column (Phenomenex) operating at a flow rate of 0.7 ml/min. Lactate was detected using an Agilent variable wavelength detector set to 210 nm. The retention time of lactate was confirmed by injection standard lactate solutions. Lactate levels were quantified by injection varying concentrations of standard lactate solutions and the use of Agilent ChemStation software. Extracellular Flux (XF) Analysis of Cellular Metabolic Characteristics 20,000 cells per well were plated in a XF24-well plate (Seahorse Bioscience, North Billerica, MA, USA), grown to ,90% confluency and differentiated for 7 days in HG, LG or GAL. Myotubes were then incubated for 45 min at 37uC at ambient CO2 in HCO3-free DMEM (pH 7.4) containing 4 mM glutamine, 1 mM pyruvate and 25 mM glucose (HG), or 5 mM glucose (LG) or 10 mM galactose (GAL). Oxygen consumption rates (OCR) were determined in situ using a Seahorse Extracellular Flux Analyzer. Baseline oxygen consumption rate was measured 3 times for 4 min each separated by a 2 min wait and a 2 min mix. Following the measurement of basal respiration, oligomycin (600 ng/ml) (Sigma–Aldrich, Oakville, ON, Canada) was injected into each well, followed by 3 cycles of: 2 min mix, 2 min wait and 4 min measurement to measure state 4 (non-phosphorylating) respiration. Then, FCCP (1 mM) (Sigma–Aldrich, Oakville, ON, Canada) was injected into each well, followed by 3 cycles of: 2 min mix, 2 min wait and 2.5 min measurement to measure maximal respiration. Finally, antimycin A (4 mM) (Sigma–Aldrich, Oakville, ON, Canada) was injected into each well, followed by 3 cycles of: 2 min mix, 2 min wait and 3 min measurement to measure extramitochondrial O2 consumption. The cells were then collected for determinations of protein content (Bradford assay). The data are presented as: mitochondrial OCR (basal, oligomycin or FCCP OCR are reported following the subtraction of OCR in the presence of antimycin) and expressed per mg of total cellular protein. Mitochondrial yield, Citrate Synthase (CS) and COX Activity Assays Myoblasts were grown in a 75-cm2 flask to ,90% confluency and then differentiated in their respective differentiation media (HG, LG or GAL) for 7 days. Intact mitochondria were isolated using a kit (MITOISO; Sigma–Aldrich, Oakville, ON, Canada). Mitochondrial and total cellular protein levels were determined by a BCA (bicinchoninic acid) assay. Mitochondrial yield was References 1. Bouzakri K, Roques M, Gual P, Espinosa S, Guebre-Egziabher F, et al. (2003) Reduced activation of phosphatidylinositol-3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes. Diabetes 52: 1319–1325. 2. Jackson S, Bagstaff SM, Lynn S, Yeaman SJ, Turnbull DM, et al. (2000) Decreased insulin responsiveness of glucose uptake in cultured human skeletal muscle cells from insulin-resistant nondiabetic relatives of type 2 diabetic families. Diabetes 49: 1169–1177. 3. Nikoulina SE, Ciaraldi TP, Carter L, Mudaliar S, Park KS, et al. (2001) Impaired muscle glycogen synthase in type 2 diabetes is associated with diminished phosphatidylinositol 3-kinase activation. J Clin Endocrinol Metab 86: 4307–4314. 4. Gaster M, Petersen I, Hojlund K, Poulsen P, Beck-Nielsen H (2002) The diabetic phenotype is conserved in myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen synthase activity. Diabetes 51: 921–927. 5. Gaster M, Rustan AC, Aas V, Beck-Nielsen H (2004) Reduced lipid oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin: evidence from cultured myotubes. Diabetes 53: 542–548. 6. Gaster M, Rustan AC, Beck-Nielsen H (2005) Differential utilization of saturated palmitate and unsaturated oleate: evidence from cultured myotubes. Diabetes 54: 648–656. Galactose Effects on Human Muscle Cell Metabolism expressed as mg of mitochondrial protein per mg of total cellular protein. CS activity was assayed in lysed mitochondria using a kit (CSO720; Sigma–Aldrich, Oakville, ON, Canada) with 0.5 mmol/l oxaloacetate, 0.3 mmol/l acetyl-CoA, 0.1 mmol/l of 5, 59-dithiobis 2-nitro-benzoic acid. Enzyme activity was moni- tored by recording the changes in absorbance at 412 nm over 1.5 min. COX activity was measured in intact mitochondria using a kit (Cytocox1; Sigma–Aldrich, Oakville, ON, Canada) based on the decrease in absorbance over 3 min at 550 nm of ferrocyto- chrome C caused by its oxidation to ferricytochrome C by COX. Enzyme activities were normalized to protein content. Cardiolipin staining and quantification 10N-nonyl acridine orange (NAO, Sigma–Aldrich, Oakville, ON, Canada) staines the mitochondrial phospholipid cardiolipin [44]. NAO is used as a mitochondrial content marker since it is essentially independent of the mitochondrial membrane potential [45]. After differentiation in HG, LG and GAL for 7 days, myotubes were fixed with paraformaldehyde at 4% in PBS for 10 min, and then cardiolipin was stained by NAO (1 mM) for 30 min, in the dark, at room temperature. The dye was then washed 3 times with PBS, and mitochondrial content quantified in a fluorometer (emission: 535 nm, excitation: 485 nm). Statistical analyses Data are presented as mean 6SEM. Statistical analyses were performed using Statview 5.0 (SAS institute Inc, Cary, NC, USA). One-way or 2-ways ANOVAs with Fisher’s protected least significant difference post-hoc test were used to assess statistical differences. P,0.05 was considered to be significant. 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Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y (2007) Circumventing the Crabtree effect: replacing media glucose with galactose increases PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28536