The hypothalamus, defined by the posterior margin of the optic chiasm and the anterior margin of the mammillary bodies (to the depth of ∼2 mm), was dissected out and immediately homogenated on ice to preserve phosphorylated protein levels (8,18,26). Hypothalamic total protein lysates were subjected to SDS-PAGE, electrotransferred on a polyvinylidene fluoride membrane, and probed with the following antibodies: ACC, pACC-Ser79, AMPKα1, and AMPKα2 (Upstate Biotechnology, Temecula, CA); FAS (BD Biosciences, Franklin Lakes, NJ), pAMPK-Thr172, and phospho-liver kinase B1 (pLKB1)-Ser428 (Cell Signaling, Danvers, MA); PP2Cα and Ca2+/calmodulin-dependent protein kinase kinase α and β (CaMKKα and CaMKKβ) (Santa Cruz Biotechnology, Santa Cruz, CA); and β-actin (Sigma-Aldrich) as described previously (8,18,26). Values were expressed in relation to β-actin levels.
