Galactose Effects on Human Muscle Cell Metabolism Figure 2. Effect of replacing a glucose medium with a galactose medium on myotube aerobic capacity. A. Basal mitochondrial oxygen consumption rate. *, p,0.05, GAL vs HG and LG. 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. p = 0.06, GAL vs LG. C. Percentage of basal OCR due to proton leak was calculated from the data shown in Figure 2A and B. Data are presented as means 6 SEM, n = 7, in which each condition was assessed in 5–6 replicates. D. Maximal mitochondrial oxygen consumption capacity. After basal and state 4 respiration measurements, cells were treated with FCCP (1 mM) to determine maximal oxygen consumption. *, p,0.05, GAL vs LG. E. Non-mitochondrial oxygen consumption rate. After basal, state and maximal respiration measurements, cells were treated with antimycin (4 mM) to determine non-mitochondrial oxygen consumption. **, p,0.01, GAL vs LG. F. Lactate concentration in the extracellular media of 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 = 7, in which each condition was assessed in duplicate. ***, p,0.0001, GAL vs HG and LG. doi:10.1371/journal.pone.0028536.g002 from obese post-diabetic patients were responsive to GAL at the level of OCR, post-diabetic myotubes and their matched obese non-diabetic myotubes were differentiated for 7 days in HG, LG or GAL. When differentiated in LG or HG, post-diabetic myotubes showed the same basal mitochondrial OCR as obese non-diabetic myotubes (Fig. 5A). However, unlike obese non- diabetic myotubes, basal mitochondrial OCR in post-diabetic myotubes showed no response to GAL, leading to a significant difference between groups (Fig. 5A; p,0.05). This interesting result highlights a defect in mitochondrial function in post- diabetic myotubes. Mitochondrial state 4 OCR (Fig. 5B) and maximal mitochondrial capacity (Fig. 5C) were however not differentially affected by GAL, or different between post-diabetic myotubes and obese non-diabetic myotubes. Interestingly, non- mitochondrial OCR (in the presence of saturating antimycin) was significantly lower in post-diabetic myotubes compared to obese non-diabetic myotubes in the 3 different conditions (Fig. 5D). Post-diabetic myotubes show no increases in COX activity or P-AMPK when differentiated in galactose medium compared to low or high glucose media To identify why post-diabetic myotubes are incapable of increasing oxidative metabolism in response to GAL, we measured mitochondrial content, and COX expression and activity (Fig. 6). Surprisingly, we found a significant increased mitochondrial yield in post-diabetic myotubes differentiated in LG compared with myotubes differentiated in both HG (p = 0.05) and GAL (p,0.01) (Fig. 6A). However, COX activity was not significantly different between conditions due to the high variability in activity between post-diabetic samples (Fig. 6B). Furthermore, COX expression was not significantly increased when post-diabetic cells were differen- tiated in GAL compared to LG or HG (Fig. 6C). We also measured the level of P-AMPK in post-diabetic myotubes differentiated in HG, LG or GAL. In contrast to control myotubes (Figure 3G), post-diabetic myotubes did not show increased AMPK phosphorylation in response to GAL medium (Fig. 6D). PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28536