Chemicals and reagents
Culture media α-MEM (α-minimum essential medium), DMEM (Dulbecco’s Modified Eagle’s medium), Medium 199, foetal bovine serum, horse serum, Mitosox, prolong diamond antifade mountant, and mitotracker dyes were all purchased from Thermo Fisher Scientific (UK). α-MEM media-lacking glucose was purchased from PAN Biotech, UK, Mitoquinone (Mito Q) was obtained from Cambridge biosciences, UK). BI605906 was a generous gift from Professor Sir Philip Cohen (MRC Protein Phosphorylation Unit, University of Dundee), but also purchased from Tocris (Bristol, UK), MitoSpy™ Green FM was from BioLegends, UK and MitoPYI, Mitotempo, hepatocyte growth factor, dexamethasone, basic FGF, gelatine, vitamin B12, retinoic acid, VAS2870, palmitate, oligomycin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), rotenone, antimycin-A, hygromycin B, apo-transferrin human, SYBR® Green JumpStart Taq Ready Mix, and Polyberen were all purchased Sigma-Aldrich, UK).
Rat and human skeletal muscle cell culture, transfection and fatty acid treatment
L6 muscle cells were cultured to myotubes as described previously  in α-minimal essential media (αMEM) containing 2% (v/v) foetal bovine serum (FBS) and 1% (v/v) antibiotic/antimycotic solution (100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B) at 37 °C with 5% CO2. In some experiments, L6 myotubes were infected with adenovirus harbouring a mutated IκBαS32A/S36A construct. This virus was kindly provided by Dr Harry Heimberg (Vrije Universiteit Brussel, Belgium) and was initially propagated in HEK293 cells and stored at – 80 °C. The viral titre was determined by standard plaque assay in HEK293 cells. Confluent mononucleated L6 myoblasts were infected with the adenovirus at 5.5 pfu/cell for IκBαS32A/S36A in serum free α-MEM for 2 h at 37 °C. Cells were subsequently maintained in fresh α-MEM containing 2% FBS at 37 °C and allowed to differentiate into myotubes prior to experimental use. For some studies, we also used human (LHCN-M2) myotubes. These were cultured in DMEM/M199 medium (4:1) supplemented with penicillin streptomycin (100 μg/ml), FBS 15% (v/v) HEPES (20 mM), Zinc sulphate (30 ng/ml), vitamin B12 (1.4 μg/ml), dexamethasone (55 ng/ml), hepatocyte growth factor, recombinant human (2.5 ng/ml), and basic FGF (10 ng/ml) on plastic ware that had been coated overnight with gelatin (0.1% (w/v) at room temperature. Confluent myotubes were differentiated by change of culture media to DMEM/medium 199 containing zinc sulphate, HEPES, vitamin B12, insulin (10 μg/ml), and apotransferrin (100 μg/ml) and refreshing this every 48 h for 9–10 days until myotubes were 70–80% differentiated.
Prior to use in experiments, L6 myotubes were incubated in serum-containing media that either had or lacked d-glucose (5 mM) as indicated in the figures. For palmitate (PA) treatments, a 100 mM stock solution of the fatty acid was prepared in absolute ethanol as previously reported [28, 29]. This stock was subsequently diluted to a final concentration as indicated in the figure legends by addition to culture media containing 2% (w/v) fatty acid-free BSA and allowed to precomplex for 1 h at 37 °C before being applied onto myotubes for the periods indicated in the figure legends.
Quantitative real-time PCR, mitochondrial DNA quantification, and analysis of citrate synthase activity
Myotubes were incubated with glucose, palmitate, and 2-dexoglucose (2DG) or with inhibitors and/or fluorescent dyes as indicated in the figure legends and prepared for RNA extraction, qPCR analysis, and immunoblotting as described previously [17, 25, 30, 31]. Briefly, total RNA was extracted from L6 myotubes using the TRizol extraction protocol (Thermo Fisher Scientific, UK). RNA samples were used to prepare cDNA using a qScript cDNA synthesis kit as per manufacturer’s instructions and cDNA quantified using the real-time PCR Syber Green based method to establish mRNA abundance. Analysis of mitochondrial DNA (mtDNA) and citrate synthase activity were used as a proxy for mitochondrial mass. For mtDNA quantification, total DNA was extracted from L6 myotubes using a Qiagen DNaesy kit. The mtDNA was quantified by qPCR using primers directed against the mitochondrial ND4 gene and the nuclear-encoded COX4 gene and using the Syber Green method. Data were expressed as a ratio of the ∆∆Ct ND4 to the ∆∆Ct of COX4. The forward and reverse primer sequences for the different gene targets are detailed in Table 1. Citrate synthase (CS) activity was measured using a kit purchased from Sigma-Aldrich/UK (MAK193). Myotubes were treated as indicated in the figure legend and whole cell extracts prepared at the end of the appropriate treatments. 20 μg protein from the cell extract was used for each enzymatic analysis (with measurements being conducted in triplicate for each experimental determination at room temperature). Enzyme activity was measured spectrophotometrically (using an absorbance wavelength of 412 nm) using a µQUNT BIOTEK plate reader from LabTech UK with readings taken every 3 min over a 60 min assay period. CS activity was calculated as per manufacturer’s instructions.
A mitochondrial-enriched membrane fraction was isolated from L6 myotubes using a mitochondria isolation kit (#89874, Thermo Fisher Scientific) as per manufacturer’s instructions. The methodological protocol involves homogenisation of myotubes that have been harvested from 10 cm tissue culture plates having undergone prior experimental treatments as indicated in the appropriate figure legends in lysis buffer [10 mM HEPES, PH 7.5, 10 mM KCl, 0.1 mM EDTA, 0.1 mM DTT, 0.5% (v/v) Nonidet-P40 and 0.5 mM PMSF and protease inhibitor cocktail]. The homogenised cell material was subject to two differential centrifugation steps and within the final centrifugation step the resulting mitochondrial pellet was washed twice prior to being solubilised in RIPA buffer. The supernatant from the final spin (cytosolic fraction) and the solubilised mitochondrial membrane pellet were stored at – 20 ºC until required.
For isolation of nuclear membranes, L6 myotubes were grown on 10 cm dishes as described above and, after treatments, washed three times in PBS before being harvested and spun down in a microfuge (100 g for 5 min). The cell pellet was resuspended in lysis buffer and held on ice for 20 min with intermittent mixing prior to being centrifuged at 10,000 g for 5 min. The resulting supernatant represents a cytosolic fraction. The pelleted nuclei were washed three times in lysis buffer before being resuspended in nuclear extraction buffer (20 mM HEPES PH 7.5, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF with protease inhibitor cocktail) and re-spun at 10,000g for 15 min at 4 °C. The resulting nuclear pellet was resuspended in fresh extraction buffer and stored at – 20 °C until required.
SDS-PAGE and immunoblotting
Cell lysates, cytosolic, nuclear, or mitochondrial-enriched fractions (20 μg protein) from L6 myotubes and human LHCN-M2 myotubes were subjected to SDS/PAGE on 10% resolving gels and transferred onto nitrocellulose membranes (Millipore, Harts, UK), as described previously . Membranes were probed with the following primary antibodies for immunoblot analysis: actin (#A5060) and tubulin (#T6074) were obtained from Sigma: ANT-1 (#ab180715) and PGC1α (#ab54481) were from Abcam; IkBα (#SC-371), SDHA (#SC98253), and GAPDH (#SC32233) were purchased from Santa Cruz; p65 (#8242), Akt (#9272), p-AktSer473 (#9271S), TOM20 (# 42406S), HA (#2367S), COX4 (#4580S), and GPX1 (# 3286S) and SOD2 (#D9V9C) were all purchased from Cell Signalling Technology; DLP1/Drp1 (#611112) and OPA1 (#612607) were from BD Biosciences; and UCP3 (#GTX112699) from Genetex. Primary antibody detection was performed using appropriate horse-radish peroxidase (HRP) conjugated secondary mouse (#7076S) or rabbit (#7074S) antibodies were purchased from Cell Signalling Technology and visualised using enhanced chemiluminescence (Pierce-Perbio Biotech, Tattenhall, UK) on Kodak X-OMAT film (Eastman-Kodak, Rochester, UK). The immunoreactive protein bands were quantified using ImageJ software.
L6 myotubes were incubated with glucose, palmitate and BI605906 for times and at concentrations indicated in the figure legends prior to assaying uptake of 10 μM 2-deoxy-d-[3H]-glucose as described previously . Non-specific binding was determined by quantifying cell-associated radioactivity in the presence of 10 μM cytochalasin B. Cells were washed and subsequently lysed in 50 mM NaOH and radioactivity quantified by scintillation counting. Protein concentration in cell lysates was determined using the Bradford reagent .
For analysis of superoxide, L6 myotubes were subject to experimental treatments as indicated in the figure legends prior to being treated with 5 μM Mitosox at 37°C in a 5% CO2 incubator for 30 min. Mitosox is a fluorogenic dye that is specifically targeted to mitochondria in live cells, and whose oxidation by superoxide produces red fluorescence that was quantified using a Clario Star plate reader with absorption/emission maxima: 510/585 nm. In some experiments, L6 myotubes were also treated with Mitotempo (a mitochondrial targeted anti-oxidant) prior to analysis of superoxide.
For determination of hydrogen peroxide (H2O2) under live cell conditions, L6 myotubes were incubated with 5 μM MitoPYI (a mitochondrial targeted H2O2 probe) and 1 μM deep red cell tracker at 37 °C in a 5% CO2 incubator for 45 min. Myotubes were subsequently imaged using a Zeiss confocal microscope with excitation/emission maxima for MitoPYI set to 488/530 nm and that for the cell tracker at 633/647 nm. Captured images were analysed to quantify the fluorescent signal generated by MitoPYI from at least 8–10 different visual fields (40–50 myotubes) per condition per experiment using ImageJ software.
Analysis of cellular respiration and mitochondrial energetics
For analysis of cellular respiration and mitochondrial energetics in L6 and LHCN-M2 myotubes, we used a Seahorse XF24 analyser. L6 myotubes were cultured on Seahorse culture plates in serum-containing media supplemented with 5 mM d-glucose and/or palmitate at concentrations indicated in the figure legends for 16 h. In some experiments, the culture media were also supplemented with 5 mM 2-deoxyglucose (2-DG), BI605906 (IKKβ inhibitor) or 2 mM carnitine as indicated prior to analysis of basal respiration, ATP-linked respiration, H+ (proton) leak, maximal respiratory capacity and non-mitochondrial respiration using modulators of cellular respiration (i.e., oligomycin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), rotenone, and antimycin as previously described . The various mitochondrial parameters were normalised to protein content/well within the Seahorse plate. For Seahorse XF analyser studies, data points per experimental condition were collected from a minimum of three replicates with each experiment being conducted at least three times.
Mitochondria morphology and live cell mitochondrial imaging
For analyses of mitochondrial morphology, we stained L6 myotubes with Mitospy Green FM (BioLegend, UK); a green-fluorescent stain that localizes to mitochondria. L6 myotubes were grown on 15 mm2 glass coverslips and following the experimental treatments specified in the figure legends were washed with fresh media and subsequently incubated in medium containing 300 nM Mitospy for 30 min at 37°C in a 95% O2/5% CO2 environment. After this incubation period, myotubes were washed with PBS prior to being fixed with 2% (w/v) paraformaldehyde and mounted in prolonged diamond antifade before being visualised using a Zeiss confocal microscope. Live cell imaging was also used in some of our studies. For these, L6 or LHCN-M2 myotubes were grown and differentiated in eight well chamber slide plates (Ibidia, UK) and having been treated (as indicated in the figure legends) were washed with fresh phenol red-free media prior to incubation with Mitospy. Mitochondrial morphology was then visualised in real time using Zeiss confocal microscope 37 °C in a 5% CO2 chamber with excitation/emission set at 480 nm and 520 nm, respectively. For real-time recording of mitochondrial length, we used the ZEISS ZEN microscope software or Image J. Within each experimental condition, at least 50 myotubes were randomly selected from between 10 and 12 visual fields. Mitochondrial morphology within myotubes was categorised as either spheroid/fragmented in which mitochondria were equal to or less than 1 μm in length or tubular/elongated (including being part of a network), where mitochondrial length was greater than 1 μm. The number of mitochondria in each category within the fields being visualised was then determined and expressed as a percentage.
Analysis of mitophagy
Mitophagy was quantified using the mitophagy QC approach , which involves stable expression of a tandem mCherry-GFP tag attached to the outer mitochondrial membrane localization signal of Fis1 (residues 101–152) . The retrovirus harbouring this construct was introduced into L6 myotubes using the approach detailed previously . The L6-GFP–mCherry cells were grown and differentiated on 15 mm2 cover slips and subjected to the treatments detailed in the figure legends prior to being washed and fixed with 3.7% cell culture grade paraformaldehyde and mounted in prolonged diamond antifade. Cells were visualised using Zeiss 710 confocal microscope. Myotubes expressing the mCherry-GFP construct fluoresce red and green (yellow when confocal images are merged). However, upon increased mitophagy, mitochondria are delivered to lysosomes, where the low pH quenches the GFP signal but not mCherry. Consequently, some of the mitochondria form punctate structures and fluoresce red only and the degree of mitophagy calculated by quantitating their increase using the volocity software.
Statistical analysis was performed using the GraphPad Prism version 7 software using one-way analysis of variance (ANOVA) and Tukey post hoc test for multiple comparisons. Values were considered significant at P < 0.05.