post-diabetic myotubes (data not shown). Studying the mechanism underlying this phenomenon was not the purpose of our study and needs further investigation. To our knowledge, this is the first study to test the impact of different carbohydrate sources on human myotube bioenergetics. Furthermore, this is the first study that has directly assessed oxygen consumption rate in vitro in myotubes derived from post-diabetic patients and demonstrated decrease oxygen consumption rates in post-diabetic myotubes compared to obese non-diabetic myotubes. Moreover, we have shown that differentiating cells in GAL is an excellent model system to investigate the mitochondrial bioener- getics of human myotubes derived from patients with a history of T2DM. The use of this model could enable further research to better understand the molecular mechanisms leading to mito- chondrial dysfunction during the development of insulin resistance and other metabolic disorders in skeletal muscle. Materials and Methods Ethics statement All participants gave informed consent and the experimental protocol was approved by the Human Research Ethics Commit- tees of the Ottawa Hospital and the University of Ottawa Heart Institute. Muscle biopsy and primary human muscle cell culture Biopsies of vastus lateralis were obtained from 7 lean and healthy participants (male/female: 6/1, age: 41 years 66.7, BMI (Body Mass Index): 20.8 kg/m2 60.5) and from 5 obese non- diabetic persons (male/female: 4/1, age: 55 years 62.1, BMI: 39.2 kg/m2 61.3) and 5 obese post-diabetic patients (male/ female: 4/1, age: 54 years 62.3, BMI: 36.6 kg/m2 61.3) after local anesthesia, using a 5 mm Bergstrom needle (Opitek, Glostrup, Denmark) as described previously by Costford et al [40]. The post-diabetic patients had a documented history of obesity-associated T2DM prior to a 26-week standard clinical weight loss program at the Ottawa Hospital Weight Management Clinic and were no longer diabetic after the weight loss program (blood glucose ,6.1 mmol/l and HbA1c ,5.7%). Characteriza- tion of primary muscle cells from the obese non-diabetic and the obese post-diabetic participants was previously published by our lab [40]. Subjects were fasted for 12 hours and refrained from physical activity for three days prior to the biopsy. 40 mg of the muscle biopsy were processed for satellite cell isolation and culture. The muscle biopsy was minced, subjected to a 30 minutes trypsin digestion and plated in Ham’s F-10 medium supplemented with 15% Foetal Bovine Serum (FBS, Gibco, Burlington, ON, Canada), 0.5 mg/ml BSA, 1 mM dexamethazone (Invitrogen, Burlington, ON, Canada), 10 ng/ml Epidermal Growth Factor (EGF, Invitrogen, Burlington, ON, Canada) and 25 pM insulin (Sig- ma–Aldrich, Oakville, ON, Canada), and maintained in 37uC in humidified 95% air 5% CO2. Muscle satellite cells were isolated at ,80% confluency using an immuno-based magnetic sorting technique as previously described [40]. Isolated primary muscle cells were grown in Ham’s F-10 (6.11 mM glucose) (Gibco, Burlington, ON, Canada) supplemented with 12.5% FBS, 1 mM dexamethazone, 10 ng/ml EGF, 25 pM insulin, 16 antibiotic- antimycotic (Gibco, Burlington, ON, Canada) and 2.5 mg/ml gentamycin (Growth Medium, GM) to ,90% confluency prior to differentiation in either low glucose DMEM (5.5 mM glucose, supplemented with 1 mM sodium pyruvate, 25 pM insulin, 2% horse serum, 16 antibiotic-antimycotic and 2.5 mg/ml gentamy- cin) (LG, Gibco, Burlington, ON, Canada), galactose DMEM Galactose Effects on Human Muscle Cell Metabolism (10 mM galactose (Sigma–Aldrich, Oakville, ON, Canada), 1 mM sodium pyruvate, supplemented with 25 pM insulin, 2% horse serum, 1% antibiotic-antimycotic and 2.5 mg/ml gentamycin) (GAL) or high glucose DMEM (25 mM glucose, supplemented with 1 mM sodium pyruvate, 25 pM insulin, 2% horse serum, 1% antibiotic-antimycotic and 2.5 mg/ml gentamycin) (HG) for 7 days prior to experimentation. The choice of 10 mM galactose was based on previous published studies on other cell lines [13,14,15]. For acute treatments (Figure 4), myotubes were differentiated in LG for 7 days, and then incubated for 45 min in HG, LG or GAL prior to experimentation. Immunofluorescence staining and fusion index In order to calculate the fusion index, cells differentiated in HG, LG or GAL for 7 days were subjected to a desmin immunoflu- orescence staining as previously described [8]. The fusion index was determined as the number of nuclei in differentiated myotubes (desmin positive cells with more than 2 nuclei) as a percentage of the total number of nuclei in muscle cells (desmin positive cells) [43]. Western Blots Cell lysates were quantified for protein content and 30 mg of protein were separated by 8% SDS-polyacrylamide gel electro- phoresis and then transferred to a nitrocellulose membrane. Following primary antibodies were used: monoclonal anti- troponin T (Sigma–Aldrich, Oakville, ON, Canada), anti-complex III (Invitrogen, Burlington, ON, Canada), anti-SDHA (succinate dehydrogenase, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-complex IV (MitoSciences, Eugene, OR, USA), anti- AMPK a2 (Upstate, Lake Placid, NY, USA) and polyclonal anti-b- actin (Cell signaling, Pickering, ON, Canada) diluted 1/1000. Polyclonal anti-phospho-AMPK (Cell signaling, Pickering, ON, Canada) was diluted at 1/500. The secondary antibodies were anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase, respectively. Proteins were visualized using an enhanced luminescent reagent and exposed to autoradiograph film. b-actin and Troponin T were used as a loading control and a marker of myotube differentiation, respectively. Expression of proteins was quantified by density analysis using ImageJ Launcher Software. Cell redox environment Cell redox environment following 7 days of differentiation in HG, LG or GAL was assessed using the MTT Assay Kit (Millipore, Billerica, MA, USA). ATP assay ATP content in myotubes differentiated in HG, LG or GAL for 7 days was assessed using the StayBrite ATP assay kit (Biovision, Mountain View, CA, USA). HPLC Determination of Cellular Lactate Release Myoblasts were seeded at 200,000 cells per well of a 6-well plate. Upon reaching ,90% confluency, cells were differentiated in their respective differentiation medium (HG, LG or GAL) for 7 days. On the 6th day post differentiation the medium was changed and then collected 24 h later for analysis. For experiments, 1 ml of media was treated with 50 ml of 0.5% (v/v) perchloric acid solution. Samples were then centrifuged at 12,000 g for 10 min and subsequently filtered with a sterile syringe and 0.2 mm filtration unit. Samples were injected into an Agilent 1100 Series HPLC system equipped with a C18 reverse phase hydrophilic PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28536