Figure 5.

Metformin induces widespread inhibition of protein synthesis. (A) Metformin abrogates the expression of p‐4EBP1 and p‐eIF4E at 24 h, and the effect is partially rescued by coincubation of cells with the mTOR activator MHY1485, in HCT116 cells. (B) The AMPK activator AICAR reduces MYC expression, similar as metformin in HCT116 cells. (C) Metformin blocks protein synthesis, as shown by reduced FITC signal, in HT29 cells stained with OPP, a puromycin analog that incorporated only to newly synthesized proteins. (D) Left panel shows the rational of using ribopuromycylation (puromycin labeling) to monitor protein synthesis, after which the newly synthesized protein can be detected by antibody against puromycin. Metformin strongly blocks protein synthesis, shown by reduced puromycin labeling. AICAR induces a similar yet weaker effect. HT29 cells were treated with metformin (2.5 mm) or AICAR (0.5 mm) for 24 h, and then, the cells were incubated with puromycin (10 μg/mL) for 20 min. Total proteins were isolated and detected by western blot using puromycin antibody. (E) Metformin causes an increase of mRNA association with polysomes and a reduction of mRNA association with monosomes.