Glucose uptake/consumption in 3T3-L1 cells

Glucose uptake/consumption studies used the glucose oxidase/peroxidase assay to measure the ability of insulin glargine preparations to stimulate glucose uptake in differentiated murine 3T3-L1 adipocyte cells. Briefly, 3T3-L1 cells (Cat No: CL-173, ATCC, Manassas, VA) were seeded at a density of 25,000 cells/well in 200 μL of complete L1 medium (Cat No: 11995, Invitrogen) in a 96-well plate and incubated for 70 ± 2 hours at 37°C and 5% CO₂. The medium was replaced with 200 μL of differentiation medium, incubated for 94 ± 2 hours at 37°C and 5% CO₂ and then replaced with 200 μL of complete L1 medium and further incubated for 70 ± 2 hours at 37°C and 5% CO₂. During this incubation, drug doses were prepared at a 2× concentration in low-glucose assay medium (Cat No: D-5921, Sigma-Aldrich) to be diluted to final concentrations of 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 ng/mL. After the 70-hour incubation in L1 medium was complete, the medium was removed, cells were rinsed with DPBS, and the medium was replaced with 100 μL of DPBS and 100 μL of the diluted insulin glargine concentrations in low-glucose medium. Cells were incubated for 22 ± 2 hours at 37°C and 5% CO₂; then, 10 μL of cell culture supernatant were transferred to a fresh, clear 96-well plate, and 85 μL of water, 40 μL of peroxidase substrate, and 65 μL of glucose oxidase/peroxidase reagent mix (Cat No: G-3660, Sigma-Aldrich) were added. A final incubation at 37°C for 10 ± 2 minutes was followed by reading absorbance at 550 nm in a spectrophotometer.