Figure 4.

Effect of C75 on hypothalamic neuropeptide mRNA levels in “lean” (BALBc) mice. BALBc mice were weighed and then fasted (n = 6), injected i.p. with vehicle (n = 6) (control) or injected i.p. with C75 (30 mg/kg body weight) (n = 8) 3 h before “lights off”; the latter two groups were fed ad libitum. Twenty-four hours after injection, mice were weighed (a), food consumption was measured (b), and hypothalamic tissue was dissected and frozen quickly. Total RNA (5 μg) extracted from paired hypothalami was mixed with probe mixtures containing [³²P]UTP-labeled cRNAs for AgRP (c), CART (d), NPY (e), POMC (f), MCH (g), and MCHR (h) mRNAs, and subjected to the RNase protection protocol. Protected fragments were separated by urea-PAGE and exposed to an imaging plate for 24 h. Radioactivity in the gel bands was quantified, normalized to the cyclophilin signal, and expressed as percent relative to the mRNA level of control mice. Values are shown as means ± SEM. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 vs. control group; †, P < 0.05, ††, P < 0.01, and †††, P < 0.001 vs. fasted group.