Fig. 4.

Expression of AMPK-dominant negative (DN) prevents the insulin-sensitizing effect of serum starvation. Uninfected (control) myotubes and myotubes that were adenovirally transduced with green fluorescent protein (GFP) or AMPK-DN were incubated in serum-containing or serum-free medium for 3 h. A: Western blot was performed using antibodies specific to AMPKα1, AMPKα2, AMPK, and myc-tag, and P-ACC. The use of myc-tag here is to demonstrate that the AMPK-DN (which is AMPKα2 containing a mutation of a single amino acid) was effectively expressed. B: representation of a glucose transport assay with cells expressing AMPK-DN or GFP. Differentiated cells were incubated in serum-free medium for 3 h. They were further incubated in HBS containing 5 mM nonradiolabeled d-glucose in the presence or absence of 100 nM insulin for 20 min before glucose transport assay (n = 12/group). *Significant difference from corresponding control not treated with insulin (P < 0.05); †significant inhibition of the insulin effects by expression of AMPK-DN. C: ATM and ACC (n = 9/group). D: P-ACC and ACC (n = 9/group). *Serum-starvation-related increase (P < 0.05), which does not occur (†P < 0.05 vs. serum-starved cells expressing GFP) in cells expressing AMPK-DN (C and D). E: myotubes were incubated in the absence (C, control) or presence (A, AICAR) of 2 mM AICAR (an AMPK activator) for 1 h and assessed for ATM (n = 5/group) or P-AMPK and AMPK (n = 5–6/group). *Effect of AICAR (P < 0.05). Data represent means ± SE.