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1.
J Biol Chem ; 289(28): 19279-93, 2014 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-24876382

RESUMO

Peroxisome proliferator-activated receptors (PPARs) play major roles in the regulation of hepatic lipid metabolism through the control of numerous genes involved in processes such as lipid uptake and fatty acid oxidation. Here we identify hypoxia-inducible lipid droplet-associated (Hilpda/Hig2) as a novel PPAR target gene and demonstrate its involvement in hepatic lipid metabolism. Microarray analysis revealed that Hilpda is one of the most highly induced genes by the PPARα agonist Wy14643 in mouse precision cut liver slices. Induction of Hilpda mRNA by Wy14643 was confirmed in mouse and human hepatocytes. Oral dosing with Wy14643 similarly induced Hilpda mRNA levels in livers of wild-type mice but not Ppara(-/-) mice. Transactivation studies and chromatin immunoprecipitation showed that Hilpda is a direct PPARα target gene via a conserved PPAR response element located 1200 base pairs upstream of the transcription start site. Hepatic overexpression of HILPDA in mice via adeno-associated virus led to a 4-fold increase in liver triglyceride storage, without any changes in key genes involved in de novo lipogenesis, ß-oxidation, or lipolysis. Moreover, intracellular lipase activity was not affected by HILPDA overexpression. Strikingly, HILPDA overexpression significantly impaired hepatic triglyceride secretion. Taken together, our data uncover HILPDA as a novel PPAR target that raises hepatic triglyceride storage via regulation of triglyceride secretion.


Assuntos
Lipogênese/fisiologia , Fígado/metabolismo , Proteínas de Neoplasias/metabolismo , PPAR alfa/metabolismo , Triglicerídeos/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Linhagem Celular , Humanos , Lipogênese/efeitos dos fármacos , Fígado/citologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , PPAR alfa/genética , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Elementos de Resposta/fisiologia , Triglicerídeos/genética
2.
BMC Genomics ; 16: 822, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26482353

RESUMO

BACKGROUND: The transcription factor farnesoid X receptor (FXR) governs bile acid and energy homeostasis, is involved in inflammation, and has protective functions in the liver. In the present study we investigated the effect of Fxr deficiency in mouse precision cut liver slices (PCLS) exposed to a model hepatotoxicant cyclosporin A (CsA). It was anticipated that Fxr deficiency could aggravate toxicity of CsA in PCLS and pinpoint to novel genes/processes regulated by FXR. METHODS: To test this hypothesis, PCLS obtained from livers of wild type mice (WT-PCLS) and Fxr-knockout mice (FXRKO-PCLS) were treated with 40 µM CsA for 24 h and 48 h. ATP and histological assays were applied to assess the viability of PCLS. DNA microarrays combined with bioinformatics analysis were used to identify genes and processes that were affected by CsA in WT-PCLS and/or FXRKO-PCLS. In addition, WT-PCLS and FXRKO-PCLS were exposed to the endogenous FXR ligand chenodeoxycholic acid (CDCA) and subjected to q-PCR to determine whether subsets of known FXR-targets and the identified genes were regulated upon FXR activation in an FXR-dependent manner. RESULTS: No difference in viability was observed between WT-PCLS and FXRKO-PCLS upon CsA treatment. Transcriptomics data analysis revealed that CsA significantly upregulated stress-response and inflammation and significantly downregulated processes involved in lipid and glucose metabolism in WT-PCLS and FXRKO-PCLS. However, only in FXRKO-PCLS, CsA upregulated additional pro-inflammatory genes and downregulated genes related to mitochondrial functions. Furthermore, only in WT-PCLS, CDCA upregulated a subset of known FXR-target genes as well as the regulator of inflammation and mitochondrial functions peroxisome proliferator- activated receptor delta (Ppar delta). CONCLUSIONS: Although FXR governs energy metabolism, no major differences in response to CsA could be observed between WT-PCLS and FXRKO-PCLS in regulation of processes involved in lipid and glucose metabolism. This finding indicates that CsA does not directly affect FXR functions in relation to the above mentioned processes. However, the more pronounced induction of pro-inflammatory genes and the downregulation of genes involved in mitochondrial functions only in FXRKO-PCLS suggest that FXR deficiency aggravates CsA-induced inflammation and impairs mitochondrial functions. Therefore, FXR can exert its hepatoprotective functions by controlling inflammation and mitochondrial functions, possibly involving an FXR-PPAR delta cross-talk.


Assuntos
Ciclosporina/toxicidade , Fígado/metabolismo , Mitocôndrias/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Ácidos e Sais Biliares/metabolismo , Metabolismo Energético/genética , Glucose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , PPAR delta/genética , PPAR delta/metabolismo , Receptores Citoplasmáticos e Nucleares/deficiência , Ativação Transcricional/genética
3.
BMC Genomics ; 15: 1106, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25511156

RESUMO

BACKGROUND: The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that regulates lipid catabolism and inflammation and is hepatocarcinogenic in rodents. It is presumed that the functions of PPARα in liver depend on cross-talk between parenchymal (hepatocytes) and non-parenchymal (Kupffer and endothelial cells) fractions as well as inter-organ interactions. In order to determine how cellular composition and inter-organ interactions influence gene expression upon pharmacological activation of PPARα, we performed a meta-analysis of transcriptomics data obtained from mouse hepatocytes (containing only the parenchymal fraction), mouse liver slices (containing both fractions), and mouse livers exposed to a PPARα agonist. The aim was to obtain a comprehensive view of common and model-specific PPARα-dependent genes and biological processes to understand the impact of cross-talk between parenchymal and non-parenchymal fractions as well as the effect of inter-organ interactions on the hepatic PPARα transcriptome.To this end we analyzed microarray data of experiments performed in mouse primary hepatocytes treated with the PPARα agonist Wy14643 for 6 or 24 h (in vitro), mouse precision cut liver slices treated with Wy14643 for 24 h (ex vivo), and livers of wild type and Ppara knockout mice treated with Wy14643 for 6 h or 5 days (in vivo). RESULTS: In all models, activation of PPARα significantly altered processes related to various aspects of lipid metabolism. In ex vivo and in vivo models, PPARα activation significantly regulated processes involved in inflammation; these processes were unaffected in hepatocytes. Only in vivo models showed significant regulation of genes involved in coagulation, carcinogenesis, as well as vesicular trafficking and extracellular matrix. CONCLUSIONS: PPARα-dependent regulation of genes/processes involved in lipid metabolism is mostly independent of the presence of non-parenchymal cells or systemic factors, as it was observed in all liver models. PPARα-dependent regulation of inflammatory genes requires the presence of non-parenchymal cells, as it was observed only ex vivo and in vivo. However, the full spectrum of PPARα biology at the level of lipid metabolism, immunity, carcinogenesis, as well as novel aspects of PPARα signaling such as coagulation, vesicular trafficking and the extracellular matrix, seems to require systemic factors, as it was observed exclusively in vivo.


Assuntos
Hepatócitos/metabolismo , PPAR alfa/genética , Animais , Células Cultivadas , Feminino , Hepatócitos/citologia , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Modelos Biológicos , PPAR alfa/metabolismo , Pirimidinas/farmacologia , Transcriptoma/efeitos dos fármacos
4.
PLoS One ; 9(1): e86795, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489787

RESUMO

Although drug induced steatosis represents a mild type of hepatotoxicity it can progress into more severe non-alcoholic steatohepatitis. Current models used for safety assessment in drug development and chemical risk assessment do not accurately predict steatosis in humans. Therefore, new models need to be developed to screen compounds for steatogenic properties. We have studied the usefulness of mouse precision-cut liver slices (PCLS) as an alternative to animal testing to gain more insight into the mechanisms involved in the steatogenesis. To this end, PCLS were incubated 24 h with the model steatogenic compounds: amiodarone (AMI), valproic acid (VA), and tetracycline (TET). Transcriptome analysis using DNA microarrays was used to identify genes and processes affected by these compounds. AMI and VA upregulated lipid metabolism, whereas processes associated with extracellular matrix remodelling and inflammation were downregulated. TET downregulated mitochondrial functions, lipid metabolism, and fibrosis. Furthermore, on the basis of the transcriptomics data it was hypothesized that all three compounds affect peroxisome proliferator activated-receptor (PPAR) signaling. Application of PPAR reporter assays classified AMI and VA as PPARγ and triple PPARα/(ß/δ)/γ agonist, respectively, whereas TET had no effect on any of the PPARs. Some of the differentially expressed genes were considered as potential candidate biomarkers to identify PPAR agonists (i.e. AMI and VA) or compounds impairing mitochondrial functions (i.e. TET). Finally, comparison of our findings with publicly available transcriptomics data showed that a number of processes altered in the mouse PCLS was also affected in mouse livers and human primary hepatocytes exposed to known PPAR agonists. Thus mouse PCLS are a valuable model to identify early mechanisms of action of compounds altering lipid metabolism.


Assuntos
Amiodarona/farmacologia , Inibidores Enzimáticos/farmacologia , Fígado Gorduroso/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Tetraciclina/farmacologia , Ácido Valproico/farmacologia , Animais , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , PPAR alfa/agonistas , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , PPAR beta/agonistas , PPAR beta/genética , PPAR beta/metabolismo , Técnicas de Cultura de Tecidos , Transcriptoma
5.
BMC Med Genomics ; 6: 39, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24112857

RESUMO

BACKGROUND: Unexpected cholestasis substantially contributes to drug failure in clinical trials. Current models used for safety assessment in drug development do not accurately predict cholestasis in humans. Therefore, it is of relevance to develop new screening models that allow identifying drugs with cholestatic properties. METHODS: We employed mouse precision cut liver slices (PCLS), which were incubated 24 h with two model cholestatic compounds: cyclosporin A (CsA) and chlorpromazine (CPZ). Subsequently, transcriptome analysis using DNA microarrays and q-PCR were performed to identify relevant biological processes and biomarkers. Additionally, histology was carried out and levels of triglycerides (TG) and bile acids (BA) were measured. To verify the ex vivo mouse data, these were compared with publically available human data relevant for cholestasis. RESULTS: Whole genome gene expression analysis showed that CsA up-regulated pathways related to NF-κB, ER stress and inflammation. Both CsA and CPZ down-regulated processes related to extracellular matrix (ECM) remodelling, BA homeostasis, Fxr signalling, and energy metabolism. The differential expression of a number of characteristic genes (e.g. Abcg5, Abcg8, Klf15, and Baat) could be confirmed by q-PCR. Histology revealed that CsA but not CPZ induced "ballooning" of hepatocytes. No effects on TG and BA levels were observed after incubation of PCLS with CsA and CPZ. A substantial number of processes altered in CsA- and CPZ-treated mouse PCLS ex vivo was also found to be affected in liver biopsies of cholestatic patients. CONCLUSION: The present study demonstrated that mouse PCLS can be used as a tool to identify mechanisms of action of cholestatic model compounds. The induction of general stress responses and down-regulated Fxr signalling could play a role in the development of drug induced cholestasis. Importantly, comparative data analysis showed that the ex vivo mouse findings are also relevant for human pathology. Moreover, this work provides a set of genes that are potentially useful to assess drugs for cholestatic properties.


Assuntos
Colestase/induzido quimicamente , Citotoxinas/toxicidade , Regulação para Baixo/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Animais , Biomarcadores/metabolismo , Clorpromazina/toxicidade , Ciclosporina/toxicidade , Perfilação da Expressão Gênica , Humanos , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos
6.
Toxicol In Vitro ; 27(5): 1513-24, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23531554

RESUMO

Precision cut liver slices (PCLSs) are widely used as a model to study hepatotoxicity. For culturing of PCLS diverse protocols are used which could affect slices viability and results. We aimed to identify the most optimal culture protocol for mouse PCLS. Slices were cultured for 24h under different concentrations of serum, glucose, insulin, and oxygen. Thereafter, slices viability was assessed by biochemical methods. Transcriptome analysis was performed to identify changes introduced by culture at different oxygen concentrations (20%, 40%, 60%, and 80% of oxygen). Medium composition did not affect the slices viability. Although metabolic competence was unaffected by oxygen concentrations, culturing at 80% of oxygen yielded slices with the best biochemical characteristics. The comparison of uncultured vs. cultured slices revealed 2524 genes to be differentially expressed. Genes involved in drug metabolism, peroxisomal and mitochondrial functions were down-regulated while several adaptive/stress response processes were up-regulated. Moreover, 80% of oxygen was the most favorable condition with respect to maintenance of expression of genes involved in drug and energy metabolism. The outcome of this study indicates that mouse PCLS are a valuable tool in research on hepatic functions and toxicity, particularly if they are cultured under a controlled oxygen concentration of 80%.


Assuntos
Fígado/metabolismo , Oxigênio/farmacologia , Técnicas de Cultura de Tecidos , Trifosfato de Adenosina/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Testosterona/metabolismo , Triglicerídeos/metabolismo
7.
Mol Cell Biol ; 33(7): 1303-16, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23339868

RESUMO

Angiopoietin-like protein 4 (ANGPTL4/FIAF) has been proposed as a circulating mediator between the gut microbiota and fat storage. Here, we show that transcription and secretion of ANGPTL4 in human T84 and HT29 colon adenocarcinoma cells is highly induced by physiological concentrations of short-chain fatty acids (SCFA). SCFA induce ANGPTL4 by activating the nuclear receptor peroxisome proliferator activated receptor γ (PPARγ), as demonstrated using PPARγ antagonist, PPARγ knockdown, and transactivation assays, which show activation of PPARγ but not PPARα and PPARδ by SCFA. At concentrations required for PPARγ activation and ANGPTL4 induction in colon adenocarcinoma cells, SCFA do not stimulate PPARγ in mouse 3T3-L1 and human SGBS adipocytes, suggesting that SCFA act as selective PPARγ modulators (SPPARM), which is supported by coactivator peptide recruitment assay and structural modeling. Consistent with the notion that fermentation leads to PPAR activation in vivo, feeding mice a diet rich in inulin induced PPAR target genes and pathways in the colon. We conclude that (i) SCFA potently stimulate ANGPTL4 synthesis in human colon adenocarcinoma cells and (ii) SCFA transactivate and bind to PPARγ. Our data point to activation of PPARs as a novel mechanism of gene regulation by SCFA in the colon, in addition to other mechanisms of action of SCFA.


Assuntos
Adenocarcinoma/metabolismo , Angiopoietinas/biossíntese , Neoplasias do Colo/metabolismo , Ácidos Graxos Voláteis/metabolismo , PPAR gama/metabolismo , Células 3T3-L1 , Adenocarcinoma/genética , Adipogenia/genética , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Linhagem Celular Tumoral , Colo/metabolismo , Neoplasias do Colo/genética , Células HT29 , Humanos , Inulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/agonistas , PPAR gama/genética , Transcrição Gênica , Ativação Transcricional
8.
Peptides ; 32(5): 938-45, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21334410

RESUMO

GIP receptor knockout mice were shown to be protected from the development of obesity on a high fat diet, suggesting a role of GIP in the development of obesity. In our study we aimed to test the hypothesis if excess of GIP could accelerate development of obesity and to identify GIP gene targets in adipose tissue. Therefore, mice were kept on a chow or a high fat diet and during the last 2 weeks D-Ala(2)-GIP or PBS injections were performed. Afterwards, serum LPL activity and several biochemical parameters (TG, FFA, cholesterol, glucose, insulin, resistin, IL-6, IL-1ß, TNFα, GIP) were measured. Fat tissue was isolated and QPCR was performed for a set of genes involved in energy metabolism and inflammation. A DNA-microarray was used to identify GIP gene targets in adipose tissue of the chow diet group. We found that the D-Ala(2)-GIP injections caused a significant decrease in both body weight and LPL activity compared to controls. Serum biochemical parameters were not affected by D-Ala(2)-GIP, with an exception for resistin and insulin. The set of inflammatory genes were significantly decreased in adipose tissue in the D-Ala(2)-GIP injected animals on a chow diet. A DNA-microarray revealed that APO-genes and CYP-genes were affected by D-Ala(2)-GIP treatment in adipose tissue. These results suggest that the body weight-reducing effect of D-Ala(2)-GIP may be explained by lower LPL activity and insulin serum level. Moreover, the identified GIP candidate gene targets in adipose tissue link GIP action to lipid metabolism exerted by APO and CYP genes.


Assuntos
Peso Corporal/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/farmacologia , Lipase Lipoproteica/sangue , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Gorduras na Dieta/toxicidade , Polipeptídeo Inibidor Gástrico/análogos & derivados , Polipeptídeo Inibidor Gástrico/sangue , Insulina/sangue , Interleucina-6/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipase Lipoproteica/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Resistina/sangue , Fator de Necrose Tumoral alfa/sangue
9.
BMC Med Genomics ; 4: 71, 2011 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-21978410

RESUMO

BACKGROUND: Insulin resistance (IR) is accompanied by chronic low grade systemic inflammation, obesity, and deregulation of total body energy homeostasis. We induced inflammation in adipose and liver tissues in vitro in order to mimic inflammation in vivo with the aim to identify tissue-specific processes implicated in IR and to find biomarkers indicative for tissue-specific IR. METHODS: Human adipose and liver tissues were cultured in the absence or presence of LPS and DNA Microarray Technology was applied for their transcriptome analysis. Gene Ontology (GO), gene functional analysis, and prediction of genes encoding for secretome were performed using publicly available bioinformatics tools (DAVID, STRING, SecretomeP). The transcriptome data were validated by proteomics analysis of the inflamed adipose tissue secretome. RESULTS: LPS treatment significantly affected 667 and 483 genes in adipose and liver tissues respectively. The GO analysis revealed that during inflammation adipose tissue, compared to liver tissue, had more significantly upregulated genes, GO terms, and functional clusters related to inflammation and angiogenesis. The secretome prediction led to identification of 399 and 236 genes in adipose and liver tissue respectively. The secretomes of both tissues shared 66 genes and the remaining genes were the differential candidate biomarkers indicative for inflamed adipose or liver tissue. The transcriptome data of the inflamed adipose tissue secretome showed excellent correlation with the proteomics data. CONCLUSIONS: The higher number of altered proinflammatory genes, GO processes, and genes encoding for secretome during inflammation in adipose tissue compared to liver tissue, suggests that adipose tissue is the major organ contributing to the development of systemic inflammation observed in IR. The identified tissue-specific functional clusters and biomarkers might be used in a strategy for the development of tissue-targeted treatment of insulin resistance in patients.


Assuntos
Tecido Adiposo/metabolismo , Biomarcadores/metabolismo , Inflamação/induzido quimicamente , Inflamação/genética , Lipopolissacarídeos , Fígado/metabolismo , Transcriptoma , Adolescente , Adulto , Células Cultivadas , Análise por Conglomerados , Feminino , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Resistência à Insulina/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Proteômica , Transdução de Sinais/genética
10.
J Clin Endocrinol Metab ; 94(8): 3051-7, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19454585

RESUMO

CONTEXT: Resistin is an adipokine correlated with inflammatory markers and is predictive for cardiovascular diseases. There is evidence that serum resistin levels are elevated in obese patients; however, the role of resistin in insulin resistance and type 2 diabetes remains controversial. OBJECTIVE: We addressed the question of whether inflammation may induce expression of resistin in organs involved in regulation of total body energy metabolism, such as liver and adipose tissue (AT). METHODS: Human liver tissue, sc AT, and omentum were cultured in the absence/presence of lipopolysaccharide (LPS). The resistin and cytokine mRNA and protein expression levels were determined by real-time PCR, ELISA, and Multiplex Technology, respectively. The localization of resistin in human liver was analyzed by immunohistochemistry. RESULTS: Resistin gene and protein expression was significantly higher in liver than in AT. Exposure of human AT and liver tissue in culture to LPS did not alter resistin concentration; however, concentrations of IL-1beta, IL-6, and TNFalpha were significantly increased in these tissues. In liver, resistin colocalizes with markers for Kupffer cells, for a subset of endothelial and fibroblast-like cells. CONCLUSIONS: High level of resistin gene and protein expression in liver compared to AT implies that resistin should not be considered only as an adipokine in humans. LPS-induced inflammation does not affect resistin protein synthesis in human liver and AT. This suggests that elevated serum resistin levels are not indicative for inflammation of AT or liver in a manner similar to known inflammatory markers such as IL-1beta, IL-6, or TNFalpha.


Assuntos
Tecido Adiposo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Resistina/genética , Adolescente , Adulto , Proteína C-Reativa/genética , Citocinas/genética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Omento/metabolismo , Resistina/análise , Regulação para Cima
11.
Biotechnol Prog ; 23(1): 217-24, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17269691

RESUMO

The early factors inducing insulin resistance are not known. Therefore, we are interested in studying the secretome of the human visceral adipose tissue as a potential source of unknown peptides and proteins inducing insulin resistance. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry is a high-throughput proteomics technology to generate peptide and protein profiles (MS spectra). To obtain good quality and reproducible data from SELDI-TOF, many factors in the sample pretreatment and SELDI protocol should be optimized. To identify the optimal combination of factors resulting in the best and the most reproducible spectra, we designed an experiment where factors were varied systematically according to a fractional factorial design. In this study, seven protein chip preparation protocol factors were tested in 32 experiments. The main effects of these factors and their interactions contributing to the best quality spectra were identified by ANOVA. To assess the reproducibility, in a subsequent experiment the eight protocols generating the highest quality spectra were applied to samples in quadruplicates on different chips. This approach resulted in the development of an improved chip protocol, yielding higher quality peaks and more reproducible spectra.


Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , Proteoma/análise , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise Serial de Tecidos/métodos , Análise Fatorial , Humanos , Técnicas de Cultura de Órgãos
12.
Mol Cell Proteomics ; 6(4): 589-600, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17255083

RESUMO

Adipose tissue is an endocrine organ involved in storage and release of energy but also in regulation of energy metabolism in other organs via secretion of peptide and protein hormones (adipokines). Especially visceral adipose tissue has been implicated in the development of metabolic syndrome and type 2 diabetes. Factors secreted by the stromal-vascular fraction contribute to the secretome and modulate adipokine secretion by adipocytes. Therefore, we aimed at the characterization of the adipose tissue secretome rather than the adipocyte cell secretome. The presence of serum proteins and intracellular proteins from damaged cells, released during culture, may dramatically influence the dynamic range of the sample and thereby identification of secreted proteins. Part of the study was therefore dedicated to the influence of the culture setup on the quality of the final sample. Visceral adipose tissue was cultured in five experimental setups, and the quality of resulting samples was evaluated in terms of protein concentration and protein composition. The best setup involved one wash after the 1st h in culture followed by two or three additional washes within an 8-h period, starting after overnight culture. Thereafter tissue was maintained in culture for an additional 48-114 h to obtain the final sample. For the secretome experiment, explants were cultured in media containing L-[(13)C(6),(15)N(2)]lysine to validate the origin of the identified proteins (adipose tissue- or serum-derived). In total, 259 proteins were identified with > or =99% confidence. 108 proteins contained a secretion signal peptide of which 70 incorporated the label and were considered secreted by adipose tissue. These proteins were classified into five categories according to function. This is the first study on the (human) adipose tissue secretome. The results of this study contribute to a better understanding of the role of adipose tissue in whole body energy metabolism and related diseases.


Assuntos
Tecido Adiposo/metabolismo , Proteínas/metabolismo , Metabolismo Energético , Feminino , Humanos , Lisina/metabolismo , Pessoa de Meia-Idade , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Técnicas de Cultura de Tecidos
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