Characterization of the Human Visceral Adipose Tissue Secretome

Alvarez-Llamas, Gloria; Szalowska, Ewa; Vries, Marcel P. de; Weening, Desireé; Landman, Karloes; Hoek, Annemieke; Wolffenbuttel, Bruce H. R.; Roelofsen, Han; Vonk, Roel J.

doi:10.1074/mcp.m600265-mcp200

Cited by 209 publications

(200 citation statements)

References 39 publications

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“…At this point, three dishes also received 60 nM insulin. The tissues were cultured for an additional 72 h. In previous experiments, this period showed sufficient label incorporation in secreted proteins (11). After the incubation was completed, media were collected and prepared separately for LC-MS/MS analyses.…”

Section: Resultsmentioning

confidence: 99%

“…The adipose tissue culture protocol is described in Alvarez-Llamas et al (11). Briefly adipose tissue explants were transported from the operating room to the laboratory in transport buffer (PBS, 5.5 mM glucose, 50 g/ml gentamicin) at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Also the relevance of identified proteins may be difficult to assess if the source (secreted, serum, or intracellular) is not clear. We have shown previously that incorporation of 13 C-labeled lysine in secreted proteins can be used for validation of the origin (tissue or serum) of detected proteins (11). Proteins that contain label are synthesized by the tissue and are not derived from serum.…”

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confidence: 99%

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Comparison of Isotope-labeled Amino Acid Incorporation Rates (CILAIR) Provides a Quantitative Method to Study Tissue Secretomes

Roelofsen

Dijkstra

Weening

et al. 2009

Molecular & Cellular Proteomics

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Adipose tissue is an endocrine organ involved in regulation of whole-body energy metabolism via storage of lipids and secretion of various peptide hormones (adipokines). We previously characterized the adipose tissue secretome and showed that [ 13 C]lysine incorporation into secreted proteins can be used to determine the origin of identified proteins. In the present study we determined the effect of insulin on the secretome by comparing incorporation rates of 13 C-labeled lysine in the presence and absence of insulin. Human visceral adipose tissue from one patient was divided over six dishes. After subsequent washes to remove serum proteins, [13 C]lysinecontaining medium was added. Three dishes also received 60 nM insulin. The other three were controls. After 72 h of culture, media were collected and processed separately, involving concentration by ultrafiltration and fractionation by SDS-PAGE followed by in-gel digestion of excised bands and LC-MS/MS analyses. The obtained spectra were used for database searching and calculation of heavy/light ratios. The three control data sets shared 342 proteins of which 156 were potentially secreted and contained label. The three insulin-derived data sets shared 361 proteins of which 141 were potentially secreted and contained label. After discarding secreted proteins with very low label incorporation, 121 and 113 proteins remained for control and insulin data sets, respectively. The average coefficient of variation for control triplicates was 10.0% and for insulin triplicates was 18.3%. By comparing heavy/light ratios in the absence and presence of insulin we found 24 up-regulated proteins and four down-regulated proteins, and 58 proteins showed no change. Proteins involved in the endoplasmic reticulum stress response and in extracellular matrix remodeling were up-regulated by insulin. In conclusion, comparison of isotope-labeled amino acid incorporation rates (CILAIR) allows quantitative assessment of changes in protein secretion without the need for 100% label in-

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Comparison of Isotope-labeled Amino Acid Incorporation Rates (CILAIR) Provides a Quantitative Method to Study Tissue Secretomes

Roelofsen

Dijkstra

Weening

et al. 2009

Molecular & Cellular Proteomics

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“…[20][21][22] Other studies have applied SILAC to study cell function or tissue characterization. [23][24][25] Our study is the first one applying SILAC in proteomic studies of cartilage or chondrocytes. The validation experiments by Western blot included in our study were in full accordance with the results achieved by SILAC proteomics, thus further confirming the robustness and reproducibility of the method.…”

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confidence: 99%

Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

Polacek

Bruun

Johansen

et al. 2010

Journal Orthopaedic Research

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ABSTRACT:The main goal of our study was to analyze and compare the profiles of secreted proteins from adult human articular chondrocytes in monolayers, and cartilage explants in culture, using a de novo protein labeling approach. Stable isotope labeling of proteins in culture was used to differentiate between chondrocyte-derived proteins and other preexisting matrix-derived components, or proteins coming from serum or synovial fluids. Proteins in culture supernatants were resolved by one-dimensional SDS-PAGE electrophoresis, and analyzed in tandem with LC/MS-MS (liquid chromatography/double mass spectrometry). Results from stable isotope labeling with amino acids in cell culture (SILAC) were validated by specific immunoblotting of four relevant proteins identified in the secretion media. After 8-10 days of culture, over 90% of proteins secreted during monolayer growth contained 13 C 6 -Arg and 13 C 6 -Lys. Nonlabeled proteins corresponded mostly to plasma-associated proteins, indicating background contamination of medium with serum remnants. The majority of the secreted proteins in 2D cultures were extracellular matrix components and matrix regulators, along with some inflammatory agents and metabolic enzymes. In explants, only 25%-30% of proteins were labeled with heavy amino acids, corresponding to matrix regulators and carrier molecules. Nonlabeled proteins corresponded primarily to structural matrix components. In qualitative terms, all labeled proteins coming from cartilage explants were also found in chondrocytes supernatants. In summary, our results show differences in the labeling pattern of proteins found in supernatants from explants and monolayers. Most proteins found in the media of explants were subproducts of matrix turnover rather than newly synthesized. To our knowledge, this study is the first one so far applying SILAC technology in the context of cartilage and chondrocytes physiology. ß

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“…In this case, detection of low-abundance secreted proteins may be impeded by the presence of high-abundance serum-contaminating proteins and intracellular ones, so that it is important to validate the identified proteins as truly secreted by the tissue of interest. As a valid approach, adipose tissue explants were cultured in presence of stable-isotope labeled aminoacids and, in this way, proteins that incorporate the label are validated as derived from adipose tissue and not from an external source [49]. Such approach could be extended to other particular studies.…”

Section: The Secretome Of Atheroma Plaquesmentioning

confidence: 99%

Vascular proteomics

Vivanco

Mas

Dardé

et al. 2007

Proteomics Clinical Apps

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The characterization of patients with acute coronary syndromes (ACS) at the molecular and cellular levels provides a novel vision for understanding the pathological and clinical expression of the disease. Recent advances in proteomic technologies permit the evaluation of systematic changes in protein expression in many biological systems and have been extensively applied to cardiovascular diseases (CVD). The cardiovascular system is in permanent intimate contact with blood, making blood-based biomarker discovery a particularly worthwhile approach. Thus, proteomics can potentially yield novel biomarkers reflecting CVD, establish earlier detection strategies, and monitor response to therapy. Here we review the different proteomic strategies used in the study of atherosclerosis and the novel proteins differentially expressed and secreted by atherosclerotic lesions which constitute novel potential biomarkers (HSP-27, Cathepsin D). Special attention is paid to MS-Imaging of atheroma plaque and the generation, for the first time, of 2-D images of lipids, showing the distribution of these molecules in the different areas of the atherosclerotic lesions. In addition new potential biomarkers have been identified in plasma (amyloid A1a, transtherytin), circulating cells (protein profile in monocytes from ACS patients) and individual cells constituents of atheroma plaques (endothelial, VSMC, macrophages) which provide novel insights into vascular pathophysiology.

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Characterization of the Human Visceral Adipose Tissue Secretome

Cited by 209 publications

References 39 publications

Comparison of Isotope-labeled Amino Acid Incorporation Rates (CILAIR) Provides a Quantitative Method to Study Tissue Secretomes

Comparison of Isotope-labeled Amino Acid Incorporation Rates (CILAIR) Provides a Quantitative Method to Study Tissue Secretomes

Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

Vascular proteomics

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