FIGURE 5.

TRB3 regulation of AMPK phosphorylation in mouse primary hepatocytes. A, WT and ALKO mice were fed a control (−EtOH) or ethanol-containing diet (+EtOH) for 28 days. B–E, mouse primary hepatocytes were infected with Ad-scrambled (−Ad-shTRB3) or Ad-shTRB3 (+Ad-shTRB3) for 72 h or transfected with control vector (−MYC-TRB3) or Myc-TRB3 (+MYC-TRB3) at different doses, without (−HA-LKB1 or −HA-CaMKK2) or with HA-tagged LKB1 (+HA-LKB1) or CaMKK2 (+HA-CaMKK2) for 48 h. Means ± S.E. (error bars) shown are representative of at least three independent in vitro experiments or at least two independent in vivo experiments, with the number of mice included in each group in each experiment indicated (n = 8). Statistical significance was determined by Student's t test for the effect of with versus without ethanol in WT or ALKO mice in A or Ad-shTRB3, Myc-TRB3, HA-LKB1, or HA-CaMKK2 versus corresponding controls in B–E (*, p < 0.05); ALKO versus WT under the same treatment (with or without ethanol) in A, with versus without Myc-TRB3 in the presence of HA-LKB1 or HA-CaMKK2 in D and E (#, p < 0.05). A–E, TRB3, p-AMPK, AMPK, p-ACC, and ACC proteins (top or left, Western blotting; bottom or right, quantitative measurements of TRB3, p-AMPK, and p-ACC relative to their total proteins or actin).