An Adipose-Specific Secretome Revealed Through Mass Spectrometry-Based Proteomic Analysis.

Obesity is a major risk factor for a number of disorders ranging from type II diabetes to cardiovascular disease, however, the molecular basis for these associations has not been be fully clarified. A number of adipokines, cytokines expressed exclusively or at an elevated level in adipose tissue, have been identified that play key roles in regulating energy homeostasis by affecting insulin sensitivity, glucose and lipid metabolism and satiety. Dysregulated expression of adipokines may link obesity with its related metabolic disorders. We have developed a proteomics platform that enables the direct identification and functional characterization of adipokines from cells derived from human subcutaneous and visceral adipose depots. Adipocytes isolated from different fat depots (subcutaneous and visceral) from subjects with a spectrum of BMI (19-42) and diabetic progression, were subjected to Mass Spectrometric (MS) analyses. Preadipocytes isolated from adipose tissue were differentiated for 2 weeks, before being incubated in conditioned medium. Secreted and shed proteins released into the medium were labeled with ICAT and digested with trypsin. The cysteine-containing peptides were subjected to Liquid Chromatography (LC) / MS analysis. Peptide maps representing different adipocyte populations were compared and candidates that were differentially expressed were selected. Sequence-composition was resolved through tandem MS and database search. The functionality of a number of these adipokines was evaluated in a series of RNAi-based assays, established in human adipocytes, to determine the possible involvement of these secreted proteins in key metabolic transitions including: lipolysis and glucose uptake. Our systematic proteomic analysis has resulted in the identification of > 20 adipokines. A number of previously characterized secreted proteins, including visfatin and complement C1r were identified. In addition, a number of novel candidates that included: a protease inhibitor, a hydrolase and a molecule involved in cell-adhesion, were also resolved. Interestingly, subsequent comparison of the MS maps within the adipocyte samples revealed significant differences in expression of secreted / shed proteins isolated from: (i) subcutaneous and visceral depots, (ii) lean and obese subjects and (iii) normal and diabetic patients. [ABSTRACT FROM AUTHOR]