Department of Physiology, The University of Melbourne, Parkville, Victoria 3010, Australia.
J Appl Physiol (1985). 2010 Mar;108(3):589-95. doi: 10.1152/japplphysiol.00377.2009. Epub 2009 Dec 31.
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.
5-氨基咪唑-4-甲酰胺核苷酸（AICAR）和咖啡因分别通过激活 AMP 激活的蛋白激酶（AMPK）和引起肌浆网钙离子释放来增加 L6 肌管中的线粒体生物发生。一氧化氮（NO）供体也增加线粒体生物发生。由于神经元和内皮型一氧化氮合酶（NOS）依赖于钙离子，并且还被 AMPK 磷酸化，因此我们假设 NOS 抑制会减弱对 AICAR 和咖啡因反应的线粒体生物发生的激活。L6 肌管要么不处理（对照），要么急性暴露或每天处理 5 小时，持续 5 天，用 100μM N（G）-硝基-L-精氨酸甲酯（L-NAME，NOS 抑制剂）、100μM S-亚硝基-N-乙酰青霉胺（SNAP）（NO 供体）±100μM L-NAME、2mM AICAR±100μM L-NAME 或 5mM 咖啡因±100μM L-NAME（n = 12/处理）。急性 AICAR 给药增加（P < 0.05）磷酸化（P-）AMPK，但也增加 P-CaMK，导致过氧化物酶体增殖物激活受体γ共激活因子-1α（PGC-1α）、细胞色素-c 氧化酶（COX）-1 和 COX-4 蛋白表达与对照细胞相比慢性增加。NOS 抑制对 AICAR 刺激的 P-AMPK 没有影响，但出人意料地增加了 P-CaMK，并减弱了 AICAR 诱导的 COX-1 和 COX-4 蛋白增加。咖啡因给药增加了 P-CaMK 而不影响 P-AMPK，增加了 COX-1、COX-4、PGC-1α 和柠檬酸合酶活性。NOS 抑制出人意料地大大减弱了咖啡因对 P-CaMK 的影响，并减弱了 COX-1 和 COX-4 蛋白的增加。SNAP 增加了所有线粒体生物发生的标志物，并且还增加了 P-AMPK 和 P-CaMK。总之，AICAR 和咖啡因通过与 NOS 的相互作用至少部分地增加 L6 肌管中的线粒体生物发生。
