(667c) Quantitative Analysis of the 3T3-L1 Adipocyte Proteome During Hypertrophic Enlargement and Its Integration with Metabolic Flux Models

The increase in adipose tissue mass arises through either an increase in cell number or an increase in the size of individual adipocytes. The latter, termed hypertrophy, occurs in part due to progressive lipid loading and triglyceride accumulation in adipocytes. Hypertrophic adipocytes produce the highest levels of pro-inflammatory molecules and reactive oxygen species (ROS), and not surprisingly, hypertrophy plays an important role in obesity and associated metabolic disorders. Since mitochondria is the site for major metabolic processes such as the TCA cycle and fatty acid oxidation that govern the extent of triglyceride accumulation, and the primary site of ROS generation, we first quantitatively investigated changes in the adipocyte mitochondrial proteome during different stages of differentiation and lipid loading-driven enlargement. Mitochondrial proteins from undifferentiated 3T3-l1 preadipocytes and adipocytes at different stages of lipid accumulation (days 4 through 18) were isolated, digested, and labeled using the iTRAQ 8-plex kit. The labeled peptides were fractionated using an in-house developed liquid phase isoelectric fractionation system (MSWIFT) to increase the depth of proteome coverage, and analyzed using LC-MS/MS. Our data show that the majority of enzymes and transporters involved in the TCA cycle, fatty acid oxidation, and ATP synthesis were up-regulated upon differentiation, and maintain or increase these levels during hypertrophy. Our results also suggest that cultured adipocytes enter a state of metabolic-overdrive where increased flux through the TCA cycle and increased fatty acid oxidation occur simultaneously. The observed increase in ROS was likely compensated by a concurrent increase in anti-oxidant and detoxification enzymes. Since the effects of mitochondrial oxidative stress also impact the cellular enzymatic machinery in the cytosol, we also profiled changes in the cytosolic proteome. A targeted proteomic approach was used wherein enzymes involved in pathways identified as being important using a metabolic flux model (e.g., glycolysis, palmitate biosynthesis) were quantitatively profiled using mass spectrometry. The combination of mitochondrial and cytosolic protemic profiling with metabolic flux models provides a systems-level understanding of processes during hypertrophic enlargement and lipid-loading in adipocytes.