Figure 5. Hypothalamic NDN production is potentially involved in the leptin signaling pathway and the control of energy homeostasis.

(a,b) Western blot analysis of hypothalamic protein lysates from 18 hr-fasted mice icv-injected with aCSF (control) or leptin (Leptin; 2 µg) 2 hr before sacrifice, performed using ACBD7 and β-actin specific antibodies. Quantification of relative ACBD7 protein levels performed using β-actin signal as loading control (n=4). Data are expressed as mean ± SEM. Unpaired Student’s t test: ***p<0.001. (c) MRM-MS relative quantification of NDN levels in MBH explants harvested from 18 hr-fasted mice and icv-injected with aCSF, or with leptin (Leptin, 2 µg), 2 hr before sacrifice. Relative quantity was determined using exogenous peptide as an internal control (n=3). Data are expressed as mean ± SEM. Unpaired Student’s t test: *p<0.05. (d) Ad libitum-fed mice received icv injection of leptin (2 µg) with or without the endozepine metabotropic receptor antagonist cyclo_1-8[DLeu⁵]ODN_(11-18) (LV-1075; 1 µg) and/or the MC4R antagonist HS024 (100 ng), 2 hr before the beginning of the dark period, and food intake was measured after 4 hr (n=6). Data are expressed as mean ± SEM. One-way ANOVA followed by a post-hoc multiple comparison Bonferroni test using aCSF injected mice as control ***p<0.001; *p<0.05, or leptin injected mice as control # p<0.05, ### p<0.001. (e,f) Effect of two daily injections of NDN (100 ng/day) and LV-0175 (1 µg/day) on food intake (e) and body weight (f). Mice were injected each day at ZT2 and ZT10. Daily food intake (e) and body weight (f) were measured each day at ZT2 for 5 days (n=6). Data are expressed as mean ± SEM. Two-way ANOVA followed by a post-hoc multiple comparison Bonferroni test: *p< 0.05; **p<0.01, ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.11742.029