Cell culture

C2C12 murine myoblasts [35] between passage 8 and 10 were incubated in separate T75 flasks in a humidified environment (37 °C, 5% CO₂) with growth media (GM) containing Dulbecco’s Modified Eagle Serum (DMEM) (D6429, Sigma-Aldrich, UK), 1% Penicillin Streptomycin (Pen-strep), 10% New born calf serum (NBCS) and 10% Foetal Bovine Serum (FBS) until 80% confluency was attained. Cells were trypsinized and cell counts were preformed using a haemocytometer in the presence of Trypan Blue dye. For studies in differentiating myoblasts, 6-well plates were pretreated with 0.2% porcine gelatin for 10 min at room temperature (RT) and 10 min in a humidified, 37 °C/5% CO₂ environment. The excess gelatin was aspirated and cells were seeded at 8 × 10⁴ cells/ml in 2 ml of GM per well, these were then incubated for 24 h until 80% confluency was attained. Experiments were initiated by removing GM, washing twice with phosphate buffered saline (PBS) followed by the addition of low serum differentiation media for a total period of 7 days, containing: 8.3 g/L of DMEM (D5030, Sigma-Aldrich, UK), 0.584 g/L l-Glutamine, 3.7 g/L Sodium Bicarbonate, 0.11 g/L Sodium Pyruvate, 0.0159 g/L Phenol red, 2% horse serum and 1% Pen-strep and either 0.6 g/L or 3.3 mM (glucose restricted/GR) or 4.5 g/L (25 mM) high-glucose differentiation medium (DM) universally used for myoblast proliferation and differentiation and used extensively in previous studies assessing the role of resveratrol in C2C12 cells [17, 31–34], whilst also allowing relevant comparisons with the existing literature. The reduction in serum content causes C₂C₁₂ myoblasts to undergo spontaneous differentiation without requiring the addition of growth factors to initiate the process [35]. Time point zero was defined as an incubation of 30 min after transfer to DM and is denoted as 0 h (0 h). To assess the effect of resveratrol treatment and SIRT1 inhibition in myoblasts that were glucose restricted (GR) versus high-glucose differentiation media (DM), cells were incubated in either 0.6 g/L / 3.3 mM (GR) versus 4.5 g/L / 25 mM (DM) in the absence or presence of resveratrol (RES) at a concentration of 10 µM and SIRT1 inhibitor (EX-527) at 100 nM. Morphological analysis (myotube number, diameter and area), creatine kinase assays, and RNA extraction/ isolation for gene expression of genes associated with muscle cell differentiation (myogenin) and myotube maturation (Myhc 1, 2, 4, 7) were conducted at 0, 72 h and 7 days.

For studies in differentiated myotubes, myoblasts at passages 12–15 were washed in PBS and transferred into 2 ml of DM in 37 °C at 5% CO₂ for 7 days in order to differentiate. Once myotubes had been formed over 7 days, cells were dosed in the below experimental conditions for a further 72 h (total time 10 days in culture) to assess the impact of resveratrol and EX-527 treatment during glucose restriction (GR) conditions in existing myotubes: GR differentiation media (0.6 g/L / 3.3 mM glucose alone), High-glucose differentiation media (DM) (4.5 g/L glucose alone), GR + Resveratrol (RES) (0.6 g/L glucose + 10 μM RES), DM + RES (4.5 g/L glucose + 10 μM RES), GR + EX-527 (0.6 g/L glucose + 100 nM EX-527) and DM + EX-527 (4.5 g/L glucose + 100 nM EX-527). Resveratrol/EX-527 was purchased from Merck Millipore (cat no 554325/566322, respectively, Nottingham, UK). Resveratrol was manufactured by Calbiochem (cat no CAS 501-36-0, San Diego, CA, USA). Resveratrol/EX-527 was reconstituted in DMSO and this stock was stored in -20 °C for up to three/six months, respectively, according to the manufacturer’s instructions. Morphological analysis of myotube number, diameter and area were performed 24, 48 and 72 h. For these experiments, the 0 h (0 h) baseline control condition was 7 days in DM to promote myotube formation, after which time cells were washed × 2 in PBS and then placed in fresh DM for 30 min containing (denoted DM 0 h). Protein activity of AMPK and P70S6K was analysed from protein lysates extracted at 0-, 15- and 30-min, 2- and 24-h time points after dosing occurred in myotubes in order to investigate energy sensing vs. protein synthetic/growth-associated cellular signalling following resveratrol and EX-527 administration in existing myotubes in GR versus DM glucose conditions. Gene expression for later differentiation and myotube maturation (Mrf4, Myhc1, 2, 4, 7) and genes associated with myotube hypertrophy (Igf-I, Igf-Ir, Igf-II, Igf-IIr, Igfbp2, Mtor), myotube atrophy (Tnf-α, Tnfrsflb, Myostatin, Mur1f, Mafbx, Musa1, Fox01, 3, Nf-kb, p53) as well as Sirt1 gene expression were completed at 0, 24 and 72 h.