Carnitine

Flies were maintained as previously described (Yoo et al, 2016 (link)). The fly food is composed of the following ingredients: 0.8% agar, 10% glucose, 4.5% corn flour, 3.72% dry yeast, 0.4% propionic acid, 0.3% butyl p‐hydroxybenzoate. For acetate supplementation experiments, acetate (FUJIFILM Wako) was added to the fly food to a final concentration of 333 or 500 mM. Carnitine (Tokyo Chemical Industry) was also added to the fly food to a final concentration of 100 mM.

C2C12 cell mitochondrial respiration, basal respiratory capacity, coupling efficiency, proton leakage, and spare respiratory capacity were profiled by an XF24 metabolic flux analyzer (Seahorse Bioscience, Billerica, MA, USA). Furthermore, the oxygen consumption rate (OCR) was assessed in C2C12 cells that had been induced to undergo differentiation while being continuously treated with the ATP synthase inhibitor oligomycin (Wako Pure Chemicals), the uncoupling agent carbonyl cyanide p-(trifluoromethoxyl)-phenyl-hydrozone (FCCP; StressMarq Biosciences Inc., Victoria, BC, Canada), and a mitochondrial complex inhibitor rotenone (Sigma-Aldrich)/antimycin (Enzo Life Sciences, Plymouth Meeting, PA, USA). Fatty acid β-oxidation was evaluated by measuring the OCR when differentiated C2C12 cells were treated with bovine serum albumin–conjugated palmitate after 1 h of equilibration in Krebs-Henseleit buffer containing 0.5 mM carnitine (Tokyo Chemical Industry, Tokyo, Japan) and 2.5 mM glucose.