Glucose uptake

Primary EDL myotubes or transfected C2C12 myotubes were used for glucose uptake experiments. For SAFit2 experiments, a toxicity assay was initially performed to determine the appropriate SAFit2 concentration for subsequent experiments. Based on a lethal dose of 15 (LD 15), the cells were incubated with 0.6 µM SAFit2 or DMSO overnight before inducing glucose uptake.

Basal and insulin-stimulated glucose uptake in primary EDL muscle cells and differentiated C2C12 myotubes was examined. Briefly, the cells were serum-starved in low glucose (1000 mg L⁻¹) DMEM for 4 h, and then incubated in Krebs–Ringer-HEPES (KRH) buffer (136 mM NaCl, 4.7 mM KCl, 10 mM sodium phosphate buffer, 1 mM MgSO₄, 1 mM CaCl₂, and 10 mM HEPES, pH 7.4, 0.2% BSA) for 10 min. The cells were stimulated with insulin (100 nM) or left unstimulated for 1 h. Glucose uptake was induced by the addition of KRH buffer containing 100 µM 2-deoxy-D-[1,2-3 H]glucose, 2 µCi ml⁻¹ (Perkin Elmer) to each well. After 4 min, the reactions were terminated by washing the cells with ice-cold PSB containing 10 µM cytochalasin B (inhibitor of membrane transporter-dependent glucose transport), and then 2 additional washes with ice-cold 1x PBS. Cells were lysed with 0.1 M NaOH for 30 min, and the incorporated radioactivity was determined by liquid scintillation counting. 2-deoxy-D-[1,2-3 H]glucose uptake was furthermore normalized to total protein content assessed by the BCA assay (BCA Protein Assay Kit, Life Technologies, Darmstadt, Germany).