WATsignal peptide WATno signal peptide Endothelial cells signal peptide Endothelial cells no signal peptide -6 -5 -4 -3 -2 -1 Log e (visceral/subcutaneous ratio) C Increased in subcutaneous No change Increased in visceral Preadipocytes signal peptide Preadipocytes no signal peptide DEPOT-SPECIFIC SECRETOME OF ADIPOSE TISSUE A 25 Increased in subcutaneous No change Increased in visceral -6 -5 -4 -3 -2 -1 0 1 2 3 Log e (visceral/subcutaneous ratio) 4 5 6 B 12 Increased in subcutaneous No change Increased in visceral 20 y cn15 e u q e F10 r 5 0 10 y8 c n e u6 q e r F 4 2 0 10 9 8 7 y c 6 n e 5 u q r4 e F 3 2 1 0 -6 -5 -4 -3 -2 -1 0 1 2 3 Log e (visceral/subcutaneous ratio) 4 5 6 FIG. 2. A: White adipose tissue (WAT) explants secretory proteins are secreted in greater amounts from VAT. B: Preadipocyte secreted proteins are secreted at greater amounts from visceral-derived prea- dipocytes. C: Endothelial cell secretory proteins are secreted at greater amounts from visceral-derived endothelial cells. WAT ex- plants, preadipocytes, and microvascular endothelial cells from vis- ceral and subcutaneous WAT depots were cultured in media containing two different isotopes of arginine and lysine. LAC–CILAIR mass spec- trometry was performed, and isotopic ratios were calculated. The histogram shows distribution of visceral-to-subcutaneous ratios for proteins detected. Note the use of the natural log scale. (27). Of the 86 proteins identiﬁed in the whole adipose tissue secretome, 26 were shared with the 3T3-L1 adi- pocyte secretome and 21 were shared with the isolated rat adipocyte secretome (Fig. 4). These data conﬁrm that adipocytes alone are not the principal source of secreted proteins. It is possible that other cells types in the stromovascular fraction such as macrophages and neurons are the source of secreted proteins. To interro- gate this further, we compared the secretome of whole adipose tissue explants to the secretome of human macrophages, and only three common proteins were identiﬁed (37). 3012 DIABETES, VOL. 59, DECEMBER 2010 DISCUSSION In this study, we have made three important observations. First, the overall secretory capacity of VAT is considerably higher than SAT, and second, this enhanced secretory capacity of VAT extends to cells of the stromovascular fraction—preadipocytes and MVECs. Third, adipose tissue as a whole secretes more proteins than its individual component parts, suggesting the adipocyte is not the principal secretory component of adipose tissue. The secretome of adipose tissue is likely due to the complex interplay between its component cell types. To interrogate the adipose tissue secretome, we com- bined two techniques, CILAIR (29) and LAC (30). Using this streamlined method excluded contaminating proteins from entrapped serum and intracellular contents from damaged cells. Metabolic labeling identiﬁes proteins that are newly synthesized during the culture period. Proteins already in the secretory pathway will remain unlabeled as will contaminants from serum and cell debris (38). LAC selectively enriches for proteins targeted for secretion, thus overcoming contamination from dead or damaged cells. Abundant serum proteins, such as albumin, that are nonglycosylated are removed. This obviates the need for multiple washing steps to dilute these contaminating se- rum proteins, decreasing sample processing time and the negative impact of prolonged tissue handing. Despite these measures, unlabeled proteins will remain in the medium. As conditioned media from visceral and subcu- taneous samples are mixed, nonlabeled (“light”) proteins form a “common denominator” that both isotopes are measured against. This eliminates any potential confound- ing from unlabeled contaminants differentially distributed between the two depots. The technique of LAC–CILAIR is a useful and valid tool for interrogation of the adipose tissue secretome, indicated by the detection of well- known adipokines including adiponectin, adipsin, and plasminogen activator inhibitor-1 (PAI-1). First, we compared rates of protein secretion from VAT and SAT explants. VAT explants secreted quantitatively more proteins than SAT. Of proteins secreted in 2-fold abundance, 59% were from VAT explants compared with 21% from SAT explants. This upregulation of protein secretion from VAT was consistent across cells of the stromovascular fraction with 68% of proteins secreted in 2-fold abundance from VAT preadipocytes compared with 0% from SAT and 62% of proteins secreted in 2-fold abundance from VAT MVECs compared with 15% from SAT (Table 4). Preadipocytes and MVECs were cultured in vitro for at least one week prior to application of labeled media so were no longer subject to paracrine inﬂuences from their depot of origin. This suggests increased protein secretion is an intrinsic characteristic of components of VAT. The detection of a small number of proteins secreted in twofold greater abundance from subcutaneous depots provides evidence that our technique does not selectively detect enhanced protein secretion from the visceral depot. Cluster analysis of proteins secreted in greater abun- dance from VAT revealed members of the acute phase response and innate immune system. These data raise two interesting hypotheses. The ﬁrst is that VAT is more pro-angiogenic than SAT, as many members of the acute phase response, for example, haptoglobin (39), and innate immune system (40) are involved in angiogenesis. The capacity for angiogenesis is a crucial requirement for adipose tissue expansion, and cells of the adipose tissue diabetes.diabetesjournals.org