Comparative proteome analysis of secreted proteins from insulin-resistant C2C12 cells

Skeletal muscle accounts for majority of insulin-stimulated glucose disposal and has a high capacity to metabolize fatty acids. Impaired glucose metabolism and lipid metabolism in skeletal muscle are hallmark features of insulin resistance associated with obesity, type 2 diabetes, and metabolic syndrome. Elevated plasma free fatty acids (FFA) are thought to be responsible for development of insulin resistance however the mechanisms by which FFA cause insulin resistance are not clear. Skeletal muscle has been suggested to be source of secreted proteins which can influence metabolism and other biological processes in a systemic manner. Here we report a secretome of an insulin- resistant muscle cell line. To analyze proteins secreted from insulin-resistant murine C2C12 skeletal muscle cells, we applied a stable isotope labeling by amino acids in cell culture (SILAC) based quantitative proteomics platform. Exposure of isotopicaly labeled C2C12 cells to 0.5 mM palmitate results in development of insulin resistance (confirmed by impairment in insulin signaling). Thereafter, we compared the secretome of control vs insulin-resistant cells. Protocols were optimized to efficiently derive secreted proteins from cells in culture. In supernatants, we identified and quantified 2205 proteins including 36 cytokine signaling proteins, 60 growth factors and 46 metallopeptidase. We found that 182 of these proteins were significantly different in palmitate treated cells compare to non-treated cells. In addition to previously reported secreted protein, we identified many novel proteins that have not been shown to be released from skeletal muscle. These proteins may act as signaling mediators to other cells and tissues and supporting a role of skeletal muscle as an important secretory organ.