Glucose uptake measurements in cellular models

Glucose uptake was determined in primary human skeletal muscle cells as described⁴². Cells were starved of serum for 4 h prior to treatment with insulin. Glucose uptake was determined following incubation of cells for 1 h in absence or presence of insulin, prior to addition of 2-[1,2-³H]deoxy-D-glucose (Moravek Inc., CA) and 10 mM unlabeled 2-deoxy-D-glucose for 15 min. Cells were homogenized with 0.03% SDS and the amount of 2-[1,2-³H]deoxy-D-glucose in the homogenates was determined by scintillation counting.

Glucose uptake in primary mouse skeletal muscle cells was determined as described⁴⁸. Cells were serum-starved for 4 h before incubation in the absence or presence of insulin for 20 min. Thereafter, cells were rinsed twice with HEPES-buffered saline (140 mM NaCl, 20 mM Na-HEPES, 2.5 mM MgSO₄, 1 mM CaCl₂, 5 mM KCl, pH 7.4) and incubated for 5 min (with 2-[1,2-³H]deoxy-D-glucose and 10 mM unlabeled 2-deoxy-D-glucose.

C2C12 myotubes were serum-starved for 4 h and then incubated in the absence or presence of insulin. Thereafter, glucose uptake was determined for 15 min in cells incubated with 2-[1,2-³H]deoxy-D-glucose and 10 mM unlabeled 2-deoxy-D-glucose. Cells were homogenized with 0.03% SDS and the amount of 2-[1,2-³H]deoxy-D-glucose in homogenates was determined by scintillation counting.

Normalization of glucose uptake was performed to total protein content (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific) and scintillation counting was determined using a 1414 WinSpectral Liquid Scintillation Counter (Wallac, Perkin-Elmer, MA). All radioisotopes were acquired from Perkin-Elmer if not otherwise stated.