MC4R regulates energy expenditure during leucine deprivation. Mice receiving daily intracerebroventricular injection of 1 μL of 0.1 mmol/L SHU9119 (+SHU) or 1 μL PBS (−SHU) via cannula implantation were maintained on a control (+leu) or leucine-deficient (−leu) diet for 7 days. Data are means ± SEM of at least two independent experiments (n = 6 for each group). Statistical significance was determined by one-way ANOVA followed by the SNK test. *P < 0.01, for the effect of −leu vs. control diet within the same intracerebroventricular group; #P < 0.01, for the effect of with vs. without SHU under −leu diet; &P < 0.01, for the effect of with vs. without SHU under control diet. A: Hypothalamic MC4R protein (top, Western blot; bottom, quantitative measurements of MC4R relative to actin). B: Body weight change. C: Abdominal fat mass in proportion to body weight. D: Rectal temperature. E: Ucp1 mRNA in BAT. F: UCP1 protein in BAT (top, Western blot; bottom, quantitative measurements of UCP1 protein relative to actin).