avoids these issues but introduces the presence of non- adipose-derived contaminating serum proteins. To circum- vent this problem, we used comparison of isotope-labeled amino acid incorporation rates (CILAIR) (29). Cells or tissues are incubated with isotopically labeled amino acids, which become incorporated into newly synthesized proteins. This method allows quantitative assessment of differences in protein secretion as a protein with a higher incorporation of isotope is synthesized at a greater rate or in greater abundance. The quality of the secretome sample can also be dimin- ished by contamination with cytosolic, nonsecretory pro- teins released from damaged cells. Secreted proteins commonly undergo N-glycosylation, whereas cytosolic proteins do not. We exploited differential N-glycosylation, using lectin afﬁnity chromatography (LAC) to selectively enrich proteins targeted for secretion. We used LAC followed by CILAIR (LAC-CILAIR) to quantitate relative differences in the secretomes of murine VAT and SAT. Because adipose tissue is composed of multiple cell types, which may contribute to depot-speciﬁc differences in secretion, we isolated preadipocytes and microvascular endothelial cells (MVECs) and compared their secretomes to that from whole adipose tissue. Al- though we did not observe any discrete differences in secretion proﬁle between each depot, VAT exhibited an overall higher level of protein secretion than SAT, for whole adipose tissue, preadipocytes, and MVECS. Inter- estingly, a large proportion of the adipose tissue secre- tome was factors that play a role in angiogenesis. The increased production of these factors from VAT leads to the inescapable speculation that VAT represents a more readily expandable adipose tissue depot than SAT. RESEARCH DESIGN AND METHODS Materials and buffers. All tissue culture reagents were from Life Technol- ogies unless otherwise stated. Transfer medium. Dulbecco’s modiﬁed eagle medium (DMEM)/Ham’s F-12 1:1 containing 1% BSA and supplemented with 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 g/ml amphotericin B (antibiotic–antimycotic, Invitrogen). SILAC medium. Low-glucose DMEM without leucine, lysine, and arginine (Sigma Aldrich) was supplemented with 0.105 g/l leucine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 0.0159 g/l phenol red, and 100 nmol/l insulin (DMEM/K-/R-). Glucose was added to a ﬁnal concentration of 4.5 g/l. “Heavy” or “medium” isotopes of arginine (Cambridge Laboratories U-13C6 units–15N4 arginine CNLM-539 or U-13C6 arginine CLM-2265 0.021 g/l) and lysine (Cambridge Laboratories U-13C6 units-15N2 lysine CNLM-291 or 4,4,5,5-D4 lysine CNLM-2640 0.0365 g/l) were added to produce “heavy” and “medium” SILAC medium, respectively. Endothelial cell growth medium. Endothelial cell growth medium (ECGM) is low-glucose DMEM supplemented with 100 units/ml penicillin, 0.1 mg/ml streptomycin, 20 mmol/l HEPES, 1% nonessential amino acids, 50 mmol/l 2-mercaptoethanol, 20% heat-inactivated FCS, 12 units/ml heparin, and 150 g/ml endothelial cell growth supplement (Sigma). Endothelial cell SILAC medium. Endothelial cell SILAC medium is DMEM/ K-/R- supplemented with 20 mmol/l HEPES, 1% nonessential amino acids, and 50 mmol/l 2-mercaptoethanol. SILAC isotopes were added as above. Preadipocyte growth medium. Preadipocyte growth medium is DMEM/ Ham’s F-12 1:1 supplemented with 100 units/ml penicillin, 0.1 mg/ml strepto- mycin, and 10% FCS. Animals. Male C57BL/6J mice were from Animal Resources Centre (Perth, Australia). Animals were kept in a temperature-controlled room (22 1°C), 80% relative humidity, on a 12-h light/dark cycle with free access to food and water. Experiments were carried out with approval of Garvan Institute Animal Experimentation Ethics Committee, following guidelines issued by the Na- tional Health and Medical Research Council of Australia. For each experi- ment, 2–5 mice were used as tissue donors. diabetes.diabetesjournals.org S.L. HOCKING AND ASSOCIATES Preparation of anti-CD31 (anti-PECAM-1) antibody-coated magnetic beads. Sheep anti-rat IgG Dynabeads (Invitrogen) were precoated with rat anti-mouse monoclonal antibody to CD31 (PECAM-1) (BD Pharmingen) according to the manufacturer’s instructions at a concentration of 1 g antibody per 1 107 Dynabeads. Dynabeads were stored in PBS with 0.1% BSA at 4°C at 4 108 beads/ml. Adipose tissue explants. Paired VAT (epididymal) and SAT (inguinal) depots were obtained from 6-week old C57BL/6J mice. Fat pads were immediately transported in warm transfer medium to a tissue culture hood, where they were placed in fresh transfer medium and minced into 1 mm3 pieces. Minced explants were rinsed in PBS and centrifuged at 250g to remove cell debris and erythrocytes. Adipose tissue explants (200 l) were incubated in 1 ml SILAC medium. After 48 h of incubation at 37°C in 5% CO2, conditioned medium was collected and centrifuged at 2,000g to pellet cell debris. Lectin afﬁnity chromatography and subsequent mass spectrometry were performed. Preadipocyte and endothelial cell isolation and culture. Preadipocytes and microvascular endothelial cells (MVECs) were isolated from the same biopsies. Paired VAT and SAT depots were obtained from 6-week-old C57BL/6J mice. Fat pads were immediately transported in warm transfer medium to a tissue culture hood, where they were placed in fresh transfer medium and minced into 1 mm3 pieces. Medium was removed and replaced with fresh transfer medium containing 1 mg/ml collagenase type I and incubated with gentle shaking at 37°C for 1 h. Adipose tissue to digest solution ratio was 4:1. Resulting material was ﬁltered through 250 m mesh, and adipocytes and free oil were separated from stromovascular components by centrifugation at 250g for 5 min at room temperature. The stromovascular pellet was resuspended, washed in PBS, and centrifuged at 250g for 5 min at room temperature. This washing step was repeated twice. The stromovascular pellet was resuspended and plated in 2% gelatin-coated 10 cm dishes in ECGM. This mixed cell population was cultured for 5– 6 days at 37°C in 5% CO2. Cells were detached using 0.25% trypsin containing 3.42 mmol/l EDTA and trypsin neutralized by addition of Hank’s balanced salt solution containing 5% FCS (HBSS-FCS). The cell solution was centrifuged at 600g for 5 min at room temperature. The cell pellet was resuspended in 0.5 ml HBSS-FCS and incubated with 25 l of anti-CD31 (anti-PECAM-1) coated dynabeads for 15 min at 4°C under constant rotation. The cell/bead suspension was brought to a total volume of 10 ml with HBSS-FCS and MVECs selected by magnet. Nonselected cells in the wash were transferred to a fresh tube. The wash and selection procedure was repeated ﬁve times. Selected cells were resuspended in ECGM and transferred to 2% gelatin-coated culture dishes. After 3–5 days, cells were routinely passaged and plated at equal density in ECGM. Nonse- lected cells in wash medium were centrifuged at 600g for 5 min at room temperature. The resultant pellet was resuspended in preadipocyte growth medium and plated at equal density in 10 cm culture dishes. MVECs and preadipocytes were grown until conﬂuent. At conﬂuence, medium was replaced with endothelial cell SILAC medium and SILAC medium, respec- tively. After 48 h, conditioned medium was collected and centrifuged at 2,000g to pellet cell debris. Lectin afﬁnity chromatography and subsequent mass spectrometry were performed. Lectin afﬁnity chromatography. Conditioned media from paired visceral and subcutaneous depots were mixed in 1:1 ratio, and glycoprotein puriﬁca- tion was performed as previously described (30). Brieﬂy, MnCl2 was added to conditioned media to a ﬁnal concentration of 1 mmol/l. Samples were incubated overnight at 4°C under constant rotation with 50 l/ml of 50% slurry of Concanavalin A Sepharose beads (GE Healthcare). Beads were washed extensively with ConA binding buffer (0.5 mol/l NaCl, 0.1 mol/l Tris, 1 mmol/l MnCl2, and 1 mmol/l CaCl2, pH 7.4). Proteins were eluted with 0.3 mol/l methyl- -D-mannopyranoside, 0.5 mol/l NaCl, 0.1 mol/l Tris, 10 mmol/l EDTA, and 10 mmol/l EGTA, pH 7.4. Eluted proteins were precipitated using chloroform/methanol (31). Mass spectrometry. Peptide extraction and mass spectrometry were as previously described (30,32) using a Waters Ultima tandem mass spectrome- ter. Mascot distiller software (Matrix Sciences) was used for data analysis. SwissProt database was used for protein identiﬁcation, taxonomy restricted to Mus musculus, mass spectrometry tolerance set at 0.5 Da, and mass spectrometry/mass spectrometry tolerance set at 0.05 Da. A minimum ion score of 60 was chosen as the criterion for positive identiﬁcation. “Heavy (U-13C6 units-15N4 arginine, U-13C6 units-15N2 lysine) to light (naturally occurring lysine and arginine)” (H/L) and “medium (U-13C6 arginine, 4,4,5,5-D4 lysine) to light” (M/L) isotope ratios were used to determine relative abun- dance of proteins from visceral or subcutaneous samples, respectively. As visceral and subcutaneous samples are mixed, nonlabeled proteins form a “common denominator” that both isotopes are measured against (Fig. 1). Isotope ratios below 0.05 were discounted as “no label incorporation.” Signal peptides were determined using SignalP 3.0 (33). DIABETES, VOL. 59, DECEMBER 2010 3009