Hypoxia Promotes Glycogen Accumulation through Hypoxia Inducible Factor (HIF)-Mediated Induction of Glycogen Synthase 1 Nuria Pescador 1,2 , Diego Villar 1,2. , Daniel Cifuentes 3.¤ , Mar Garcia-Rocha 3 , Amaya Ortiz-Barahona 1,2 , Silvia Vazquez 4 , Angel Ordon ˜ ez 6 , Yolanda Cuevas 1,2 , David Saez-Morales 5 , Maria Laura Garcia- Bermejo 5 , Manuel O. Landazuri 4 , Joan Guinovart 3 , Luis del Peso 1,2 * 1 Departamento de Bioquı ´mica, Universidad Auto ´ noma de Madrid, Madrid, Spain, 2 Instituto de Investigaciones Biome ´ dicas ‘‘Alberto Sols’’, Consejo Superior de Investigaciones Cientı ´ficas, Madrid, Spain, 3 Institute for Research in Biomedicine (IRB Barcelona), University of Barcelona, Barcelona, Spain, 4 Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain, 5 Servicio de Inmunologı ´a Hospital de la Princesa, Universidad Auto ´ noma de Madrid, Madrid, Spain, 6 Servicio de Anatomı ´a Patolo ´ gica Hospital Ramo ´ n y Cajal, Madrid, Spain Abstract When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1a or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-a glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia. Citation: Pescador N, Villar D, Cifuentes D, Garcia-Rocha M, Ortiz-Barahona A, et al. (2010) Hypoxia Promotes Glycogen Accumulation through Hypoxia Inducible Factor (HIF)-Mediated Induction of Glycogen Synthase 1. PLoS ONE 5(3): e9644. doi:10.1371/journal.pone.0009644 Editor: Laszlo Tora, Institute of Genetics and Molecular and Cellular Biology, France Received October 13, 2009; Accepted January 27, 2010; Published March 12, 2010 Copyright: ß 2010 Pescador 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 work was supported by grants from Ministerio de Ciencia y Tecnologı ´a/Ministerio de Ciencia e Innovacio ´ n (SAF2005-00180 and SAF2008-03147 to L. P. and SAF2004-0824 to M. O. L.), Red Cardiovascular (RECAVA) and Comunidad Auto ´ noma de Madrid (S-SAL-0311_2006). 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: luis.peso@uam.es . These authors contributed equally to this work. ¤ Current address: Yale Medical School, Yale University, New Haven, Connecticut, United States of America Introduction Aerobic glycolysis yields sixteen times more ATP than glucose fermentation. Thus, metazoans are heavily dependent on the highly energy-efficient aerobic metabolism to meet their large ATP demands. Since oxygen is the final electron acceptor during mitochondrial respiration, this element is essential for metazoan metabolism. In fact, most of the oxygen consumed by animals is required to sustain oxidative phosphorylation. However, several animal species are able to adapt to reduced oxygen tensions during variable periods of time [1]. These include diving animals, fossorial mammals, animals living at high altitude and species of fish that live in stagnant waters, among others [1]. In nearly all these cases, the adaptation to hypoxia involves systemic and cellular responses aimed to reduce oxygen consumption and optimize its utilization. At the cellular level, the reduction in oxygen consumption is mediated by a shift from oxidative to fermentative glucose metabolism [2,3,4,5], the fine-tuning of mitochondrial respiration [6] and an adjustment of mitochondria number. This hypometa- bolic state is achieved by the induction of a specific gene expression program under the control of the Hypoxia-Inducible Factor (HIF) family of transcription factors. This family comprises three transcription factors (HIF-1, HIF-2 and HIF-3) that are heterodimers of a constitutively expressed b subunit and an oxygen-sensitive a subunit (HIF1a, HIF2a or HIF3a) [7,8,9]. In the presence of oxygen, HIFa is extremely unstable due to an oxygen-dependent posttranslational hydroxylation that targets it for proteosomal degradation [10,11,12,13,14]. A family of prolyl hydroxilases (EGLNs), that require molecular oxygen as a reaction cosubstrate, catalyzes the hydroxylation of HIFa [15,16,17]. Hence, under hypoxia, hydroxylation is compromised leading to PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9644