Targeted assessment of the glycolytic enzyme content of extracellular vesicles in the context of exercise

Anghileri, Genevieve (2022). Targeted assessment of the glycolytic enzyme content of extracellular vesicles in the context of exercise. University of Birmingham. M.Sc.

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Abstract

Exercise, capable of greatly increasing intracellular glycolytic rate, has frequently been shown to also induce an increase in circulating exosome-like-vesicles (ELVs) (Frühbeis et al, 2015; Whitham et al, 2018; Brahmer et al, 2019). However, the physiological relevance of ELVs in this context remains elusive. Attempts to unravel the cargo proteome, and therefore potential function, of exercise-mediated ELVs have unexpectedly revealed the expression, and dynamic regulation via exercise, of multiple glycolytic enzymes (Whitham et al, 2018; Vanderboom et al, 2021). Though, some data has contradicted this finding (Jeppesen et al, 2019), and the physiological function of such ELVs enriched with glycolytic enzymes remains unclear. The aim of the present study was therefore to confirm the presence of glycolytic enzymes in ELVs isolated from both murine C2C12 myotube conditioned media and human plasma and test the hypothesis that their expression is modulated by glucose availability and high intensity exercise.

Western blots confirmed the presence of multiple glycolytic enzymes alongside classic ELV markers ALIX, TSG101, Syntenin 1 and CD9 in ELV lysates from conditioned media of C2C12 myotubes following both ultracentrifugation (UC) and size exclusion chromatography (SEC) isolation. Furthermore, culturing C2C12 myotubes in high glucose (HG) versus low glucose (LG) media, to assess the role of glucose availability on ELV glycolytic enzyme content, induced a significant (p<0.05) reduction in the expression of GAPDH, PGK1, PMK1, PKM2 and PD in ELVs isolated via UC. Subsequently, in human tetraspanin-positive ELV samples in vivo, we observed the presence of GAPDH at single EV resolution microarray level and dynamic regulation of glycolytic enzymes in response to an exhaustive bout of exercise following immunomagnetic isolation.

These data overall demonstrate that glycolytic enzymes are detected in murine muscle cell-derived ELVs in vitro, irrespective of isolation method and are responsive to glucose availability. Furthermore, glycolytic enzymes GAPDH and HKII are present in tetraspanin-positive ELVs in human plasma and are increased in circulation with exercise; supporting the proposition that ELVs transport glycolytic enzymes during the metabolic stress of exercise.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):