Fig. 4.

Untargeted phosphoproteomic analysis reveals that 4-HPAA activates the AMPK pathway and downstream effectors to reduce de novo hepatic lipogenesis. (A) 4-HPAA concentration in the liver and peripheral plasma, 10 min after animals received an intraportal injection of 150 µg 4-HPAA or saline control. (B) Volcano plot of label-free quantitation (LFQ) ratio vs. −log P value for 4-HPAA/control hepatic phosphoproteomic analysis following portal vein injection of 4-HPAA or a saline vehicle control. (C) Pathway enrichment analysis of all hepatic phosphopeptides down-regulated following 4-HPAA/control portal vein injections. (D) Pathway enrichment analysis of top 125 up-regulated hepatic phosphopeptides following portal vein injection of 4-HPAA or a saline vehicle control, n = 5 per group for A–D. (E) Western blot analysis of pAMPKα (T172), total AMPKα, and β-actin with densitometric quantification, n = 6 per group. (F) Western blot analysis of pACC (S79), total ACC, and β-actin with densitometric quantification, n = 6 per group. (G) Primary murine hepatocytes were treated with either DMSO vehicle control (0 μM) or 0.01, 0.1, 1, and 10 μM 4-HPAA for 30 min at which point the protein expression of pACC (S79), total ACC, pAMPKα (T172), total AMPKα, and β-actin was measured via western blot analysis with densitometric quantification, with dots representing the mean of two biological replicates. Statistical analysis for panels A and B was performed using unpaired two-tailed Student’s t test. Individual points represent individual mice, and bars represent group means.