2.5. Fatty acid oxidation

Incubations were carried out as previously described (33). Briefly, soleus muscle strips (∼25 mg) were excised tendon to tendon and placed in sealed vials to equilibrate for 30 min in a 30 °C shaking water bath in a pre-gassed (95% O₂, 5% CO₂) incubation media of DMEM supplemented with 8 mM glucose and 4% BSA. Remaining soleus muscle from each animal was frozen at −80 °C to assess proteins (COXIV, CS, GLUT4, FAT/CD36, GHS-R1a, CRF-2R). For 5d diet experiments, muscles were incubated in the presence of 1 or 2 mM palmitate (with or without 150 ng/mL AG or UnAG) as we were only interested in the assessment of FAO as a marker of ghrelin response. In experiments examining the effect of 6w of diet and exercise training, muscles were incubated in buffer containing low (0.2 mM, baseline) or high (2 mM) palmitate, with or without 150 ng/mL AG or UnAG. This was designed to test the ability of ghrelin to stimulate FAO and protect the muscle from becoming less responsive to insulin during exposure to 2 mM palmitate (see below for determination of glucose uptake). Incubations lasted for 4 h. After the first 2 h, muscles were transferred to vials for an additional 2 h containing the identical palmitate and ghrelin concentrations with the addition of 0.5 mCi/mL ¹⁴C-palmitic acid for the assessment of FAO. At the end of the incubation, sulfuric acid was injected into the vial and liberated ¹⁴CO₂ was trapped in a 500 μL Eppendorf tube containing benzethonium hydroxide. The tube containing the trapped gas was placed into a 5 mL liquid scintillation vial containing CytoScint™-ES, allowed to quench overnight in darkness, and counted for 5 min per sample using a PerkinElmer Tri-Carb LSC 4910 TR liquid scintillation counter.