DEPOT-SPECIFIC SECRETOME OF ADIPOSE TISSUE TABLE 4 Summary of numbers of proteins detected and relative abun- dance between visceral and subcutaneous adipose tissue samples WAT explants Endothelial cells Preadipocytes Incorporated label Signal peptide Signal peptide, visceral/subcutaneous ratio 2 Signal peptide, subcutaneous/visceral ratio 2 No signal peptide, visceral/subcutaneous ratio 2 No signal peptide, subcutaneous/visceral ratio 2 % of proteins identiﬁed with signal peptide % of total ion score from signal peptide containing proteins 145 86 51 18 23 22 59 88 66 34 21 5 10 14 52 76 23 22 15 0 1 0 96 99 proteins secreted more abundantly from visceral preadi- pocytes revealed proteins involved in extracellular matrix production. This, together with our ﬁnding that VAT secretes pro-angiogenic factors, suggests that visceral preadipocytes are better adapted for rapid adipose tissue expansion than subcutaneous preadipocytes. The second hypothesis is that VAT secretes proinﬂammatory cyto- kines. This is consistent with recent data that VAT, partic- ularly in obesity, develops chronic low-grade inﬂammation with macrophage inﬁltration. This is associated with insu- Macroglobulins, collagen, gelsolin, decorin, thrombospondin Collagens, cathepsin B, osteonectin, TIMP, PAI-1, C1 inhibitor WAT explants 67 proteins 7 proteins Endothelial cells 16 proteins 8 proteins 3 proteins 4 proteins Preadipocytes 7 proteins Collagen, biglycan, PEDF Haptoglobin, Cathepsin L1, fibronectin, EGF receptor FIG. 3. Venn diagram showing overlapping proteins detected from samples. A total of 116 secretory proteins were detected across all samples. PEDF, pigment epithelial derived factor; TIMP, tissue inhib- itor of metalloproteinase 1. The diagram was composed using 3-Venn applet (48). (A high-quality color representation of this ﬁgure is available in the online issue.) 3014 DIABETES, VOL. 59, DECEMBER 2010 WAT explants 50 proteins 14 proteins 3T3-L1 adipocytes 69 proteins 12 proteins 10 proteins 7 proteins Rat isolated adipocytes 55 proteins FIG. 4. Venn diagram showing overlap of protein secretion from WAT explants, 3T3–L1 adipocytes, and isolated rat adipocytes (from ref 27). The 12 proteins detected in all three samples were adiponectin, adipsin, angiotensinogen (SerpinA8), cathepsin B, cathepsin D, colla- gen -1(IV), collagen -2(IV), complement C1s, haptoglobin, laminin subunit -2, osteonectin, and thrombospondin-1. The diagram was composed using 3-Venn applet (48). (A high-quality color representa- tion of this ﬁgure is available in the online issue.) lin resistance. Proteins involved in macrophage recruit- ment were secreted in greater abundance from visceral MVECs. This suggests that adipose tissue MVECs play a pivotal role in enticing circulating macrophages into adi- pose tissue. However, adipose tissue used for this study was derived from young, lean animals. In this setting, the upregulation of these cytokines is more likely targeted for angiogenesis and adipose tissue proliferation rather than a proinﬂammatory milieu. Furthermore, recruitment of mac- rophages into adipose tissue is not always associated with inﬂammation as macrophages in adipose tissue from lean mice express IL-10, which protects adipocytes from insu- lin resistance (43). This study represents the ﬁrst survey of the endothelial cell secretome. In comparing MVECs from VAT and SAT, signiﬁcant differences in protein secretion were demon- strated. Among the endothelial secretome were throm- bospondins 1, 2, and 3, known to be secreted by endothelial cells, as well as a number of proteins not previously documented as endothelial secreted factors. Surprisingly, many were known adipocyte secretory pro- teins, including growth arrest speciﬁc 6, PAI-1, periostin, pentraxin-related protein 3, and SPARC. Of particular interest was identiﬁcation of AEBP1, a secreted protein proposed to act as a transcriptional repression factor through binding of the enhancer sequence of the aP2/ FABP4 gene in adipocytes, as well as PPAR and LXR sites in macrophages (44,45) Preadipocytes deﬁcient in AEBP1 are hyperproliferative, with enhanced adipogenesis (46). AEBP1 can be detected in whole adipose tissue samples, but not when adipocytes are puriﬁed from adipose tissue (47). Given our discovery of AEBP1 secretion from endo- thelial cells, we consider it likely that the source of AEBP1 in adipose tissue is endothelial cells. In this study, we found that MVECs from VAT secreted far more AEBP1 diabetes.diabetesjournals.org