Briefly, 5 µL DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) dissolved in chloroform and ethanol (final concentration 80 mM) was mixed with 20 µL jetSI™ to form Solution A. Solution A was diluted first with ethanol (Solution B) and then with 25% glucose in order to obtain the solution C with a final glucose concentration of 5%. 5,6 µg negative control siRNA or siRNA targeting rat Sirt1 were diluted into a final glucose concentration of 5%. Solution C was added into the siRNA solution in a 1∶1 (v/v) ratio. After 30 min incubation at room temperature and in less than one hour, 1 µL of the siRNA complexes were injected into each side of the third ventricle into the ARC of rats under anesthesia. The animals were individually caged, and food intake and body weight were measured every day. 2, 5 or 10 days after the injection some animals were sacrificed and the ARC Sirt1 protein levels were determined by western blotting.

Sirt1 is an evolutionarily conserved NAD(+) dependent deacetylase involved in a wide range of processes including cellular differentiation, apoptosis, as well as metabolism, and aging. In this study, we investigated the role of hypothalamic Sirt1 in energy balance. Pharmacological inhibition or siRNA mediated knock down of hypothalamic Sirt1 showed to decrease food intake and body weight gain. Central administration of a specific melanocortin antagonist, SHU9119, reversed the anorectic effect of hypothalamic Sirt1 inhibition, suggesting that Sirt1 regulates food intake through the central melanocortin signaling. We also showed that fasting increases hypothalamic Sirt1 expression and decreases FoxO1 (Forkhead transcription factor) acetylation suggesting that Sirt1 regulates the central melanocortin system in a FoxO1 dependent manner. In addition, hypothalamic Sirt1 showed to regulate S6K signaling such that inhibition of the fasting induced Sirt1 activity results in up-regulation of the S6K pathway. Thus, this is the first study providing a novel role for the hypothalamic Sirt1 in the regulation of food intake and body weight. Given the role of Sirt1 in several peripheral tissues and hypothalamus, potential therapies centered on Sirt1 regulation might provide promising therapies in the treatment of metabolic diseases including obesity.