Fig. 3: DPI stimulates a rapid increase in glycolytic activity through the formation of GPR3- β-arrestin2-GAPDH-PKM2 enzymatic super complex.

a Co-IP of β-arrestin2 with ERK1/2, enolase, GAPDH, and PKM2. ImKCs were transfected with β-arrestin2 and then treated with or without 50 nM DPI for 6 h. Cell lysates were immunoprecipitated with anti-β-arrestin2 and the precipitates were analyzed by Western blotting for the indicated proteins. Shown are representative data from one of the three experiments. b DPI-stimulated glycolysis requires PKM2. BMDMs were prepared from wildtype and Pkm2^−/− mice, seeded, and incubated with or without DPI (50 and 500 nM) for 24 h, and ECAR was measured by a Seahorse analyzer. Data are presented as the mean ± sd from three independent experiments (n = 15 biological replicates). c WT and Pkm2^−/− BMDMs were seeded and incubated with or without DPI (50 and 500 nM) for 24 h in the presence of 2-NBDG to measure glucose uptake. Data are presented as the mean ± sd (n = 4 independent experiments). d, e DPI stimulates enzymatic activities of PKM2 and GAPDH. Wildtype and Arrb2^−/− ImKCs were treated with vehicle (black line) or 500 nM DPI (red line) for 6 h and the enzymatic activities of PKM2 (d) and GAPDH (e) were measured by colorimetric assay kits (Biovision). Data are presented as the mean ± sd from n = 3 independent experiments. P values were calculated by the two-sided student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file.