Extended Data Fig. 9: Intracolonic flagellin modulates food intake.

Mice were fasted overnight and received flagellin (1 μg/mL) or PBS enemas prior to gaining ad libitum access to standard chow for 60 min. Flagellin reduced food intake compared to PBS in (a) PyyCre mice (n = 6 mice); (b) 5-week-old mice (n = 5 mice); and 10-week-old mice (n = 5 mice; *P < 0.05, enema*time interaction by rmANOVA with post-hoc two-tailed Tukey HSD). (c) (Left) PolyI:C (1 μg/mL) enema was not sufficient to alter food intake compared to PBS enema (n = 5 mice; P < 0.05, enema*time interaction by rmANOVA with post-hoc two-tailed Tukey HSD). (Right) Effects of flagellin dissipated after 180-minutes post-enema (n = 9 mice; P > 0.05 at 180 min by post-hoc Tukey HSD). (d) Neither vehicle enema (dimethyl sulfoxide, DMSO; n = 8 mice) nor vehicle intraperitoneal injection (saline; n = 10 mice) altered feeding response to flagellin enema (P < 0.05 by two-tailed t-test. (e) Crunch Master analysis of bites for number of bites, bite time, which is the average time of a single feeding bout, and feeding time which is the number of minutes that mice engaged in feeding, did not change during the 1-hour recording session (n = 7 mice per treatment group; P > 0.05 by two-tailed unpaired t-test). Violin plots show median indicated by thick line and mean indicated by thin line. (f) Relative flagellin concentration was measured in stool collected from wildtype mice following an 18-hour fast (n = 5 mice), ad libitum feeding (n = 5 mice), or an 18-hour fast plus enema of PBS (n = 6 mice) or flagellin [1 μg/mL] (n = 6 mice). Feeding significantly increased stool flagellin, however, flagellin enema was not sufficient to rescue flagellin levels to the fed state (*P < 0.05 by rmANOVA with post-hoc two-tailed Tukey HSD). Error bars indicate S.E.M.