Assessing Insulin-Stimulated Metabolic Flexibility

Mice were fasted for 2 h and were injected intraperitoneally with 0.75 units/kg Humulin R insulin as previously described [19 (link)]. Islet histology procedures have been described [19 (link), 20 (link)]. Briefly, the antibodies used were as follows: insulin (Invitrogen; 18-0067; used at 1 : 800), glucagon (Cell Signaling Technologies; 8233; used at 1 : 400), Aldh1a3 (Novus Biologicals; NBP2-15339; used at 1 : 100), and Nkx6.1 (F55A12; 1 : 200; [21 (link)]). All antibodies were incubated overnight at 4°C and detected with either Alexa Fluor secondary conjugation (Alexa 488, 594), except for Nkx6.1, which was detected using a biotin/streptavidin exposure method. Substrate oxidation was measured in the liver, and gastrocnemius muscle homogenates were prepared and used as described previously [22 (link), 23 (link)]. Briefly, using [1-¹⁴C] palmitate (100 μM), both complete (¹⁴CO₂) and incomplete (¹⁴C-acid soluble metabolites (ASMs)) fatty acid oxidation were assessed ± varying doses of unlabeled pyruvate (0, 0.1, 1, and 10 mM) to assess metabolic flexibility.