Galactose Enhances Oxidative Metabolism and Reveals Mitochondrial Dysfunction in Human Primary Muscle Cells Ce´ line Aguer1, Daniela Gambarotta1, Ryan J. Mailloux1, Cynthia Moffat1, Robert Dent2, Ruth McPherson 3 , Mary-Ellen Harper1* 1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ontario, Canada, 2 Ottawa Hospital Weight Management Clinic, Ottawa, Ontario, Canada, 3 Division of Cardiology, University of Ottawa Heart Institute, Ottawa, Ontario, Canada Abstract Background: Human primary myotubes are highly glycolytic when cultured in high glucose medium rendering it difficult to study mitochondrial dysfunction. Galactose is known to enhance mitochondrial metabolism and could be an excellent model to study mitochondrial dysfunction in human primary myotubes. The aim of the present study was to 1) characterize the effect of differentiating healthy human myoblasts in galactose on oxidative metabolism and 2) determine whether galactose can pinpoint a mitochondrial malfunction in post-diabetic myotubes. Methodology/Principal Findings: Oxygen consumption rate (OCR), lactate levels, mitochondrial content, citrate synthase and cytochrome C oxidase activities, and AMPK phosphorylation were determined in healthy myotubes differentiated in different sources/concentrations of carbohydrates: 25 mM glucose (high glucose (HG)), 5 mM glucose (low glucose (LG)) or 10 mM galactose (GAL). Effect of carbohydrates on OCR was also determined in myotubes derived from post-diabetic patients and matched obese non-diabetic subjects. OCR was significantly increased whereas anaerobic glycolysis was significantly decreased in GAL myotubes compared to LG or HG myotubes. This increased OCR in GAL myotubes occurred in conjunction with increased cytochrome C oxidase activity and expression, as well as increased AMPK phosphorylation. OCR of post-diabetic myotubes was not different than that of obese non-diabetic myotubes when differentiated in LG or HG. However, whereas GAL increased OCR in obese non-diabetic myotubes, it did not affect OCR in post-diabetic myotubes, leading to a significant difference in OCR between groups. The lack of an increase in OCR in post-diabetic myotubes differentiated in GAL was in relation with unaltered cytochrome C oxidase activity levels or AMPK phosphorylation. Conclusions/Significance: Our results indicate that differentiating human primary myoblasts in GAL enhances aerobic metabolism. Because this cell culture model elicited an abnormal response in cells from post-diabetic patients, it may be useful in further studies of the molecular mechanisms of mitochondrial dysfunction. Citation: Aguer C, Gambarotta D, Mailloux RJ, Moffat C, Dent R, et al. (2011) Galactose Enhances Oxidative Metabolism and Reveals Mitochondrial Dysfunction in Human Primary Muscle Cells. PLoS ONE 6(12): e28536. doi:10.1371/journal.pone.0028536 Editor: Raul M. Luque, University of Cordoba, Spain Received June 29, 2011; Accepted November 10, 2011; Published December 15, 2011 Copyright: 2011 Aguer 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 research was funded by a grant from the Canadian Institutes of Health Research (Institute of Nutrition, Metabolism and Diabetes). The funder 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: mharper@uottawa.ca Introduction Human primary muscle cells are a widely used model system to study muscle metabolism and its related disorders. Cell culture of human primary myotubes offers not only an excellent and dynamic model for studying metabolism under standardized conditions, but provides an excellent system for studying metabolic disorders. Indeed, a number of studies have shown that human primary muscle cells retain the same metabolic phenotype as those previously evidenced in vivo in muscle [1,2,3,4,5,6,7,8,9,10,11]. The oxidative capacity of skeletal muscle is highly influenced by various genetic and environmental factors; cultured myotubes offer a unique model that separates these two influences on metabolic phenotype [7]. Although a valuable model system, cultured muscle cells are highly glycolytic when grown and differentiated under high glucose conditions relative to muscle tissue in vivo [12], complicat- ing the study of mitochondrial dysfunction. Indeed, myoblasts are routinely cultured in a standard high glucose medium which can diminish mitochondrial function. In the same way, rapidly proliferating cancer cells are known to be highly glycolytic when grown in high glucose conditions [13,14,15], a phenomenon known as the Crabtree effect, when glucose inhibits oxidative phosphorylation [16]. This phenomenon is not restricted to cancer cells. Other types of cells including embryonic tissues [17] and proliferative thymocytes [18] show diminished oxidative metabo- lism when grown under high glucose conditions. Whereas the production of pyruvate via glycolytic metabolism of glucose yields 2 net ATP, the production of pyruvate via glycolytic metabolism of galactose yields no net ATP, forcing cells to have an increased reliance on oxidative phosphorylation PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28536