Galactose Effects on Human Muscle Cell Metabolism Figure 1. Effect of replacing a glucose medium with a galactose medium on myotube differentiation, redox environment and ATP content. A. Fusion index was measured in myotubes differentiated in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose) media for 7 days. The fusion index has been measured as the number of nuclei in differentiated myotubes (.2 myonuclei) as the percentage of the total number of nuclei in muscle cells (determined by a desmin immunofluorescence staining). Data are shown as mean 6 SEM, n = 3. B. Myotube differentiation was assessed by Troponin T expression after 7 days of differentiation in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose) media. Top panel: representative Western blot of Troponin T expression in myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Beta-actin was used as a loading control. Bottom panel: quantification by densitometry of Troponin T expression. Results are normalized to beta-actin expression. Data are shown as mean 6 SEM, n = 4. *, p,0.05, LG and GAL vs HG. C. Myotube redox environment in response to differentiation in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose) was assessed using the MTT assay as described in the Methods section. Data are presented as mean 6SEM, n = 3, in which each condition was assessed in 6 replicates. D. ATP content in myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Results are presented as means 6 SEM, n = 4, in which each condition was assessed in duplicate. doi:10.1371/journal.pone.0028536.g001 method corroborates that differentiating the cells in HG, LG or GAL do not affect mitochondrial content. These results were confirmed by measuring the protein levels of the mitochondrial markers, complex III and SDHA. The levels of each protein were not significantly affected by the different carbohydrate sources (Fig. 3D). The impact of differentiating cells in galactose on the activity of citrate synthase and cytochrome C oxidase (COX) was also examined. The activities were measured on isolated mitochondria to determine whether the capacity of the TCA (tricarboxylic acid) cycle or the electron transport chain was increased in GAL myotubes, respectively. Citrate synthase activity was not changed by GAL medium compared to either HG or LG medium (Fig. 3C). However, COX activity was significantly higher in GAL myotubes compared to both HG and LG myotubes (p,0.01, Fig. 3E). This higher COX activity was in relation with a higher COX expression level in myotubes differentiating in GAL compared to LG or HG (p,0.05, Fig. 3F). AMPK is a major metabolic sensor that is activated by an increase in the ratio of AMP/ATP in order to restore energy status of the cell trough the stimulation of ATP-producing processes (e.g., glucose uptake, fatty acid oxidation, and mitochondrial biogenesis) [33,34] and the inhibition of ATP-consuming processes (fatty acid synthesis, glycogen synthesis, and protein synthesis) [35,36,37]. To determine if the differentiation of cells in GAL medium also affects AMPK activity, we estimated AMPK activity by measuring AMPK phosphorylation (P-AMPK) in cells differentiated in either HG, LG or GAL for 7 days. As shown on Figure 3G, P-AMPK was higher in cells differentiated in GAL compared to LG or HG (p,0.05 for P-AMPK/b-actin; p = 0.07 for P-AMPK/AMPK). Acute exposure to galactose does not affect myotube oxidative capacity In order to determine if an acute treatment was sufficient to improve the aerobic capacity of the myotubes, cells were differentiated for 7 days in LG, and then incubated for 45 min before measuring oxygen consumption in HG, LG or GAL media. As shown in Figure 4, basal mitochondrial (Fig. 4A), mitochondrial state 4 (Fig. 4B), and maximal mitochondrial OCR (Fig. 4C) were unaffected by an acute treatment with GAL. Hence, these data indicate that in order to induce a metabolic shift towards increased oxidative metabolism, cells have to be exposed to GAL for a prolonged period. Post-diabetic myotubes show incapacity to increase their oxidative metabolism when differentiated in galactose medium The observed effect of galactose on oxidative metabolism prompted us to test the effect of this carbohydrate on the aerobic function of muscle cells collected from patients with a history of diabetes (post-diabetic patients). To test if myotubes derived PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28536