Lin and 100 mg/ml streptomycin. After three washes with PBS, the media was replaced with serum-free -MEM (0.1 BSA) containing antibiotics and the indicated treatments. Final vehicle (DMSO or EtOH) concentrations did not exceed 0.05 . Oleic acid was purchased already complexed with BSA (Sigma). RG7666 supplement Palmitate was conjugated to fatty acid-free BSA by the method described by Sinha et al [22]. Media was collected after 24 hours. Parallel flasks and plates were treated for 48 hr before media collection and cell protein extraction, glucose uptake, and fatty acid oxidation. Media was centrifuged (800 x g, 10 min, 4 ) to remove debris, and stored at -80 . Culture in serum-free -MEM for up to 48 hr had no effect on cell viability, protein content, or extent of differentiation [23].Glucose uptake assayUptake of the non-metabolized analog 2-deoxyglucose (final concentration = 0.01 mM) was measured in triplicate over 10 minutes at room temperature [17]. An aliquot of the suspension was removed for protein analysis. The uptake of L-glucose was used to correct each sample for the contribution of diffusion. All results are adjusted for total cellular protein content determined by the method of Bradford [24].Free Fatty Acid (FFA) oxidationFatty acid metabolism was assayed by b-oxidation of the long chain fatty acid palmitate. Cells were incubated in serum-free -MEM containing [9,10-3H] palmitic acid (final concentration = 20 M) in a 95 O2:5 CO2 incubator at 37 for 3h. The final reaction volume was 500 mL. After incubation, an aliquot (100 mL) of the culture medium was placed over an ion-exchange resin and the column washed with 1.5 mL of water. Intact FFA (charged state) was retained by the resin, whereas the oxidized portion of FFA passed freely through the column in the form of water. Results were adjusted for total cellular protein content.Protein extractionMuscle cells were rapidly washed 5x with 4 PBS and then lysed in extraction buffer [25]. Protein concentration was determined by the Bradford assay and extracts stored at -80 until analyzed.Assay for circulating and secreted factorsSerum insulin levels were determined by RIA (Millipore Corp, Billerica, MA), with intra-assay coefficient of variation (CV) less than 7 . The majority of myokines in serum and conditionedPLOS ONE | DOI:10.1371/journal.pone.0158209 July 25,3 /Myokine Secretion in Type 2 Diabetesmedia were analyzed with MILLIPLEX MAP kits (Millipore) using a BioPlex 200 instrument (Bio-Rad Corp, Hercules, CA). Sensitivities (in pg/mL), inter- and intra-assay CVs for each analyte are as follows: IL1b (0.4, 7 , 6 ), IL6 (0.3, 12, 8), IL8 (0.2, 12, 7), IL10 (0.3, 9, 5), IL15 (0.4, 10, 7), GROa (10.1, 12, 5), TNFa (0.1, 16, 10), interferon-g (IFNg) (0.1, 6, 5), MCP-1 (0.9, 12, 6), VEGF (5.8, 8, 6). Follistatin was measured by ELISA (R D Systems, Minneapolis, MN) (83, 6.4, 2.5).Electrophoresis and Western blottingProcedures for the electrophoresis, transfer and western blotting of proteins are similar to standard methods. For analysis of protein phosphorylation, membranes were probed with antibodies directed U0126-EtOH web against phospho- and total protein produced in different species. Equal amounts of protein were loaded in each lane. Uniformity of loading was monitored by blotting for b-actin. A sample of human skeletal muscle tissue protein was included on each gel to serve as a control for inter-gel variability. Detection and quantification of band intensity was performed using a LiCor Odyssey CxLsys.Lin and 100 mg/ml streptomycin. After three washes with PBS, the media was replaced with serum-free -MEM (0.1 BSA) containing antibiotics and the indicated treatments. Final vehicle (DMSO or EtOH) concentrations did not exceed 0.05 . Oleic acid was purchased already complexed with BSA (Sigma). Palmitate was conjugated to fatty acid-free BSA by the method described by Sinha et al [22]. Media was collected after 24 hours. Parallel flasks and plates were treated for 48 hr before media collection and cell protein extraction, glucose uptake, and fatty acid oxidation. Media was centrifuged (800 x g, 10 min, 4 ) to remove debris, and stored at -80 . Culture in serum-free -MEM for up to 48 hr had no effect on cell viability, protein content, or extent of differentiation [23].Glucose uptake assayUptake of the non-metabolized analog 2-deoxyglucose (final concentration = 0.01 mM) was measured in triplicate over 10 minutes at room temperature [17]. An aliquot of the suspension was removed for protein analysis. The uptake of L-glucose was used to correct each sample for the contribution of diffusion. All results are adjusted for total cellular protein content determined by the method of Bradford [24].Free Fatty Acid (FFA) oxidationFatty acid metabolism was assayed by b-oxidation of the long chain fatty acid palmitate. Cells were incubated in serum-free -MEM containing [9,10-3H] palmitic acid (final concentration = 20 M) in a 95 O2:5 CO2 incubator at 37 for 3h. The final reaction volume was 500 mL. After incubation, an aliquot (100 mL) of the culture medium was placed over an ion-exchange resin and the column washed with 1.5 mL of water. Intact FFA (charged state) was retained by the resin, whereas the oxidized portion of FFA passed freely through the column in the form of water. Results were adjusted for total cellular protein content.Protein extractionMuscle cells were rapidly washed 5x with 4 PBS and then lysed in extraction buffer [25]. Protein concentration was determined by the Bradford assay and extracts stored at -80 until analyzed.Assay for circulating and secreted factorsSerum insulin levels were determined by RIA (Millipore Corp, Billerica, MA), with intra-assay coefficient of variation (CV) less than 7 . The majority of myokines in serum and conditionedPLOS ONE | DOI:10.1371/journal.pone.0158209 July 25,3 /Myokine Secretion in Type 2 Diabetesmedia were analyzed with MILLIPLEX MAP kits (Millipore) using a BioPlex 200 instrument (Bio-Rad Corp, Hercules, CA). Sensitivities (in pg/mL), inter- and intra-assay CVs for each analyte are as follows: IL1b (0.4, 7 , 6 ), IL6 (0.3, 12, 8), IL8 (0.2, 12, 7), IL10 (0.3, 9, 5), IL15 (0.4, 10, 7), GROa (10.1, 12, 5), TNFa (0.1, 16, 10), interferon-g (IFNg) (0.1, 6, 5), MCP-1 (0.9, 12, 6), VEGF (5.8, 8, 6). Follistatin was measured by ELISA (R D Systems, Minneapolis, MN) (83, 6.4, 2.5).Electrophoresis and Western blottingProcedures for the electrophoresis, transfer and western blotting of proteins are similar to standard methods. For analysis of protein phosphorylation, membranes were probed with antibodies directed against phospho- and total protein produced in different species. Equal amounts of protein were loaded in each lane. Uniformity of loading was monitored by blotting for b-actin. A sample of human skeletal muscle tissue protein was included on each gel to serve as a control for inter-gel variability. Detection and quantification of band intensity was performed using a LiCor Odyssey CxLsys.
