The Effect of 10%O2 Microenvironment and Electric Pulse Stimulation on Glucose and Lipid Metabolism

Abstract

PURPOSE: There is currently no successful cell contraction model in vitro. Electrical pulse stimulation (EPS) can induce muscle cell contraction. 10%O2 microenvironment is closer to the human skeletal muscle environment. In this study, EPS was applied to myotubes to test the changes of the metabolism of glucose and lipid, and to establish successful cell contraction model in a 10%O2 microenvironment in vitro. METHODS: After seven days of differentiation in 5% CO2(20%O2) incubator, the mouse myoblast cell line C2C12 were transferred into the 10%O2 incubator. After adaptation to the hypoxia for 12h, the myotubes were subjected to EPS. The EPS stimulation was performed for 120min each, total 4 consecutive days. And cell extracts obtained from each were prepared 3h after EPS. The content of PGC-1α,MyHCI, MyHCIIa, MyHCIIb and MyHCIIx were determined by ELISA. And RT-PCR analysis was applied to determine mRNA expression of PGC-1α, MCAD, Cpt1B, GLUT1, GLUT4, PDH, LDH and GAPDH. RESULTS: Both 10%O2 microenvironment and EPS significantly increased the protein level of PGC1-α, and the synergistic effect of hypoxia and EPS was more significantly. 10%O2 microenvironment significantly decreased the content of MyHCI, MyHCIIx and MyHCIIa in myotubes. EPS significantly decreased the content of MyHCI and MyHCIIx but significantly increased the content of MyHCIIa in myotubes. 10%O2 microenvironment significant decreased the mRNA level of GLTT1 and GLTU4.However, in stimulated myotubes, the mRNA level of GLUT1 was significantly elevated, but the RNA level of GLUT4 was decreased. Meanwhile, 10%O2 microenvironment significantly decreases the mRNA level of MCAD, CPT1B and PDH, and EPS increased the mRNA level of MCAD. CONCLUSIONS: 4 days of 10%O2 microenvironment combined with EPS successfully established myotubes cell contraction model. 10%O2 microenvironment inhibited the proliferation and glycolipid metabolism in myotubes. The usage energy of EPS-induced contraction is based on aerobic oxidation of glucose and fatty. The pulse frequency increases from 1 Hz to 2 Hz in the EPS protocol, were association with substrate from fat to glucose.