Extracellular vesicles from obese and diabetic mouse plasma alter C2C12 myotube glucose uptake and gene expression

Figures

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  FIGURE 1. Characterization of a circulating extracellular vesicle (EV) isolate. (a) Body weight of animals immediately before EV isolation. (b) Ad libitum blood glucose of animals immediately before EV isolation. (c) Content of EVs found in standard C2C12 differentiation media. (d) Average EV diameter as calculated by the ZetaSizer instrument, with a representative size trace of EVs isolated from a Db/Db mouse. (e) Western blot image of CD63 compared to myotube lysate as a negative control. (f) Western blot image of CD9 compared to myotube lysate as a negative control. (g) Western blot image of HSP70 compared to myotube lysate as a negative control. (h) Western blot image of TSG101 compared to myotube lysate as a negative control. (i) Western blot image of calnexin in isolated EV preparations compared to myotube lysate‐positive control. (j) EVs are located in extracellular spaces and are also colocalized to myotubes (phase contrast). EVs were identified with ExoGlow membrane labeling (red), and images are shown after incubation overnight with an EV concentration of 50 μg/mL (scale bar in black is 1000 μm).

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  FIGURE 2. Nanoparticle tracking analysis of plasma extracellular vesicles. (a) Representative plasma EV video frame and size trace from a control mouse. (b) Representative plasma EV video frame and size trace from a Db/Db mouse. (c) Plasma EV diameter as calculated from nanoparticle tracking analysis. (d) Plasma EV concentration as calculated from nanoparticle tracking analysis.

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  FIGURE 3. Effect of circulating extracellular vesicles (EVs) on glucose uptake, insulin signaling, and glucose transporter expression. (a) 2‐NBDG uptake in the basal and insulin‐stimulated states of myotubes exposed to plasma‐derived EVs from control or Db/Db animals. (b) Representative images of Western blots for phosphorylated AKT on Thr308 and Ser473 as well as total AKT. (c) Insulin‐stimulated phosphorylation ratio of P‐AKT (Thr308) to total AKT after exposure to circulating EVs. (d) Insulin‐stimulated phosphorylation ratio of P‐AKT (Ser473) to total AKT after exposure to circulating EVs. (e) Relative mRNA quantification genes encoding glucose transporter proteins after exposure to circulating EVs.

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  FIGURE 4. Effect of circulating extracellular vesicles (EVs) on myosin heavy chain isoforms and other determinants of muscle phenotype. (a) Relative mRNA quantification genes encoding myosin heavy chain isoforms after exposure to circulating EVs were isolated from control or Db/Db mice. (b) Relative mRNA quantification of MEF2C. (c) Relative mRNA quantification of osteopontin. (d) Relative mRNA quantification genes encoding proteins necessary for mitochondrial biogenesis and dynamics after exposure to circulating EVs. (e) Relative mRNA quantification genes encoding proteins that regulate angiogenesis in skeletal muscle after exposure to circulating EVs.

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  FIGURE 5. Effect of circulating extracellular vesicles (EVs) isolated from the plasma of control or Db/Db mice on lipid accumulation in myotubes. (a) Representative image of Oil Red O staining in control EV‐treated myotubes. (b) Representative image of Oil Red O staining in Db/Db EV‐treated myotubes. White arrows highlight dense areas of neutral lipid inclusions. (c) Elution of Oil Red O from circulating EV‐exposed myotubes. (d) Relative mRNA quantification genes that regulate lipid entry, lipolysis, and lipid synthesis in skeletal muscle after exposure to circulating EVs.