Ex vivo Muscle Stimulation Protocol

Soleus and extensor digitorum longus (EDL) muscles were excised from anesthetized mice (6 mg pentobarbital and lidocaine/100 g body weight, intraperitoneal injection) and suspended in incubation chambers (Multi Myograph System, Danish Myo-Technology, Hinnerup, Denmark) containing 30 °C Krebs Ringer Henseleit (KRH) buffer supplemented with 8 mM mannitol and 2 mM pyruvate as previously described [22 (link)]. The muscles were pre-incubated for 20–40 min with the ULK1/2 inhibitor, SBI-0206965 (#18477, Cayman Chemical Company, Ann Arbor, MI, USA) or DMSO (Sigma) as a control, and subsequently stimulated with either 60 nM insulin (Actrapid, Novo Nordisk) for 20 min or 4 mM 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR (Toronto Research Chemicals, North York, ON, Canada)) for 40 min with or without the inhibitors present. The total duration of the incubation experiments was 1 h.