Galactose Effects on Human Muscle Cell Metabolism Figure 5. Absence of an increase in basal oxygen consumption in post-diabetic myotubes differentiated in galactose media. A. Basal oxygen consumption rate. *, p,0.05, GAL vs HG and LG. #, p,0.05, post-diabetic vs obese. 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. D. Non-mitochondrial oxygen consumption rate. After basal, state and maximal respiration, cells were treated with antimycin (4 mM) to determine non-mitochondrial oxygen consumption. ##, p,0.001, post-diabetic vs obese. A–D. Myotubes were 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 = 5, in which each condition was assessed in 5–6 replicates. doi:10.1371/journal.pone.0028536.g005 the increased COX activity and oxygen consumption in GAL myotubes could be the result of an increased AMPK phosphor- ylation. Other studies will be needed to test this hypothesis. To test if an acute treatment with GAL could have the same effect as differentiating the cells for 7 days in GAL, myotubes were differentiated for 7 days in LG medium and then acutely treated for 45 min with HG, LG or GAL before OCR measurement. This acute treatment with GAL was not sufficient to increase OCR compared to LG or HG treatments. This may be attributed to the fact that glycolytic intermediates would not be completely depleted after a 45 min incubation in GAL medium. Taken together, those results show that differentiating human primary myotubes in GAL for 7 days could be an easy way to improve the aerobic capacity of these cells and decrease their reliance on anaerobic glycolysis. An unexpected and intriguing finding was the increase in non- mitochondrial OCR in myotubes differentiated in GAL compared to HG. Extra-mitochondrial sites for oxygen consumption include the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, nitric oxide synthase, and the xanthine oxidase. Measurement of the cell redox environment with the MTT assay is based on a reaction in which the formed NAD(P)H reduces tetrazolium (MTT) reagent to a blue formazan. The intensity of the reduced product colour is thus proportionate to the NAD(P)+/ NAD(P)H concentration in the cells [39]. Since the redox environment measured with the MTT assay was significantly lower in LG and GAL cells compared to HG cells, this could indicate a decrease in reduced pyridine nucleotide levels in LG and GAL myotubes compared to HG myotubes. A similar decrease in redox state was also found by others in HeLA cells grown in 10 mM galactose compared to 25 mM glucose [15]. Further studies are needed to directly assess NAD(P)H level and NAD(P)H oxidase activity to confirm this hypothesis. Galactose medium is often used to study the effect of mitochondrial toxins in cancer cells [13,23,24] and has also been used to examine mitochondrial dysfunction in primary skin fibroblasts derived from patients with defect in mitochondrial function [20]. In the present study, we have shown, not only that GAL is able to increase the oxidative metabolism of healthy human primary muscle cells, but also that GAL can be used to identify mitochondrial dysfunction in primary muscle cells derived from post-diabetic patients. Indeed, unlike myotubes derived from matched obese non-diabetic subjects, myotubes derived from post- diabetic patients were not able to increase their oxygen consumption rate when differentiated in GAL. Thus, in spite of the fact that there was no difference in OCR between obese non- diabetic myotubes and post-diabetic myotubes when differentiated in LG or HG, the GAL media was able to highlight a decreased oxidative capacity in post-diabetic myotubes compared to obese non-diabetic myotubes. Other studies have described a decrease in oxidative metabolic capacity in myotubes derived from diabetic patients as indicated by a decrease in citrate synthase activity [7] and a decrease in lipid oxidation capacity [5,9]. Myotubes derived from post-diabetic subjects are also known to have a decreased oxidative capacity when challenged with a high glucose medium (decreased mitochondrial content, decreased citrate synthase and COX activities) compared to myotubes derived from matched PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28536