S.L. HOCKING AND ASSOCIATES TABLE 1 Comparative secretion of proteins identiﬁed from visceral and subcutaneous white adipose tissue explants Description Complement C3 -2-macroglobulin Laminin subunit -1 Laminin subunit -1 Laminin subunit -2 Collagen -1(XIV) chain Fibronectin Carboxylesterase 3 Haptoglobin Collagen -2(I) chain Serotransferrin Laminin subunit -2 Complement factor B Ceruloplasmin Serine protease inhibitor A3N Serine protease inhibitor A3K Liver carboxylesterase N Gelsolin Complement C4-B Nidogen-2 Cathepsin B Antithrombin-III Decorin Signal peptide? Functional description Accession Complement cascade, innate immunity CO3_MOUSE Serine protease inhibitor, prevents coagulation and ﬁbrinolysis A2M_MOUSE Extracellular matrix LAMB1_MOUSE Extracellular matrix LAMC1_MOUSE Extracellular matrix LAMA2_MOUSE Extracellular matrix COEA1_MOUSE Extracellular matrix FINC_MOUSE Lipase CES3_MOUSE Haem binding protein, acute phase reactant HPT_MOUSE Extracellular matrix CO1A2_MOUSE Iron binding TRFE_MOUSE Extracellular matrix LAMB2_MOUSE Complement cascade, innate immunity CFAB_MOUSE Copper, iron binding CERU_MOUSE Trypsin inhibitor SPA3N_MOUSE Trypsin inhibitor, also known as contrapsin SPA3K_MOUSE Drug detoxiﬁcation ESTN_MOUSE Depolymerises extracellular actin from necrotic cells GELS_MOUSE Complement cascade, innate immunity CO4B_MOUSE Extracellular matrix NID2_MOUSE Protease activity CATB_MOUSE Serine protease inhibitor, regulation of blood coagulation ANT3_MOUSE Extracellular matrix PGS2_MOUSE Visceral or subcutaneous WAT explants were incubated in media containing different isotopes of arginine and lysine. Conditioned media were mixed, subjected to lectin afﬁnity chromatography, and subjected to quantitative mass spectrometry. Peak height ratios of isotope to endogenous (12C) amino acids and ratios between isotopes are shown. Complete dataset available in supplementary data. were cultured in vitro for at least one week before incubation in medium containing isotopic amino acids. Conditioned medium was analyzed as before (Table 2 and supplementary data). Preadipocytes secreted a more limited set of proteins than minced adipose tissue. Of 23 proteins detected, 22 were secretory proteins. Of these, 15 were upregulated 2-fold from visceral com- pared with subcutaneous preadipocytes. Not one pro- tein was upregulated 2-fold from subcutaneous as compared with visceral preadipocytes. The frequency distribution of the visceral-to-subcutaneous ratio for detected proteins is shown in Fig. 2B and is again skewed to the right. This indicates that the release of secretory proteins into conditioned medium is in- creased from visceral relative to subcutaneous preadi- pocytes. Cluster analysis of the proteins upregulated from visceral-derived preadipocytes showed an enrich- ment of extracellular matrix proteins. Next, we examined MVECs isolated from paired VAT and SAT depots. To date the secretome of endothelial cells has not been determined. In addition, it is unknown whether MVECs display depot-speciﬁc differences. We isolated MVECs using CD31 magnetic bead separation and cultured these cells in vitro. Conditioned media from cells incubated in isotopic amino acids were collected and analyzed as before (Table 3 and supplementary data). Of 66 detected proteins, 34 (52%) displayed an NH2-terminal signal peptide. Of these, 21 (62%) were increased 2-fold from visceral MVECs and only 5 were increased 2-fold from subcutaneous MVECs. The frequency distribution of the visceral to subcutaneous ratio for detected proteins is shown in Fig. 2C. Once again, these data are skewed toward increased protein secretion from visceral MVECs (Fig. 2C). This is consistent with the enhanced secretory capacity of VAT we have observed in both whole adipose diabetes.diabetesjournals.org tissue and isolated preadipocytes. The proteins identiﬁed include a number of proteins known to be secreted from endothelial cells, including thrombospondin-1 and throm- bospondin-2, and extracellular matrix proteins such as ﬁbronectin and collagens. Additionally, several proteins not previously known to be secreted from adipose tissue MVECs were identiﬁed. These include the most abun- dantly detected protein, adipocyte enhancer binding pro- tein 1 (AEBP1). This protein inﬂuences inﬂammation in macrophages via a direct interaction with PXR and PPAR sites. Multiple members of the complement pathway were also identiﬁed. Cluster analysis showed proteins more abundantly secreted from visceral MVECs were involved in extracellular matrix formation, including collagen and ﬁbronectins, and endopeptidase inhibition, including members of the Serpin family. There were no deﬁned clusters for the ﬁve secretory proteins more abundantly secreted from subcutaneous MVECs. When the secretomes of whole adipose tissue ex- plants, preadipocytes, and MVECs were compared, eight common secreted proteins were identiﬁed (Fig. 3). Seven secreted proteins were shared between the whole adipose tissue and MVEC secretomes, and four were shared between the whole adipose tissue and preadipo- cyte secretomes. These shared proteins included pro- teins involved in extracellular matrix formation, such as collagen and ﬁbronectin, and other known adipocyte proteins known such as osteonectin. Interestingly, these data suggest the majority of proteins in the whole adipose tissue secretome are secreted either by adipo- cytes themselves or by other cells in the stromovascular fraction such as macrophages and neurons. To interro- gate this further, we compared the secretome of whole adipose tissue explants to the secretomes of differenti- ated 3T3-L1 adipocytes (30) and isolated rat adipocytes DIABETES, VOL. 59, DECEMBER 2010 3011