Figure 4. Effect of an acute treatment with galactose medium on myotube aerobic capacity. A. Basal oxygen consumption rate. B. State 4 respiration (leak dependent; non-phosphorylating). After basal oxygen consumption rate measurement, cells were treated with oligomycin (600 ng/ml) to determine state 4 respiration. C. Maximal oxygen consumption capacity. After basal and state 4 respiration, cells were treated with FCCP (1 mM) to determine maximal oxygen consumption. A–C. Myotubes were differentiated for 7 days in LG (5 mM glucose). On the 7th day of differentiation, myotubes were exposed to HG (25 mM), LG (5 mM glucose) or GAL (10 mM galactose) for 45 min before measuring basal oxygen consumption rate. Results are presented as means 6 SEM, n = 4, in which each condition was assessed in 5 replicates. doi:10.1371/journal.pone.0028536.g004 Discussion The present study identifies galactose differentiation medium as a useful experimental system to analyze mitochondrial metabolism in human primary muscle cells. Our findings demonstrate that this system provides an effective method to elucidate mitochondrial dysfunction in myotubes derived from post-diabetic patients. Our main results show that OCR was 30% higher in healthy myotubes differentiating in GAL compared to LG or HG media, which is due, at least in part, to an increase in AMPK phosphorylation and COX activity in GAL treated cells. When differentiated in LG or HG media, post-diabetic myotubes had similar OCR as myotubes derived from matched obese non-diabetic volunteers. Interesting- ly, unlike obese non-diabetic myotubes, post-diabetic myotubes were unable to increase their OCR in response to GAL medium. Post-diabetic myotubes were also unable to increase AMPK Galactose Effects on Human Muscle Cell Metabolism phosophorylation and COX activity when differentiated in GAL. To our knowledge, this study is the first to show that the use of a GAL medium is a simple means to increase OCR in human primary muscle cells and to potentially identify mitochondrial dysfunction. To better characterize the effect of different carbohydrate sources and/or concentration during cell culture, we first determined the level of differentiation in myotubes differentiated for 7 days in LG (5 mM glucose), HG (25 mM glucose) or GAL (10 mM galactose). Myotubes differentiated in HG showed the highest level of differentiation as indicated by troponin T expression level, which is commonly used as a marker of muscle cell differentiation [8,38]. Thus, we have confirmed in our model (human primary muscle cells) that glucose availability can affect the level of muscle cell differentiation as previously reported by others [32] in muscle cell lines (C2C12) and mouse primary muscle cells. However, there were no differences in the levels of differentiation in myotubes differentiated in 5 mM glucose (LG) compared to 10 mM galactose (GAL), showing that the metabolic differences found between LG and GAL myotubes were not the result of alteration in levels of differentiation. As previously shown by others in different cell lines or primary fibroblasts [13,14,15], replacing glucose with galactose in the medium forces the cells to become more oxidative in order to maintain ATP levels. In accordance with an increased oxidative metabolism, differentiating the cells in GAL decreased anaerobic metabolism as shown by the diminished generation of lactate. The sharp decrease in lactate production by the myotubes differenti- ated in GAL is not surprising since the production of pyruvate through galactose metabolism yields no net ATP, forcing the cells to rely on aerobic metabolism instead of anaerobic glycolysis to produce ATP [13,19,20,21]. The increase in oxygen consumption rate in cells differentiated in GAL was not due to an increase in proton leak-dependent oxygen consumption rate as shown by the proportion of total respiration that was due to proton leak. A trend for a higher maximal respiration rate in myotubes differentiated in GAL compared to LG was also found, but this did not reach significance (p = 0.07). The higher mitochondrial OCR in GAL cells was not the result of an increase in mitochondrial content as evidenced by the similar levels of various mitochondrial markers (mitochondrial yield, cardiolipin staining, SDHA and complex III expressions). Furthermore, citrate synthase activity, commonly used as a marker of TCA cycle activity, was not affected by the different sources of carbohydrates. Interestingly, this increase in mitochondrial OCR was related to a ,70% increase in mitochondrial COX activity certainly due to an increase in COX expression. Our results on human primary muscle cells are in accordance with a previous report showing increases in COX expression and activity in HeLa cells grown in 10 mM galactose compared to 25 mM glucose [15]. We also found an increased AMPK phosphorylation in myotubes differentiated in GAL compared to LG or HG. AMPK is a major metabolic sensor in cells and is activated by increased AMP/ATP for the purpose of restoring the energy status of the cell [33,34,35,36,37]. An increase AMPK phosphorylation in response to a decrease in glucose availability (25 mM compared to 5 mM) has also been found by others in C2C12 myotubes [32]. In our primary muscle cell model, we were not able to detect an increased P-AMPK when cells were differentiated in 5 mM glucose compared to 25 mM glucose, consistent with the conclusion that 5 mM glucose is not restrictive enough in our model. However, a nice increase in P-AMPK was found when myotubes were differentiated in GAL, suggesting that GAL medium induced a deprivation of energy, which in turn did activate AMPK in order to restore energy status of the cells. Thus, PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28536