Serina/metabolismo , Soroalbumina Bovina/farmacologia
14.
Endocrinology ; 148(2): 868-77, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17082259

RESUMO

Obesity is considered a chronic low-grade inflammatory state. The white adipose tissue produces a variety of inflammation-related proteins whose expression is increased in obese subjects. The nonadipose cell fraction, which includes infiltrated macrophages, is a determinant source of inflammation-related molecules within the adipose tissue. Our working hypothesis is that macrophage infiltration affects fat expansion through a paracrine action on adipocyte differentiation. Human primary preadipocytes were then differentiated in the presence of conditioned media obtained from macrophages differentiated from blood monocytes. Preadipocytes treated by macrophage-conditioned medium displayed marked reduction of adipogenesis as assessed by decreased cellular lipid accumulation and reduced gene expression of adipogenic and lipogenic markers. In addition to this effect, the activation of macrophages by lipopolysaccharides stimulated nuclear factor kappaB signaling, increased gene expression and release of proinflammatory cytokines and chemokines, and induced preadipocyte proliferation. This phenomenon was associated with increased cyclin D1 gene expression and maintenance of the fibronectin-rich matrix. Anti-TNFalpha neutralizing antibody inhibits the inflammatory state of preadipocytes positioning TNFalpha as an important mediator of inflammation in preadipocytes. Strikingly, conditioned media produced by macrophages isolated from human adipose tissue exerted comparable effects with activated macrophages, i.e. decreased adipogenesis and increased inflammatory state in the preadipocytes. These data show that macrophage-secreted factors inhibit the formation of mature adipocytes, suggesting possible role in limiting adipose tissue expansion in humans.


Assuntos
Adipócitos/patologia , Adipogenia , Inflamação/fisiopatologia , Macrófagos/metabolismo , Células-Tronco/patologia , Adipogenia/efeitos dos fármacos , Tecido Adiposo/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Meios de Cultura/farmacologia , Ciclina D1/genética , Citocinas/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo
15.
Endocrinology ; 147(10): 4950-9, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16825321

RESUMO

We previously showed that the cysteine protease cathepsin S (CTSS), known to degrade several components of the extracellular matrix (ECM), is produced by human adipose cells and increased in obesity. Because ECM remodeling is a key process associated with adipogenesis, this prompted us to assess the potential role of CTSS to promote preadipocyte differentiation. Kinetic studies in primary human preadipocytes revealed a modest increase in CTSS gene expression and secretion at the end of differentiation. CTSS activity was maximal in preadipocyte culture medium but decreased thereafter, fitting with increased release of the CTSS endogenous inhibitor, cystatin C, during differentiation. Inhibition of CTSS activity by an exogenous-specific inhibitor added along the differentiation, resulted in a 2-fold reduction of lipid content and expression of adipocyte markers in differentiated cells. Conversely, the treatment of preadipocytes with human recombinant CTSS increased adipogenesis. Moreover, CTSS supplementation in preadipocyte media markedly reduced the fibronectin network, a key preadipocyte-ECM component, the decrease of which is required for adipogenesis. Using immunohistochemistry on serial sections of adipose tissue of obese subjects, we showed that adipose cells staining positive for CTSS are mainly located in the vicinity of fibrosis regions containing fibronectin. Herein we propose that CTSS may promote human adipogenesis, at least in part, by degrading fibronectin in the early steps of differentiation. Taken together, these results indicate that CTSS released locally by preadipocytes promotes adipogenesis, suggesting a possible contribution of this protease to fat mass expansion in obesity.


Assuntos
Adipócitos/enzimologia , Adipócitos/fisiologia , Catepsinas/fisiologia , Fibronectinas/metabolismo , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/fisiologia , Western Blotting , Catepsinas/biossíntese , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Cistatina C , Cistatinas/metabolismo , Matriz Extracelular/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Cinética , RNA/análise , RNA/biossíntese , Células-Tronco/efeitos dos fármacos
16.
J Clin Endocrinol Metab ; 91(3): 1042-7, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16394095

RESUMO

CONTEXT: Human adipose tissue produces several adipokines, including the newly identified protein cathepsin S (CTSS), a cysteine protease involved in the pathogenesis of atherosclerosis. Obesity is characterized by high levels of CTSS in the circulation and in sc white adipose tissue (scWAT). OBJECTIVE: We investigated the effect of surgery-induced weight loss on circulating CTSS and its protein expression in scWAT. DESIGN: Fifty morbidly obese women before and 3 months after surgery and 10 healthy lean women were studied. We analyzed the relationships between circulating CTSS and clinical and biological parameters. Immunohistochemistry of the CTSS protein variations in scWAT was performed. RESULTS: Weight loss decreased by 42% (P < 0.0001) the circulating CTSS levels, which correlated with changes in body weight (P = 0.03). We observed a significant decrease in CTSS enzymatic activity by 25% after weight loss (P = 0.001). Adipose tissue CTSS content was reduced by 30% (P = 0.002) after surgery. The variations in CTSS expression in scWAT after surgery correlated with changes in circulating CTSS serum levels (P = 0.03). Most of the correlations between CTSS and clinical and biological parameters disappeared after adjustment for body mass index, emphasizing the strong link between CTSS and corpulence in humans. CONCLUSIONS: Changes in CTSS scWAT might contribute to serum variations in CTSS during weight loss. The decrease in CTSS concentrations in the circulation may contribute to vascular improvement in obese subjects after weight loss.


Assuntos
Tecido Adiposo/enzimologia , Catepsinas/metabolismo , Obesidade Mórbida/sangue , Obesidade Mórbida/cirurgia , Redução de Peso , Adulto , Vasos Sanguíneos/enzimologia , Índice de Massa Corporal , Peso Corporal , Catepsinas/sangue , Técnicas de Cultura de Células , Feminino , Humanos , Obesidade Mórbida/fisiopatologia , Valores de Referência , Magreza
17.
Mol Pharmacol ; 68(3): 885-94, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15976035

RESUMO

We have developed two bioluminescence resonance energy transfer (BRET)-based approaches to monitor 1) ligand-induced conformational changes within partially purified insulin-like growth factor-1 (IGF-1) receptors (IGF1R) and 2) IGF1R interaction with a substrate-trapping mutant of protein tyrosine phosphatase 1B (PTP1B-D181A) in living cells. In the first assay, human IGF1R fused to Renilla reniformis luciferase (Rluc) or yellow fluorescent protein (YFP) were cotransfected in human embryonic kidney (HEK)-293 cells. The chimeric receptors were then partially purified by wheat germ lectin chromatography, and BRET measurements were performed in vitro. In the second assay, BRET measurements were performed on living HEK-293 cells cotransfected with IGF1R-Rluc and YFP-PTP1B-D181A. Ligand-induced conformational changes within the IGF1R and interaction of the IGF1R with PTP1B could be detected as an energy transfer between Rluc and YFP. Dose-response experiments with IGF-1, IGF-2, and insulin demonstrated that the effects of these ligands on BRET correlate well with their known pharmacological properties toward the IGF1R. Inhibition of IGF1R autophosphorylation by the tyrphostin AG1024 (3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile) resulted in the inhibition of IGF1-induced BRET signal between the IGF1R and PTP1B. In addition, an anti-IGF1R antibody known to inhibit the biological effects of IGF-1 inhibited ligand-induced BRET signal within the IGF1R, as well as between IGF1R and PTP1B. This inhibition of BRET signal paralleled the inhibition of the ligand-induced autophosphorylation of the IGF1R by this antibody. In conclusion, these BRET-based assays permit 1) the rapid evaluation of the effects of agonists or inhibitory molecules on IGF1R activation and 2) the analysis of the regulation of IGF1R-PTP1B interaction in living cells.


Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Receptor IGF Tipo 1/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Transferência de Energia , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Medições Luminescentes , Proteínas Luminescentes/genética , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Receptor IGF Tipo 1/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
18.
FASEB J ; 19(11): 1540-2, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15985526

RESUMO

The molecular mechanisms by which obesity increases the risk of cardiovascular diseases are poorly understood. The purpose of this study was to identify candidate biomarkers overexpressed in adipose tissue of obese subjects that could link expanded fat mass to atherosclerosis. We compared gene expression profile in subcutaneous adipose tissue (scWAT) of 28 obese and 11 lean subjects using microarray technology. This analysis identified 240 genes significantly overexpressed in scWAT of obese subjects. The genes were then ranked according to the correlation between gene expression and body mass index (BMI). In this list, the elastolytic cysteine protease cathepsin S was among the highly correlated genes. RT-PCR and Western blotting confirmed the increase in cathepsin S mRNA (P=0.006) and protein (P<0.05) in obese scWAT. The circulating concentrations of cathepsin S were also significantly higher in obese than in nonobese subjects (P<0.0001). Both cathepsin S mRNA in scWAT and circulating levels were positively correlated with BMI, body fat, and plasma triglyceride levels. In addition, we show that the proinflammatory factors, lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha increase cathepsin S secretion in human scWAT explants. This study identifies cathepsin S as a novel marker of adiposity. Since this enzyme has been implicated in the development of atherosclerotic lesions, we propose that cathepsin S represents a molecular link between obesity and atherosclerosis.


Assuntos
Tecido Adiposo/metabolismo , Adiposidade , Aterosclerose/etiologia , Catepsinas/genética , Adipócitos/metabolismo , Biomarcadores , Catepsinas/biossíntese , Catepsinas/sangue , Proteínas da Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Músculo Liso Vascular/química , Obesidade/complicações , Obesidade/metabolismo , RNA Mensageiro/análise
19.
Mol Pharmacol ; 67(4): 1206-13, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15630078

RESUMO

The interactions between the insulin receptor and the two highly homologous receptor-like protein tyrosine phosphatases (PTPase) PTPalpha and PTPepsilon were studied in living cells by using bioluminescence resonance energy transfer. In human embryonic kidney 293 cells expressing the insulin receptor fused to luciferase and substrate-trapping mutants of PTPalpha or PTPepsilon fused to the fluorescent protein Topaz, insulin induces an increase in resonance energy transfer that could be followed in real time in living cells. Insulin effect could be detected at very early time points and was maximal less than 2 min after insulin addition. Bioluminescence resonance energy-transfer saturation experiments indicate that insulin does not stimulate the recruitment of protein tyrosine phosphatase molecules to the insulin receptor but rather induces conformational changes within preassociated insulin receptor/protein tyrosine phosphatase complexes. Physical preassociation of the insulin receptor with these protein tyrosine phosphatases at the plasma membrane, in the absence of insulin, was also demonstrated by chemical cross-linking with a non-cell-permeable agent. These data provide the first evidence that PTPalpha and PTPepsilon associate with the insulin receptor in the basal state and suggest that these protein tyrosine phosphatases may constitute important negative regulators of the insulin receptor tyrosine kinase activity by acting rapidly at the plasma membrane level.


Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Receptor de Insulina/metabolismo , Células Cultivadas , Humanos , Insulina/farmacologia , Medições Luminescentes , Conformação Proteica , Proteínas Tirosina Fosfatases/química , Receptor de Insulina/química , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores
20.
EMBO Rep ; 4(3): 313-9, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12634852

RESUMO

The dynamics of the interaction of the insulin receptor with a substrate-trapping mutant of protein-tyrosine phosphatase 1B (PTP1B) were monitored in living human embryonic kidney cells using bioluminescence resonance energy transfer (BRET). Insulin dose-dependently stimulates this interaction, which could be followed in real time for more than 30 minutes. The effect of insulin on the BRET signal could be detected at early time-points (30 seconds), suggesting that in intact cells the tyrosine-kinase activity of the insulin receptor is tightly controlled by PTP1B. Interestingly, the basal (insulin-independent) interaction of the insulin receptor with PTP1B was much weaker with a soluble form of the tyrosine-phosphatase than with the endoplasmic reticulum (ER)-targeted form. Inhibition of insulin-receptor processing using tunicamycin suggests that the basal interaction occurs during insulin-receptor biosynthesis in the ER. Therefore, localization of PTP1B in this compartment might be important for the regulation of insulin receptors during their biosynthesis.


Assuntos
Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Células Cultivadas , DNA Complementar/genética , Humanos , Rim , Cinética , Medições Luminescentes , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/genética , Receptor de Insulina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Domínios de Homologia de src
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