L6 cells

We isolated mitochondria using a previously described method with some modifications [12 (link)]. In brief, L6 cells (ATCC; CRL-1458, Manassas, VA, USA) were homogenized using a 26 G syringe in SHE buffer (0.25 M sucrose, 20 mM HEPES [pH 7.4], 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl₂ and 0.1% defatted bovine serum albumin [BSA] with 1 × protease inhibitor) and centrifuged at 1500×g for 5 min at 4 °C. The supernatant was centrifuged at 20,000×g for 10 min at 4 °C to obtain mitochondria. To isolate mitochondria from umbilical cord mesenchymal stem cells (UC-MSCs; IRB No. 201806-BR-029-03), UC-MSCs were homogenized using a 26 G syringe in SHE buffer and centrifuged at 1100×g for 3 min at 4 °C. The supernatant was centrifuged at 12,000×g for 15 min at 4 °C. The pellet was resuspended using SHE buffer without BSA, and the suspension was centrifuged at 20,000×g for 10 min at 4 °C to obtain mitochondria. For all experiments, the isolated mitochondria were stored at 4 °C until use.

Myoblasts derived from rat skeletal muscle (L6 cells; ATCC, Manassas, Virginia, USA) were cultured in 6-well plates containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin at 37°C under a humidified 5%CO₂/95%O₂ atmosphere. When myoblasts were approximately 75% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 1% FBS. Differentiation was allowed to continue for 7 days before experimentation.
Fully differentiated L6 myotubes were treated and incubated for 24 h with recombinant rat TNFα (PeproTech, Princeton, New Jersey, USA) (10 μg/ml) and/or D-Trp(8)-γMSH (American Peptide, Sunnyvale, CA, USA) (0, 50 and 200 nM) or DMEM alone. At this concentration (10μg/ml) TNFα induces activation of NF-kB and down-regulation of IGF-I and Akt in C2C12 cells [26 (link)]. At the end of the incubation period, total RNA or proteins from cells were extracted.

Rat pancreatic beta cell line (RIN-5F) and rat L6 myoblast cell line (L6) were used in this study. RIN-5F and L6 cells were purchased from the American Type Culture Collection (ATCC, USA). RIN-5F (ATCC, CRL- 2058) cell were cultured in RPMI-1640 (Sigma–Aldrich, St. Louis, MO, USA) and L6 cells (ATCC, CRL-1458) were grown in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD, USA). The cells were supplemented with 10% fetal bovine serum (FBS, Sigma–Aldrich, St. Louis, MO, USA) and 1% antibiotics (100 IU/mL of penicillin and 100 μg/mL of streptomycin (iDNA, South America) and were maintained in a humidified 5% CO2 incubator at 37 °C. Cells were seeded in a flask at the required density per well and incubated for the desired time prior to the experiments.