AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity 
is inhibited in settings of insulin resistance. Exposure to a high glucose 
concentration has recently been shown to increase phosphorylation of AMPK at 
Ser(485/491) of its α1/α2 subunit; however, the mechanism by which it does so is 
not known. Diacylglycerol (DAG), which is also increased in muscle exposed to 
high glucose, activates a number of signaling molecules including protein kinase 
(PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in 
inhibition of AMPK by causing Ser(485/491) phosphorylation in skeletal muscle 
cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 
12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- 
and time-dependent increases in AMPK Ser(485/491) phosphorylation, which was 
associated with a ∼60% decrease in AMPKα2 activity. Expression of a 
phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in 
AMPK activity. Serine phosphorylation and inhibition of AMPK activity were 
partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the 
specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented 
Ser(485/491) phosphorylation of AMPK. Inhibition of previously identified 
kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent 
these events. PMA treatment also caused impairments in insulin-signaling through 
Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 
phosphorylated AMPKα2 at Ser(491) in cell-free conditions. These results 
identify PKD1 as a novel upstream kinase of AMPKα2 Ser(491) that plays a 
negative role in insulin signaling in muscle cells.