Figure 3. PCK1 activates AMPK^Thr172/GFAT1^Ser243 phosphorylation and inhibits UDP-GlcNAc biosynthesis.

Representative immunoblots showing AMPK^Thr172 and GFAT1^Ser243 phosphorylation in PCK1-OE cells (A) and PKO cells (B). For immunoblot analysis, PKO cells were treated with metformin (Met, 2 mM) for 12 hours (C) or PCK1-OE cells were transfected with an AMPK shRNA1/2 plasmid (D). (E–H) Hepatoma cell growth curves and colony formation capacity. PKO cells and PCK1-OE cells were treated as indicated. (I) Relative levels of UDP-GlcNAc in PKO cells treated for 24 hours with the GFAT1 inhibitor DON (20 μM), as measured by LC-MS. (J) PKO cells were treated with 20 μM DON for 24 hours. (K) Working model whereby PCK1 ablation promotes UDP-GlcNAc biosynthesis, O-GlcNAcylation, and proliferation of HCC cells through increased oxaloacetate accumulation and activation of the AMPK-GFAT axis. Data are represented as mean ± SD (n ≥ 3 experiments). *P < 0.05, **P < 0.01, ***P < 0.001, as determined using 2-tailed unpaired Student’s t test (2 groups) or 1-way ANOVA, followed by Tukey’s test (more than 2 groups). Data are representative of at least 3 independent experiments.