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. 2021 Dec 15;131(24):e140436. doi: 10.1172/JCI140436

Figure 6. Glucose uptake is restored by WWP1 inhibition through the activation of AMPKα2.

Figure 6

(A) Analysis of polyubiquitination of the AMPKα isoform in 293T cells. CA, catalytically inactive mutant. (B) Assessment of AMPK activity in Wwp1-depleted C2C12 cells. Differentiated C2C12 cells were treated with BKM120 (2.0 μM) for 17 hours with serum starvation and subjected to WB analysis. Blots are from duplicate gels run in parallel. (C) Evaluation of AMPK activation in quadriceps muscle tissue from Wwp1+/+ and Wwp1–/– mice. Mice starved for 3 hours were treated with vehicle or BKM120 (50 mg/kg) for 1 hour. Data shown are representative of 2 independent experiments. (D) Quantification of GLUT4 localized on the cell surface upon BKM120 treatment. C2C12-Myc-GLUT4-mCherry cells with stable expression of WWP1 shRNA were treated with DMSO or BKM120 for 17 hours and subjected to immunofluorescence staining with anti–Myc-tagged antibody, followed by measurement via flow cytometry. Each plot shows the mean ± SD of triplicate assays. (E) Quantitation of glucose uptake upon BKM120 treatment. C2C12-shWWP1 cells transduced with AMPK or PTEN siRNA were starved and treated with BKM120 for 2 hours followed by insulin stimulation for 15 minutes. Each plot shows the mean ± SD of triplicate assays. **P < 0.01 and ***P < 0.005, by 2-tailed t test (D) and 1-way ANOVA followed by Tukey-Kramer multiple-comparison test (E).