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. 2012 Aug 8;32(Pt 5):465–478. doi: 10.1042/BSR20120034

Figure 2. Mitochondrial function in C2C12 myotubes chronically treated with insulin.

Figure 2

(A) Oxygen consumption. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 h. Oxygen consumption was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: oligomycin A 10 μg/ml, FCCP 10 μM). Data are means±S.E.M. for five independent experiments. Statistical analysis: Student's t test (*P<0.05, **P<0.01 and ***P<0.001) (B) Cellular ATP content. C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 h. ATP content was measured using a luciferin/luciferase kit (Sigma) and following the manufacturer's instructions. The concentrations of ATP were normalized for the total protein concentrations (μmol/μg of protein) and the results presented as the percentage respective to the control. Results are means±S.E.M. for four independent experiments each measured in triplicate. (C) Mitochondrial membrane potential (Δψ). C2C12 myotubes were left untreated or treated with insulin (100 nM) for 48 h before they were serum-starved in DMEM without serum for 2 h and stained with JC-1 dye as described in the Material and Methods section. Acute treatment of insulin was for 30 min. Cells were trypsinized and analysed by flow cytometry. Data are presented as the percentage of the control; significance was analysed using a Student's t test (***P<0.001). (D) ECAR. Cells were either left untreated (controls) or treated with 100 nM insulin for 48 h. Proton excretion as a measure of glycolysis was determined using a Seahorse XF24 Flux analyser and following the manufacturer's instructions. Data were normalized by protein content. As indicated the following mitochondrial inhibitors were used: oligomycin A 10 μg/ml, FCCP 10 μM). Data are means±S.E.M. for five independent experiments. Statistical analysis: data were analysed using a Student's t test (*P<0.05). (E) ROS production. C2C12 myotubes were either left untreated or treated with insulin (100 nM, 48 h). Cells were washed in PBS and stained with DCF-DA as detailed in the Materials and Methods section. Cells were visualized in a Leica DMI6000 microscope under 488 nm excitation filter and fluorescence was quantified over a 5 min time intervals with Leica LAF software. The means±S.E.M. of fluorescence for n=41–59 cells are shown, analysed by Student's t test (***P<0.001).