Figure 4.

Receptor specificity for SPX inhibition on food consumption in mice. (A) RT-PCR for GalR2 and GalR3 expression in the liver, brain and hypothalamus. Total RNA isolated from target tissues was used for RT-PCR with primers for GalR2 and GalR3, respectively, with GAPDH as internal control and PCR with no template as negative control (“-ve Ctrl”). (B) Western blot of GalR2 and GalR3 expression in the brain and hypothalamus. Tissue lysate was prepared from mouse brain and hypothalamus and subjected to SDS- PAGE and Western blot using the antibody (Ab) for GalR2 and GalR3, respectively, with β actin as loading control. To confirm the specificity of immunoblotting, Western blot was also conducted with antibodies pre-absorbed with the synthetic peptides for GalR2/3 used to raise the antibody provided by the company. (C) GalR2 and GalR3 blockade on the inhibitory effect of SPX on food intake in mice. IP injection of SPX (5 nmol/mouse) was performed at 10:00 am with/without co-treatment of the GalR2 antagonist M871 (50 nmol/mouse) or GalR3 antagonist SNAP (50 nmol/mouse) to test the effect on food consumption occurred during the first hour in dark phase. (D) Effect of GalR2 activation on food consumption in mice. In parallel experiment, IP injection of the GalR2 agonist dN1-Qu (10 nmol/mouse) was conducted with SPX treatment (10 nmol/mouse) as positive control. For the data presented, the groups denoted by different letters represent a significant difference at p < 0.05.