Orexin Signaling in Hepatocytes

LMH cells (ATCC CRL-2117) or spontaneously immortalized chicken embryonic hepatocytes (siCEH^{22 (link)}) were grown on 0.1% gelatin-coated culture dishes in Waymouth’s media (Life Technologies, Carlsbad, CA), supplemented with 10% FBS (Life Technologies, Carlsbad, CA), and 1% penicillin–streptomycin (BioBasic, Amherst, NY) at 37 °C, in a humidified atmosphere of 5% CO₂ and 95% air. At ~ 80% confluence, cells were synchronized with serum-free media overnight and treated with different doses (0, 10, and 100 nM) of recombinant human ORX-A or ORX-B (Alpha Diagnostic International, San Antonio, TX) for 24 h. The dose of orexins was selected based on pilot and previous studies^{16 (link),20 (link),23 (link),24 (link)}. 30 min before orexin treatments, LMH cells were incubated with brefeldin A (0.3 µg/mL) for 24 h and lysates and medium were subjected to immunoblot analyses. All culture experiments were performed with cells from passages 10–15 and repeated at least twice.