Nitric oxide and AMPK cooperatively regulate PGC-1α in skeletal muscle cells

Nitric oxide (NO) induces mitochondrial biogenesis in skeletal muscle cells via upregulation of the peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha). Further, we have shown that nitric oxide interacts with the metabolic sensor enzyme, AMPK. Therefore, we tested the hypothesis that nitric oxide and AMPK act synergistically to upregulate PGC-1 alpha mRNA expression and stimulate mitochondrial biogenesis in culture. L6 myotubes treated with nitric oxide donors, S-nitroso-N-penicillamine (SNAP, 25 mu m) or diethylenetriamine-NONO (DETA-NO, 50 mu m), exhibited elevated AMPK phosphorylation, PGC-1 alpha mRNA and protein, and basal and uncoupled mitochondrial respiration (P < 0.05). Pre-treatment of cultures with the AMPK inhibitor, Compound C, prevented these effects. Knockdown of AMPK alpha 1 in L6 myotubes using siRNA reduced AMPK alpha protein content and prevented upregulation of PGC-1 alpha mRNA by DETA-NO. Meanwhile, siRNA knockdown of AMPK alpha 2 had no effect on total AMPK alpha protein content or PGC-1 alpha mRNA. These results suggest that NO effects on PGC-1 alpha expression are mediated by AMPK alpha 1. Paradoxically, we found that the AMPK-activating compound, AICAR, induced NO release from L6 myotubes, and that AICAR-induced upregulation of PGC-1 alpha mRNA was prevented by inhibition of NOS with NG-nitro-l-arginine methyl ester (l-NAME, 1 mm). Additionally, incubation of isolated mouse extensor digitorum longus (EDL) muscles with 2 mm AICAR for 20 min or electrical stimulation (10 Hz, 13 V) for 10 min induced phosphorylation of AMPK alpha (P < 0.05), which was completely prevented by pre-treatment with the NOS inhibitor, l-NG-monomethyl arginine (l-NMMA, 1 mm). These data identify the AMPK alpha 1 isoform as the mediator of NO-induced effects in skeletal muscle cells. Further, this study supports a proposed model of synergistic interaction between AMPK and NOS that is critical for maintenance of metabolic function in skeletal muscle cells.