osal and PGC1a in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity. We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal and mitochondrial biogenesis and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish. Citation: Magnoni LJ, Vraskou Y, Palstra AP, Planas JV AMP-Activated Protein Kinase Plays an Important Evolutionary Conserved Role in the Regulation of Glucose Metabolism in Fish Skeletal Muscle Cells. PLoS ONE 7: e31219. doi:10.1371/journal.pone.0031219 Editor: Alejandro Lucia, Universidad Europea de Madrid, Spain Received October 26, 2011; Accepted January 4, 2012; Published February 16, 2012 Copyright: 2012 Magnoni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the Ministerio de Ciencia e Innovacion, Spain to JVP. LJM was supported by a FP7-PIIF-2009 fellowship from the UPF 1069 chemical information European Commission with grant agreement number 235581. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: 17062696” [email protected] Current address: Institute for Marine Resources and Ecosystem Studies of the Wageningen 
University, Yerseke, The Netherlands Introduction AMP-activated protein kinase is a phylogenetically conserved enzyme which has been suggested to ” act as a `metabolic master switch’ mediating the cellular adaptation to environmental or nutritional stress factors. This fuel-sensing enzyme is activated by phosphorylation when a cellular stress increases the AMP:ATP ratio due to limited generation of ATP or increased ATP depletion and, consequently, AMP production. Activation of AMPK leads to the concomitant inhibition of energy-consuming biosynthetic pathways not required for survival and to the activation of metabolic pathways that regenerate the ATP, including glucose uptake and its subsequent utilization by the tissues. It is well recognized that in order to understand how energy balance is maintained in the organism it is important to study the mechanisms involved in the activation of AMPK in skeletal muscle. This organ, that contributes to 40% of the resting metabolic rate, undergoes an energetic challenge during exercise-induced muscle contraction, when it shows a remarkable increase in its ATP turnover rate. Furthermore, AMPK is activated in the skeletal muscle of mammals by exercise and this
