title: Nitric oxide and AMPK cooperatively regulate PGC-1 in skeletal muscle cells.
abstract: Nitric oxide (NO) induces mitochondrial biogenesis in skeletal muscle cells via upregulation of the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). 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-1α mRNA expression and stimulate mitochondrial biogenesis in culture. L6 myotubes treated with nitric oxide donors, S-nitroso-N-penicillamine (SNAP, 25 μM) or diethylenetriamine-NONO (DETA-NO, 50 μM), exhibited elevated AMPK phosphorylation, PGC-1α 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 AMPKα1 in L6 myotubes using siRNA reduced AMPKα protein content and prevented upregulation of PGC-1α mRNA by DETA-NO. Meanwhile, siRNA knockdown of AMPKα2 had no effect on total AMPKα protein content or PGC-1α mRNA. These results suggest that NO effects on PGC-1α expression are mediated by AMPKα1. Paradoxically, we found that the AMPK-activating compound, AICAR, induced NO release from L6 myotubes, and that AICAR-induced upregulation of PGC-1α mRNA was prevented by inhibition of NOS with N(G)-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 AMPKα (P < 0.05), which was completely prevented by pre-treatment with the NOS inhibitor, L-N(G)-monomethyl arginine (L-NMMA, 1 mM). These data identify the AMPKα1 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.
authors.affiliation: Center for Exercise Science, Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Vitor A
authors.initials: VA
authors.lastname: Lira
authors.forename: Dana L
authors.initials: DL
authors.lastname: Brown
authors.forename: Ana K
authors.initials: AK
authors.lastname: Lira
authors.forename: Andreas N
authors.initials: AN
authors.lastname: Kavazis
authors.forename: Quinlyn A
authors.initials: QA
authors.lastname: Soltow
authors.forename: Elizabeth H
authors.initials: EH
authors.lastname: Zeanah
authors.forename: David S
authors.initials: DS
authors.lastname: Criswell
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D051379:Mice; D008813:Mice, Inbred ICR; D008928:Mitochondria / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010101:Oxygen Consumption; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D034741:RNA, Small Interfering; D026423:S-Nitroso-N-Acetylpenicillamine; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D014226:Triazenes
chemical_list: C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; C485290:Ppargc1a protein, mouse; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; D014226:Triazenes; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D055372:AMP-Activated Protein Kinases