Central role of nitric oxide synthase in AICAR and caffeine-induced mitochondrial biogenesis in L6 myocytes.
5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and caffeine, which activate AMP-activated protein kinase (AMPK) and cause sarcoplasmic reticulum calcium release, respectively, have been shown to increase mitochondrial biogenesis in L6 myotubes. Nitric oxide (NO) donors also increase mitochondrial biogenesis. Since neuronal and endothelial NO synthase (NOS) are calcium dependent and are also phosphorylated by AMPK, we hypothesized that NOS inhibition would attenuate the activation of mitochondrial biogenesis in response to AICAR and caffeine. L6 myotubes either were not treated (control) or were exposed acutely or for 5 h/day over 5 days to 100 microM of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), 100 microM S-nitroso-N-acetyl-penicillamine (SNAP) (NO donor) +/- 100 microM L-NAME, 2 mM AICAR +/- 100 microM L-NAME, or 5 mM caffeine +/- 100 microM L-NAME (n = 12/treatment). Acute AICAR administration increased (P < 0.05) phospho- (P-)AMPK, but also increased P-CaMK, with resultant chronic increases in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), cytochrome-c oxidase (COX)-1, and COX-4 protein expression compared with control cells. NOS inhibition, which had no effect on AICAR-stimulated P-AMPK, surprisingly increased P-CaMK and attenuated the AICAR-induced increases in COX-1 and COX-4 protein. Caffeine administration, which increased P-CaMK without affecting P-AMPK, increased COX-1, COX-4, PGC-1 alpha, and citrate synthase activity. NOS inhibition, surprisingly, greatly attenuated the effect of caffeine on P-CaMK and attenuated the increases in COX-1 and COX-4 protein. SNAP increased all markers of mitochondrial biogenesis, and it also increased P-AMPK and P-CaMK. In conclusion, AICAR and caffeine increase mitochondrial biogenesis in L6 myotubes, at least in part, via interactions with NOS.
McConell
G K
GK
Department of Physiology, The University of Melbourne, Parkville, Victoria 3010, Australia. mcconell@unimelb.edu.au
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Phillips
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Ruan
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Macaulay
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Wadley
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D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D003576:Electron Transport Complex IV / Q000378:metabolism; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008931:Mitochondria, Muscle / Q000187:drug effects* / Q000201:enzymology; D032342:Muscle Cells / Q000187:drug effects* / Q000201:enzymology; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000201:enzymology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D012265:Ribonucleotides / Q000494:pharmacology*; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; C501429:Ppargc1a protein, rat; D016601:RNA-Binding Proteins; D012265:Ribonucleotides; D014157:Transcription Factors; D000620:Aminoimidazole Carboxamide; D002110:Caffeine; D026423:S-Nitroso-N-Acetylpenicillamine; D019001:Nitric Oxide Synthase; D003576:Electron Transport Complex IV; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide; D019331:NG-Nitroarginine Methyl Ester