(OXPHOS) for energy [13,19,20,21]. Indeed, different types of cells (e.g., cancer cells, primary fibroblasts) grown in a medium in which glucose is replaced with galactose show a significantly increased oxygen consumption rate compared to cells grown in medium containing a high concentration of glucose (25 mM) [13,14,15]. The more aerobic state of cells grown in galactose is mainly due to a decrease in ATP production via anaerobic glycolysis [22] but is also thought to be due to a modulation of mitochondrial structure [15] and an increase in mitochondrial oxidative capacity as evidenced by increased OXPHOS protein expression and mitochondrial enzymatic activities [15]. Since cells grown in galactose rely mostly on oxidative phosphorylation to produce their ATP, they become more sensitive to mitochondrial toxins than cells grown in high glucose medium [13,23,24]. Another study has shown that primary skin fibroblasts isolated from patients with mitochondrial deficiency (e.g., cytochrome oxidase deficiency, complex I deficiency, pyruvate dehydrogenase complex deficiency or with multiple respiratory chain defects) were not able to survive when cultured in a galactose-based medium [20]. Thus, culturing cells in a galactose medium seems to be a good alternative to high glucose medium in order to study mitochondrial dysfunction. Obesity-associated insulin resistance and type 2 diabetes mellitus (T2DM) is characterized by a decrease in mitochondrial function. Mitochondrial respiration is known to be reduced in the skeletal muscle of T2DM patients [25,26,27] and lean insulin- resistant offspring of T2DM parents [28,29]. Some studies, done on primary muscle cells derived from diabetic patients and matched controls have also shown a decrease in mitochondrial capacity: impaired ATP synthesis [30,31], perturbed palmitate beta-oxidation when exposed to high level of palmitate [9], and impaired citrate synthase activity [7]. However, to our knowledge, no study has directly assessed whether myotubes derived from T2DM patients or patients with a history of T2DM retain a decrease in mitochondrial respiration when cultured in vitro. As mentioned above, this is most likely due to the fact that in vitro, primary human myotubes are mainly glycolytic rendering difficult the study of mitochondrial dysfunction in this model. The use of galactose medium instead of glucose medium to induce a metabolic shift towards a more oxidative phenotype could be a good model to study mitochondrial dysfunction in relation to T2DM and insulin resistance. However, to date, the response of human primary muscle cells to galactose has never been assessed. Here, we characterized the effect of replacing glucose with galactose on aerobic metabolism in primary muscle cells derived from lean and healthy donors. We have shown that differentiating primary human muscle cells in a galactose-replete medium enhances aerobic metabolism without altering mitochondrial content. Moreover, we have shown that this model system can be employed to investigate the mitochondrial bioenergetics of human myotubes derived from post-diabetic patients. To our knowledge, this is the first study that tests the impact of different sources of carbohydrates on human myotube bioenergetics. The value of this model system and its importance to investigating mitochondrial dysfunction in cell culture is also discussed herein. Results Impact of different carbohydrates on cellular physiology and ATP content Glucose restriction has been shown to decrease myotube differentiation [32]. The fusion index in cells differentiated in high glucose (25 mM, HG), low glucose (5 mM, LG) or GAL was not significantly different between the 3 differentiation conditions Galactose Effects on Human Muscle Cell Metabolism (Fig. 1A). However, troponin T expression, which is another marker of myotube differentiation, was significantly decreased in cells differentiated in LG or GAL compared to cells differentiated in HG (p,0.05, Fig. 1B). Yet, cells differentiated in GAL showed the same level of differentiation than cells differentiating in LG. After 7 days of differentiation in the different mediums, the redox state of the cells was assessed using the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) assay. A significant decrease in the redox state was found in myotubes differentiated in LG or GAL compared to HG (p,0.05), with no difference between cells differentiated in LG and GAL (Fig. 1C). We also measured ATP content in myotubes differentiated for 7 days in HG, LG or GAL. As shown in Figure 1D, no significant difference was found in ATP content between myotubes differentiated in the three types of media. Hence, these observations indicate that replacing glucose with galactose does not have a negative effect on cellular physiology and metabolism. Replacing glucose with galactose in differentiation medium increases myotube aerobic capacity Primary human muscle cells differentiated in GAL showed a ,40% increase in basal mitochondrial oxygen consumption rate (basal mitochondrial OCR = basal OCR – non-mitochondrial OCR) compared to cells differentiated in either HG or LG (p,0.05, Fig. 2A). Mitochondrial state 4 respiration (proton leak dependent respiration = (OCR after oligomycin treatment – non- mitochondrial OCR)) tended to be higher in GAL myotubes compared to LG myotubes (p = 0.07) or HG myotubes (p = 0.1) (Fig. 2B) without reaching significance. However, the percentage of basal mitochondrial OCR due to proton leak was not significantly different between treatments (Fig. 2C). There was also a trend for a higher maximal mitochondrial OCR (measured after FCCP treatment) in GAL cells compared to LG cells, but this did not reach significance (p = 0.07, Fig. 2D). The non-mitochondrial OCR was measured by treating cells with antimycin A. Intriguingly, OCR after antimycin A treatment was significantly higher in myotubes differentiated in GAL compared to myotubes differentiated in HG (p,0.01, Fig. 2E). To test the metabolic changes accompanying differentiating cells in GAL further, we measured the amount of lactate released in the medium over a 24 h period. As shown in Figure 2F, cells differentiated in GAL showed a significant decrease in lactate production compared to cells differentiated in LG or HG indicating a decrease in anaerobic metabolism in cells differenti- ated in GAL (p,0.0001). No significant difference in lactate production was found between myotubes differentiated in HG and LG. Taken together, these data confirm that myotubes differentiated in presence of glucose are mostly glycolytic, and that the replacement of glucose by galactose in the differentiation medium forces cells to adopt a more oxidative metabolic state. Effect of galactose on mitochondrial content and enzyme activity In order to determine if the increase in OCR shown in Figure 2 was the result of an increase in mitochondrial biogenesis, different mitochondrial markers were measured. First, mitochondrial yield was determined as mitochondria protein content per total cellular protein content. As shown in Figure 3A, differentiating the cells in GAL did not have any effect on mitochondrial yield compared to myotubes differentiating in HG or LG. Another method to determine mitochondrial content is the staining of cardiolipin with 10N-nonyl acridine orange (NAO). As shown on Figure 3B, this PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28536