Background: Skeletal muscle plays essential roles in whole body metabolism and is the largest sink for postprandial glucose disposal (DeFronzo et al, 1981). As such, impaired muscle glucose uptake contributes significantly to whole-body insulin resistance. Physical activity is key to the maintenance of glycaemic control, with insulin resistance developing rapidly with inactivity (Burns et al. 2021). Yet the mechanisms regulating contraction/inactivity mediated glucose uptake remain unclear. The 2-deoxyglucose (2DG) tracer is commonly used to determine glucose uptake, yet over long time periods can interfere with cellular metabolism (Suginohara et al, 2021). We aimed to optimise a valid in vitro model of contraction-mediated glucose uptake.
Methods: C2C12 cells were grown in DMEM containing 10% v/v FBS, 1% L-glutamine and 1% penicillin/Streptomycin to a confluency of 90%. Media was switched for differentiation by substituting FBS with 2% horse serum, until myotubes formed. Media was changed 24hr before experiments. One hr of [pre-] electrical pulse stimulation (EPS; C-Pace) was applied at 11.5V and 1Hz with a pulse duration of 2ms to induce formation of contractile sarcomeres. 2DG was dissolved in water and applied at final concentrations of 25µM or 200µM. EPS was applied for 24hr at the same settings as pre-stimulation with samples collected at 30min, 4, 8 and 24hr (n=6). Cells were scraped into homogenisation buffer for immunoblotting or 75% methanol solution for mass spectrometry. Non-stimulated time controls were run alongside treated cells. Glucose uptake was determined by measurement of 2DG 6-phosphate (n=3). Methanol scraped cells were homogenised and derivatised to methoxime-TMS. Samples were measure on a GC-MSMS (Thermo) TRACE 1310 Gas Chromatograph connected to TSQ 8000 triple quadrupole GC-MS/MS (Thermo Scientific) alongside a standard curve of 25 to 0.78µM 2-DG6P. Simple linear regression was performed to assess glucose uptake linearity, whilst two way-ANOVA was used to analyse changes in media lactate.
Results: At 200µM, cell 2DG6P levels plateaued after 4 hr, presumably due to impaired glucose uptake. At 25µM a linear accumulation of 2DG6P over 24hr was observed (r2= 0.9797 ,P= 0.01). Further, 24 hr EPS showed a linear increase in 2DG6P over 24hr (r2=0.9479, P=0.005). In response to 24hrs EPS, media lactate increased from ~10mM at baseline to ~22mM at 24hrs (P<0.0001), with no effect of 25µM 2DG. There was no change in media lactate concentration in unstimulated cells. Finally, we observed increases in anabolic signalling over 24hr, with EPS e.g. increases in P70 phosphorylation (P=0.03).
Conclusion: At a concentration of 25µM 2-DG is suitable for use in C2C12 cells as a glucose tracer over a 24hr study period. An in vitro model of skeletal muscle glucose uptake over 24 hr has been established that is valid under conditions for EPS mediated contraction.