Animal Studies

Dosages of liraglutide were chosen on the basis of previous studies (6). For the longitudinal study, mice (n = 10 per sex and genotype) were singly housed at age 4 weeks, and body weight and food intake were monitored weekly. At age 13–15 weeks, mice were fasted at 10:00 a.m. and then subcutaneously injected with either liraglutide (400 μg/kg) or saline at 5:00 p.m. Preweighed food was given at 6:00 p.m., and food intake was measured 1, 2, 4, and 24 h later. Body weight was measured at the 24-h time point. One week later, all mice were given the opposite treatment (saline or liraglutide) in a crossover design, and the study was repeated. At age 15–17 weeks, mice were fasted at 9:00 a.m., and basal fasting blood glucose was measured at 1:00 p.m. with a glucometer (Contour Next EZ; Bayer). Mice were then injected with liraglutide (400 μg/kg) or saline as above and blood glucose measured at 3:00 p.m. The crossover treatment was performed 1 week later. At age 18 weeks, brains from the male Glp1r-flox, vGAT^ΔGlp1r, and vGlut2^ΔGlp1r mice were collected for Fos immunohistochemistry (IHC) analysis.

For the long-term liraglutide study, male Glp1r-flox, vGAT^ΔGlp1r, and vGlut2^ΔGlp1r mice (n = 6–8 per genotype and treatment) were weaned into group housing with standard rodent chow. At age 5 weeks, the food was switched to high-fat chow (D12451; Research Diets), and the mice were singly housed at age 9 weeks. At age 11 weeks, mice were randomly assigned to either the saline control or liraglutide group and subcutaneously injected once per day (5:00 p.m.) for 2 weeks using a previously described ascending dosing schedule (6). Body weight and food intake were measured daily. On day 15, mice were decapitated under anesthesia and the hypothalamus was isolated, snap-frozen on dry ice, and stored at −80°C for semiquantitative RT-PCR analysis (see above). Gonadal and perirenal fat pads were removed and weighed. To assay conditioned taste avoidance (CTA), male and female Glp1r-flox, vGAT^ΔGlp1r, and vGlut2^ΔGlp1r mice (n = 8 per genotype and treatment) were singly housed at age 10–12 weeks, and the assay was performed as previously described (20) (Supplementary Data).