Exosomes Isolated from Serum of Type 2 Diabetic Mice Increase Proliferation with no Effect on Insulin Stimulated Glucose Uptake of C2C12 Cells

Abstract

PURPOSE Exosomes are small (30-150 nm) extracellular vesicles that are of interest as a potential diagnostic and therapeutic. C2C12 cells are a murine model of muscle stem cells (MSCs) that proliferate and differentiate to myotubes similarly to the behavior of MSCs during muscle repair. Because muscle repair and insulin-stimulated glucose uptake are known to be impaired in type 2 diabetes, the purpose of this project was to test the hypothesis that exosomes isolated from the serum of diabetic mice (Db-Db mice) impair myoblast proliferation and insulin-stimulated glucose uptake in myotubes of C2C12 cells. METHODS To test this hypothesis, we used CellTrace (ThermoFisher, Waltham, MA), a flow-cytometry based measure of proliferation, and 2-NBDG (ThermoFisher, Waltham, MA), a fluorescent glucose analog that can be used to measure glucose uptake. Exoquick exosome isolation reagent (System Biosciences, Palo Alto, CA) was used to isolate exosomes from serum, and exosome protein was measured using the Bradford method. Proliferation measures were obtained after a 24-hour incubation period with exosomes from either a C57 or Db-Db mouse. To determine if secreted exosomes affected glucose uptake, myotubes were established by incubating C2C12 cells in 2% horse serum (DM). After 5 days in DM, myotubes were incubated for 24 hours with exosomes from either Db-Db mice or C57 control mice. After 24 hours of exosome incubation, myotubes were serum-starved in glucose-free DMEM for 1 hour and then incubated in glucose-free DMEM with 100 µm 2-NBDG with or without 100 nm insulin. Fluorescence was then measured with excitation/emission of 465/540 nm. RESULTS Contrary to our hypothesis, treatment of C2C12 cells with 2 µg/µl of diabetic exosomes significantly increased cell proliferation compared to control cells (One-way ANOVA P-value=0.001) with no change in proliferation observed between treatment with C57 exosomes and untreated controls. Unexpectedly, there was no difference in insulin-stimulated 2-NBDG incorporation (Student's T-test P=0.19) in myotubes incubated with diabetic or control exosomes. However, there was a tendency for an increased basal glucose uptake following incubation with diabetic exosomes (Student's T-test, P=0.09). CONCLUSION Overall, these data suggest that exosomes may modulate MSC proliferation and glucose uptake during muscle repair in the diabetic condition.