DEPOT-SPECIFIC SECRETOME OF ADIPOSE TISSUE Sample a 4,4,5,5-D4 lysine (+4 Da) y t i s n e t n i Paired tissues or primary cultures from animal Sample b Incubate 48 hours Mix in 1:1 ratio Glycoprotein purification Mass spectrometry c a b 4 Da m/z 8 Da Peak height ratios of c (endogenous unlabelled amino acids), a (isotope 1) and b (isotope 2) compared. FIG. 1. LAC–CILAIR workﬂow for comparing protein synthesis and secretion from two different samples. Paired cell cultures or tissues are incubated in media containing different isotopes of arginine and lysine, which are subsequently incorporated into newly synthesized and secreted proteins. Paired conditioned media samples are mixed in equal proportions, and proteins are subjected to mass spectrometry. Incorporation of amino acid isotopes results in a shift in molecular weight and mass to charge (m/z) ratio. Peak heights are measured, and their ratios are used to determine relative abundance of proteins from samples a and b. RESULTS We hypothesized there would be differences in protein secretion from VAT and SAT. Adipose tissue explants from epididymal (VAT) and inguinal (SAT) depots were incu- bated for 48 h in the presence of “heavy” and “medium” isotopes of arginine and lysine, respectively. Medium was collected, mixed in equal ratio, and subjected to LAC. Proteins were precipitated, resolved by SDS-PAGE, tryp- sin-digested, and subjected to quantitative mass spectrom- etry (Table 1 and supplementary data available in an online appendix at http://diabetes.diabetesjournals.org/ cgi/content/full/db10-0483/DC1). A total of 145 proteins were identiﬁed, of which 86 (60%) were predicted to contain an NH2-terminal signal peptide using SignalP. Of these, 51 proteins (59%) were present at 2-fold higher abundance in conditioned medium from VAT than SAT, whereas only 18 proteins (21%) were at 2-fold higher abundance in conditioned medium from SAT than VAT. The frequency distribution of the visceral-to-subcutaneous ratio for detected proteins is shown in Fig. 2A. Proteins without a signal peptide are nonspeciﬁcally released, newly synthesized cytosolic proteins. The distribution for these background proteins with no signal peptide resem- bles a normal distribution, whereas the distribution for proteins with a signal peptide is skewed to the right. This indicates that release of true secretory proteins into con- ditioned medium is increased from VAT relative to SAT. 3010 DIABETES, VOL. 59, DECEMBER 2010 The normal distribution of nonsecretory background pro- teins indicates a similar amount of tissue was present in VAT and SAT samples. Clustering analysis of detected proteins was performed using the database for annotation, visualization, and inte- grated discovery (DAVID) (34,35). Analysis of proteins increased from VAT revealed enrichment of proteins in- volved in endopeptidase inhibition, including members of the Serpin family, and proteins involved in innate immune system regulation, including members of the complement pathway. Cluster analysis of proteins increased from SAT showed no functional similarities. Adipose tissue is composed of adipocytes as well as preadipocytes, MVECs, and macrophages, collectively re- ferred to as the stromovascular fraction. Stromovascular cells may make a signiﬁcant contribution to the secretory capacity of white adipose tissue, perhaps even exceeding that of adipocytes (36). Interplay between cells of the stromovascular fraction and adipocytes may also modu- late the secretion proﬁle of adipose tissue. We sought to determine the relative contribution of preadipocytes and MVECs to the whole adipose tissue secretome. In addition, we examined whether depot-speciﬁc differences in protein secretion observed from whole adipose tissue explants persisted in cells from the stromovascular fraction. First we isolated preadipocytes from paired VAT and SAT biopsies. Visceral and subcutaneous preadipocytes diabetes.diabetesjournals.org