2 nbdg

Seven days after treatments, the glucose uptake was assayed by 2‐NBDG (MedChemExpress) according to the manufacturer's protocol. 2‐NBDG is a fluorescent glucose analog that has been used to monitor glucose uptake in live cells. Therefore, the intensity of 2‐NBDG immunofluorescence reflects glucose uptake and insulin sensitivity. C2C12 cells were incubated with or without media containing 10 nM insulin (Sigma Chemical Inc.) for 10 min. The media was changed to low‐glucose DMEM containing 150 μg/ml 2‐NBDG for 60 min at 37℃. The medium was removed, and cells were washed 5 times with PBS. Nikon Eclipse Ti‐s microscopy (Nikon) was used to observe fluorescence. Fluorescent data were acquired at excitation and emission wavelengths of 490 and intensity at 525 nm.

2-NBDG (Med Chem Express, USA), a fluorescent D-glucose analog, was used to detect glucose uptake level of cells following treatment with β-BA. The stock solution was diluted with basic medium to yield 20 μM working solution in advance. After treatment with 40 μM β-BA for 48 h, 2-NBDG working solution was added to cover the surface of culture cells sufficiently and incubated at 37°C for 30 min. Then, the cells were washed with PBS twice to remove residual reagent and detected as soon as possible. The intensity of fluorescence was detected using flow cytometry (Beckman, USA) with FL1.

For glucose uptake assay, cells were treated with 2-NBDG (50 µM, MedChemExpress) for 1 h, the level of glucose uptake was measured by fluorescence intensity (Varioskan™ LUX, Thermo Scientific). For lactate production assay, cells were plated on 6-well plates and cultured for 24 h. The lactate in the culture medium was measured by Lactic Acid Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The pH of the media was measured by PB-10 (Sartorius).