Quantitative proteomics analyses of the effect of insulin on the human visceral adipose tissue secretome using a metabolic labeling approach

Abstract

We previously characterized the visceral adipose tissue secretome (MCP 2007, 6, 589). In the present study we determined incorporation rates of 13C‐labeled lysine into newly synthesized secreted proteins. Comparison of the C13/C12 ratios in the absence and presence of insulin should offer quantitative information on the effect of insulin on the production of secreted proteins. To prove this concept, human visceral adipose tissue was cultured for 72 hrs in 13C‐Lys containing medium in the absence or presence of 60 nM insulin. Subsequently, media were collected, concentrated and fractionated by SDS‐PAGE, followed by in‐gel digestion and LC‐MS/MS analyses. The datasets shared 274 proteins of which 224 proteins contained label. After removing contaminating intracellular proteins and applying a threshold C13/C12 ratio of 0.5, 82 proteins remained. In the absence of insulin, highest label incorporation was found for Zinc‐alpha‐2‐glycoprotein. In the presence of insulin, CRISP‐11 showed the highest label incorporation. By comparing C13/C12 ratios in the absence and presence of insulin we found eight proteins that were up‐regulated by insulin, four proteins were down‐regulated and 70 showed no change. The highest increase (4.7 fold) was found for CRISP‐11. We developed a new proteomics method which allows quantitative assessment of effects of insulin or other agents on protein secretion by tissues in culture.