DARP regulates AMPK activity in C2C12 cells.

<p>(A) Phosphorylation of AMPK in C2C12 myotubes transfected with either negative (scramble) or DARP siRNA (DARP-KD) was assessed by immunoblotting. Silencing of DARP significantly enhanced the phosphorylation of AMPK. *P<0.05 (n = 3 each). (B) Phosphorylation of AMPK and ACC in C2C12 myotubes in the presence or absence of AICAR was assessed by immunoblotting. Silencing of DARP significantly enhanced the phosphorylation of AMPK and ACC in the absence of AICAR. Treatment with AICAR enhanced phosphorylation of AMPK and ACC in C2C12 myotubes, and the difference of their phosphorylation between scramble and DARP-KD cells disappeared when treated with AICAR. *P<0.05, **P<0.01, #Not significant (n = 3 each). (C) Glucose uptake in C2C12 myotubes was assessed by measuring 2-deoxyglucose uptake using an enzymatic photometric assay. Cells were transfected with either scramble or DARP siRNA (DARP-KD), and the glucose uptake was analyzed in the presence or absence of AICAR. Silencing of DARP significantly increased glucose uptake in C2C12 myotubes comparing to control cells. *P<0.05 (n = 5 each). Treatment with AICAR increased glucose uptake in control cells and abolished the increased glucose uptake induced by DARP-silencing. **P<0.01, #Not significant (n = 5 each). (D) Glucose oxidation in C2C12 myotubes was assessed by ¹⁴CO₂ production from ¹⁴C-D-glucose. Cells were transfected with either scramble or DARP siRNA (DARP KD). DARP-silencing enhanced the glucose oxidation in C2C12 myotubes. **P<0.01 (n = 5 for scramble, n = 4 for DARP KD). (E) Treatment with AICAR abrogated the increase in glucose oxidation induced by DARP-silencing (n = 3 each). (F) C2C12 myoblasts were infected with retroviruses carrying GFP (GFP) or DARP (DARP OE) gene, and then phosphorylation of AMPK was assessed by immunoblotting. Overexpression of DARP significantly reduced the phosphorylation of AMPK. **P<0.01 (n = 3 each).