Characterization of the Human Visceral Adipose Tissue Secretome*□S

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-[C6, N2]lysine to validate the origin of the identified proteins (adipose tissueor serum-derived). In total, 259 proteins were identified with >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. Molecular & Cellular Proteomics 6: 589–600, 2007. Adipose tissue is a key organ for the regulation of energy metabolism. Besides its function as an energy storage depot in the form of triglycerides, adipose tissue secretes a variety of peptide and protein hormones (adipokines) involved in the regulation of energy metabolism such as leptin, adiponectin, visfatin, retinol-binding protein-4, adipsin, tumor necrosis factor (TNF) and interleukin 6 (1–3). Dysregulation of the production of adipokines and free fatty acids contributes to the pathogenesis of diseases associated with energy metabolism such as insulin resistance, metabolic syndrome, and type 2 diabetes. Especially visceral adipose tissue has been implicated in the development of these diseases (2–4). Therefore, more insight into the visceral adipose tissue secretome will contribute to a better understanding of its role in energy metabolism and related diseases and may lead to the discovery of unknown peptides/proteins involved in regulation of energy metabolism and new targets for therapy. Besides adipocytes, adipose tissue contains endothelial cells, macrophages, and fibroblasts (stromal fraction) that may modulate the overall peptide and protein secretion pattern of the tissue via cross-talk between the different cell types. For example, factors secreted by macrophages have been shown to induce changes in the secretion of adipokines, free fatty acids, and glucose uptake by 3T3-L1 adipocytes (5). These interactions between cells from the stromal fraction and adipocytes are necessary for physiological functions of adipose tissue, and deregulation of this cross-talk is regarded as an important mechanism leading to insulin resistance and type 2 diabetes (6–9). Therefore, the tissue secretome provides more relevant information for the in vivo situation than the adipocyte cell secretome. To date, no studies have been published on the adipose tissue secretome. Several studies investigated the human (10), mouse (11–14), and rat (15) adipose tissue proteome, mostly using a two-dimensional gel electrophoresis approach. Celis et al. (16) analyzed the human mammary adipose tissue proteome. The secretome from adipocyte cells has been investigated by Kratchmarova et al. (17) and Wang