(575.4) Obese Animal Serum Derived Vesicles Alter Gene Expression in C2C12 Myotubes

Poster Board Number: E276

Christopher Pitzer (University of Tennessee Health Science Center, University of Tennessee Health Science Center, University of Tennessee Health Science Center, University of Tennessee Health Science Center), Hector Paez (University of Tennessee Health Science Center), Peter Ferrandi (University of Tennessee Health Science Center, University of Tennessee Health Science Center), Junaith Mohamed (University of Tennessee Health Science Center), Stephen Alway (University of Tennessee Health Science Center)

Purpose: Regulation of metabolism by extracellular vesicles (EV’s) is an emerging interest due to the ability of vesicle-bound cargo to alter gene expression. C2C12 myotubes are an in vitro model of murine skeletal muscle fibers. The skeletal muscle is the primary site of glucose disposal in the body. Obesity is known to decrease insulin-stimulated glucose uptake in the muscle. The purpose of this project was to determine the changes in gene expression caused by serum-derived extracellular vesicles of an obese animal relative to normal healthy controls. Due to the known association of obesity and dysregulated metabolism, we hypothesized that EV’s isolated from serum of an obese animal would negatively alter metabolically important gene expression.

Methods: Serum was collected from obese, but not diabetic (body weight 42 g blood glucose 131 mg/dL) and age-matched non obese/diabetic control (body weight 26g blood glucose 115 mg/dL), mice and extracellular vesicles were precipitated using a commercially available precipitation reagent (Exoquick, System Biosciences Paolo Alto CA). Fully differentiated myotubes (5 days in differentiation media) were incubated with EV’s (n=4 per group) protein matched to a concentration of 37.5 µg/ml in exosome-free differentiation media (DMEM supplemented with 1% exosome free FBS and 1% anti-anti) for 24 hours. Total RNA was isolated using Trizol and cDNA synthesis was performed prior to real-time PCR experiments.

Results: NR4A1 mRNA expression was significantly increased in obese animal exosome treated myotubes relative to controls (15% increase P=0.016). NR4A2 also tended to increase in obese animal exosome treated myotubes (31% increase P= 0.059) compared to controls. The expression of ND4, a mitochondrially encoded subunit of complex I of the electron transport system, mRNA tended to decrease (11% decrease P=0.091) in the obese animal EV treated myotubes compared to controls.

Conclusions: These data indicate a potential role of obesity-derived extracellular vesicles in altering metabolic processes of skeletal muscle. The dose of exosomes used in this work was relatively low, therefore more research is warranted to determine if additional effects would be seen at physiological exosome concentrations.