Topical Area: Energy and Macronutrient Metabolism
The metabolic requirements for muscle progenitor cell (MPC), the cell required for skeletal muscle regeneration, are ambiguous. We identified that pyruvate kinase M2 (PKM2), a glycolytic enzyme (canonical) and metabolic regulator (non-canonical) promotes MPC proliferation. Our objective was to determine the essentiality of glycolytic and mitochondrial metabolism for MPC proliferation (Experiment [EXP] 1 and 2), and how PKM2 impacts these metabolic pathways (EXP 3).
C2C12 cells, immortalized MPCs, were cultured in growth media (GM, DMEM + 10 % fetal bovine serum) for all experiments. EXP 1: MPCs were cultured in glucose free GM supplemented with/without 25 mM glucose or 25 mM galactose for 48 h. EXP 2: MPCs were cultured in GM with mitochondrial inhibitors (500 nM Rotenone or 2 μM Oligomycin) for 72 h. EXP 3: MPCs were cultured in GM and treated for 8 h with 150 μM TEPP-46 or 1.4 μM Shikonin, small molecules that differentially impact canonical and non-canonical PKM2 activity. Cell number was measured with a Celigo. Oxygen consumption (OCR, [i.e., oxidative phosphorylation]) and extracellular acidification rates (ECAR [i.e., glycolysis]) were measured with a Seahorse XF Analyzer. Glucose uptake was measured with a kit. Mitochondria membrane potential and copy number were measured with JC1 dye and qPCR, respectively.
At 48 h, glucose free media and galactose containing media decreased cell number 8.2-fold (p < 0.001) and 7.7-fold (p < 0.01), compared to cells cultured in glucose containing media. At 72 h, cell number was reduced 16.9-fold by Rotenone (p < 0.001) and 14.1-fold by Oligomycin (p < 0.01), compared to vehicle control. Both TEPP-46 and Shikonin increased ECAR (1.6- and 1.4-fold, p < 0.01) while there was a trend toward reduced glucose uptake (1.2-fold and 1.3-fold, p < 0.1). Both TEPP-46 and Shikonin reduced basal OCR (1.6- and 1.3-fold, p < 0.01) and mitochondria membrane potential (1.7- and 1.2-fold, p < 0.01), but did not affect mitochondrial copy number (p > 0.05).
Both glycolytic and mitochondrial metabolism are essential for MPC proliferation and PKM2 may regulate MPC proliferation through these pathways. Future studies will elucidate the relationship between metabolism, PKM2, and MPC proliferation.
Funding Sources :
NSF Graduate Research Fellowship