In vitro and in vivo 2-NBDG glucose uptake assay

H9C2 or SGLT1-transfected H9C2 cells were plated at a density of 1×10⁴ cells/well in a 96-well plate and used at subconfluence after 24 hours preincubation. For these experiments, all culture medium was removed from each well and replaced with 100 µL of culture medium in the absence or presence of fluorescent 2-NBDG (50 nm) or 2-NBDG with PKC activators with or without PZ at indicated concentration. Cells were treated with PZ for 30 minutes before treatment with PKC activators. Cells were incubated at 37°C with 5% CO₂ for a period of 30 minutes, and 2-NBDG uptake was measured after three washes with phosphate-buffered saline. After lysis, the fluorescence of the cells was measured at excitation/emission maxima of ~465/540 nm.14

For the in vivo study, 2-NBDG (25 µM/kg, IP) and PKC activator (Molsidomine, 10 mg/kg, IP) were administered to mice (20 g) 10 minutes after PZ injection (400 mg/kg, IP). Forty minutes later, mice hearts were excised and washed in cold saline. Hearts were homogenized with 10 volumes (weight/volume) of ice-cold 0.1 M phosphate buffer (pH 7.4). Homogenates were centrifuged at 10,000 rpm for 15 minutes, and 50 µL supernatant was used to measure 2-NBDG glucose uptake. Fluorescence was measured at excitation/emission maxima of ~465/540 nm.14