Stable Isotope Labeling of Myotubes from Lean and Very Obese Women for Quantitative Proteomics

Obesity is linked with a constellation of metabolic abnormalities that develop into cardiovascular disease and Type 2 Diabetes. A precipitating factor for the development of obesity‐associated disease states is whole‐body insulin resistance. Increased intramuscular triglycerides and depressed fatty acid oxidation are strongly associated with insulin resistance in skeletal muscle. Comparative two‐dimensional gel electrophoresis is the standard approach for protein profiling even though estimating protein abundance is difficult when spots are saturated or not well focused. Recently, Stable Isotope Labeling with Amino acids in Cell culture (SILAC) was developed to surmount these limitations and produce high‐throughput protein profiles of cultured cells. We used SILAC to compare the protein profiles of myotubes cultured from skeletal muscle of lean (BMI < 20 kg/m3) and extremely obese (BMI > 45 kg/m3) human female donors. Myotubes were also exposed to high‐physiologic levels (250 uM) of oleate to establish how obese vs lean cells respond to lipid loading. Proteins differentially expressed > 1.5 fold were then analyzed using a commercial web‐based tool for constructing networks of direct physical, transcriptional and enzymatic interactions. This analysis revealed significant clusters of proteins functionally associated with inflammation, oxidative stress and cellular death in oleate treated and obese vs lean myotubes. Furthermore, the expression of many of these proteins are regulated by pro‐inflammatory cytokines such as TNF‐alpha, INF‐gamma, TGF‐beta and their interactions with Insulin, Leptin and AKT signaling pathways.