Physiological and nutritional regulation of AMPK phosphorylation and neuropeptide expression in mouse hypothalami. Male wild-type C57BL/6J mice, at the age of 12–14 weeks, were fed ad libitum. Before sample collection, mice were fasted for overnight (16 h) with or without re-feeding for an additional 4 h. Hypothalamus lysates were prepared to examine AMPPK signaling using Western blot analyses. (A) Amount of AMPK and phospho-AMPK (Thr172). (B) Ratios of phospho-AMPK/AMPK were calculated using densitometry. AU, arbitrary unit. (C) Total RNA was prepared from mouse hypothalami and subjected to real-time RT-PCR. NPY, neuropeptide Y; AgRP, agouti-related protein; POMC, pro-opiomelanocortin; and CART, cocaine-amphetamine-related transcript. For B and C, data are means±S.E., n=4–6. *P<.05 and **P<.01 re-fed vs. fasted (in B) for the same gene (in C).