ORIGINAL ARTICLE Intrinsic Depot-Specific Differences in the Secretome of Adipose Tissue, Preadipocytes, and Adipose Tissue–Derived Microvascular Endothelial Cells Samantha L. Hocking,1 Lindsay E. Wu,1,2 Michael Guilhaus,2,† Donald J. Chisholm,1 and David E. James1 OBJECTIVE—Visceral adipose tissue (VAT) is more closely linked to insulin resistance than subcutaneous adipose tissue (SAT). We conducted a quantitative analysis of the secretomes of VAT and SAT to identify differences in adipokine secretion that account for the adverse metabolic consequences of VAT. RESEARCH DESIGN AND METHODS—We used lectin afﬁn- ity chromatography followed by comparison of isotope-labeled amino acid incorporation rates to quantitate relative differences in the secretomes of VAT and SAT explants. Because adipose tissue is composed of multiple cell types, which may contribute to depot-speciﬁc differences in secretion, we isolated preadipo- cytes and microvascular endothelial cells (MVECs) and com- pared their secretomes to those from whole adipose tissue. RESULTS—Although there were no discrete depot-speciﬁc dif- ferences in the secretomes from whole adipose tissue, preadipo- cytes, or MVECS, VAT exhibited an overall higher level of protein secretion than SAT. More proteins were secreted in twofold greater abundance from VAT explants compared with SAT explants (59% versus 21%), preadipocytes (68% versus 0%), and MVECs (62% versus 15%). The number of proteins in the whole adipose tissue secretome was greater than the sum of its cellular constituents. Finally, almost 50% of the adipose tissue secretome was composed of factors with a role in angiogenesis. CONCLUSIONS—VAT has a higher secretory capacity than SAT, and this difference is an intrinsic feature of its cellular components. In view of the number of angiogenic factors in the adipose tissue secretome, we propose that VAT represents a more readily expandable tissue depot. Diabetes 59:3008–3016, 2010 Ohowever, the distribution of body fat may be besity increases the risk of type 2 diabetes; more important than obesity per se (1). Vis- ceral adiposity is associated with an increased risk of insulin resistance (2– 4), and this relationship persists even in nonobese individuals (1). Similarly in rodents, increased adiposity in epididymal, mesenteric, From the 1Diabetes and Obesity Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia; and the 2Bioanalytical Mass Spec- trometry Facility, University of New South Wales, Sydney, NSW, Australia. Corresponding author: David E. James, d.james@garvan.org.au. Received 7 April 2010 and accepted 31 August 2010. Published ahead of print at http://diabetes.diabetesjournals.org on 14 September 2010. DOI: 10.2337/db10-0483. S.L.H. and L.E.W. contributed equally to this study. †Deceased. © 2010 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for proﬁt, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3008 DIABETES, VOL. 59, DECEMBER 2010 and perinephric depots induces insulin resistance (5). There are several differences between visceral (VAT) and subcutaneous adipose tissue (SAT) that could account for the link between VAT and insulin resistance, the most obvious being anatomical location. VAT is particularly sensitive to lipolytic stimuli and partly drains into the liver via the portal vein. Increased ﬂux of free fatty acids from VAT to liver may increase hepatic gluconeogenesis and hepatic insulin resistance (6,7). Data in both humans and rodents argue against this “portal hypothesis.” Removal of gonadal (nonportal) adipose tissue in obese mice im- proves insulin sensitivity (8). In humans, some studies show a stronger association between subcutaneous ab- dominal fat and insulin resistance (9,10). Thus, the role of anatomical location in mediating the deleterious effects of VAT remains unresolved. These divergent metabolic effects may be due to intrin- sic, cell-autonomous differences between VAT and SAT. VAT displays higher rates of fatty acid uptake and release with reduced antilipolytic response to insulin (11), in- creased lipolytic response to catecholamines (12), in- creased response to glucocorticoids with increased lipoprotein lipase activation (13), and higher glucocorti- coid receptor levels (14). Studies of gene expression have found major differences between adipocytes and preadi- pocytes from VAT and SAT (15). These data suggest intrinsic differences between cells in VAT and SAT. Adipose tissue is an endocrine organ secreting numer- ous proteins (adipokines) with potent metabolic effects (16). Metabolically beneﬁcial adipokines including leptin and adiponectin are secreted in higher amounts from SAT (17,18), whereas proinﬂammatory adipokines such as RBP4, TNF- , MCP-1, IL-8, and IL-6 are increased in VAT (19 –23). It has been suggested that this differential secre- tion of adipokines may account for the differing metabolic consequences of visceral and subcutaneous adiposity. However, it is unclear if these factors are secreted from adipocytes or other cells resident in adipose tissue such as macrophages. Whereas leptin and adiponectin are se- creted by adipocytes, other factors including resistin, visfatin, TNF- , IL-6, and MCP-1 are principally secreted by macrophages (24). Therefore, the adipocyte may not be the principal secretory component of adipose tissue. We sought to accurately characterize the secretome of adipose tissue and quantitatively compare the secretomes from VAT and SAT explants. Previous studies identiﬁed adipocyte secretory factors using 3T3-L1 cell-lines (25,26) or primary adipocytes (27,28). These studies have several limitations. Immortalized cell lines are unable to provide depot speciﬁcity, and by isolating adipocytes from non- adipose cells within adipose tissue, cross-talk between these cells is lost. Using whole adipose tissue explants diabetes.diabetesjournals.org