Bay 876

Flushed bone marrow from both femurs and tibiasof C57BL/6or Myd88^−/− mice were cultured in DMEM medium supplemented with 10% de-complemented FBS, 100 units/mL penicillin and 100 ug/mL streptomycin, 2 mM L-glutamine, and 20% of L929 conditioned media for for six days at 37°C and 5% CO₂. On day six, BMDM were trypsinized and plated in DMEM medium supplemented with 10% de-complemented FBS, 2 mM L-glutamine, 100 units/mL penicillin and 100 ug/mL streptomycin, and 10 ng/mL recombinant murine M-CSF (Peprotech, #315–02) for one day prior being stimulated for five hours with 100 ng/mL of LPS or 250 μg/mL of MSUc. 20 mM of JNK inhibitor SP600125 (Tocris, #1496), 10 mM of BHA (Sigma, B1253), 20 nM of BAY-876 (Selleckchem, #S8452), 3 μM of p38 MAPK inhibitor SB203580 (Tocris, #1202) or NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809) were added to the media one hour prior to stimulation with LPS or MSUc.

hBEC were split and seeded onto a 96-well plate (2x10⁴/0.1 mL/well) coated with 0.1% gelatin solution (Sigma-Aldrich, ES-006-B). After ~24 h, cells formed monolayer, then glucose uptake was performed. Cells were treated with vehicle (Veh; PBS, Sigma-Aldrich, D8537) or human insulin (hINS; 1.7 µM; Sigma-Aldrich, I9278) or human leptin (hLep; 62.5 nM, Thermo Fisher Scientific, RP-8634) or AAC2 (0.1 µM) which were diluted in same glucose free DMEM (Gibco, A14430, 200 µL/well) for 40 min. Then, GLUT1 inhibitor (BAY-876; 10 nM diluted in DMSO; Selleckchem, Houston, TX, S8452) or DMSO were added in 100 µL of FD-working solution per well and incubated for 50 min at 37°C. 3T3-L1 preadipocytes were seeded in a 96 well plate at a density of 4x10³ in 100 µL of culture medium per well and grow for 24 h prior to measurement of glucose uptake. Cells were pre-incubated with GLUT1 inhibitor (BAY-876; 10 nM diluted in DMSO) or DMSO diluted in DMEM not containing glucose phenol red, and L-glutamine (Gibco, A14430, 200 µL/well) for 40 min. Then, Veh or AAC2 (0.1 µM) or human insulin (hINS; 1.7 µM; Sigma-Aldrich, I9278) or mouse leptin (mLep; 12.5 nM, Peprotech, 450–31, Rocky Hill, NJ) were treated to cells with FD-working solution for 80 min.