The host circadian rhythm influences microbial cyclical fluctuations.

A: Cell counts per grams of feces in the EAM mouse (n = 8), calculated by qPCR.B: Circadian food intake and hydrogen production of the EAM mouse over two days of measurement, where measurements are taken every 24 minutes (n = 16). Curve fit was calculated using formula y∼s(x; bs= “cs). SE is illustrated with grey shading surrounding line. C: Circadian food intake and hydrogen production of the GF mouse (n = 8).

Lactulose disrupts the diurnal rhythm of microbial metabolism.

A:Hydrogen production increases after lactulose gavage, but not after PBS gavage (n = 16, 8 in each group). Treatment is indicated by a dotted line at zt= 3. An unpaired T-test was performed on final hydrogen measurements (p<0.0001). B: Hydrogen production temporarily increases after lactulose gavage, but not after PBS gavage in EAM mice (n = 11, Lactulose = 6, PBS = 5}. Treatment is indicated by a dotted line at zt= 3. C: Bacterial population after gavage (n = 22, lactulose = 12, PBS = 10). E. rectale population increases in lactulose treated mice compared to PBS treated mice (p<0.05). D: Cecum content pH of EAM (n = 17, lactulose = 10, PBS = 7) and GF (n = 16, 8 in each group) mice after treatment. Unpaired T-test revealed a decrease in pH in EAM mice (p<0.0001) and GF mice (p<0.05). E: SCFA concentration in EAM (n = 17, lactulose = 10, PBS = 7) mice after treatment. Unpaired T-test revealed an increase in acetate, butyrate, and succinate concentration after multiple testing correction (“**” = p<0.01, “***” = p<0.001, “****” = p<0.0001). E: SCFA concentration in GF (n = 8, 4 in each group) mice after treatment. There is no difference in SCFA concentration after treatment.

Inducing microbial metabolism acutely alters circadian clock gene expression in small intestinal tissue.

A: Schematic of the clock gene network in mice. B: Clock gene expression in ileum tissue after EAM mouse treatment (n = 17, lactulose = 10, PBS = 7) and GF mouse treatment (n = 16, 8 in each group) measured via RT-PCR. Unpaired T-test revealed increases in EAM gene expression after multiple testing correction(“*”= p<0.05, “**” = p<0.01). C: Clock gene expression in EAM mouse ileum tissue over 24 hours after treatment. All gene expression was normalized to samples collected from untreated mice at 9AM (control). Unpaired T-test revealed changes in expression after multiple testing correction (“*”= p<0.05, “**” = p<0.01).

Inducing microbial metabolism decreases host food intake in following active cycle.

A: EAM mouse (n = 15, lactulose = 8, PBS = 7) food intake rate in dark phase prior to treatment (control) and following treatment (treatment). Paired T-test displays a reduction in food intake rate after lactulose treatment ( p<0.01). B: GF mouse (n = 14, 7 in each group) food intake rate. Paired T-test displays no difference in food intake rate after either treatment. C: Clock gene expression in hypothalamus and liver tissues 8 hours after EAM mouse treatment (n = 9, lactulose = 5, PBS = 4) measured via RT-PCR. Unpaired T-test revealed no significant changes in gene expression.

Disruption of circadian food intake patterns is due to systemic effects of microbial metabolic products.

A: EAM mouse (n = 5) food intake rate in dark phase prior to FP treatment (control) and following treatment (treatment). Paired T-test displays a reduction in food intake rate after FP treatment (p<0.01). B: EAM mouse total caloric intake after accounting for calories provided by the FP treatment. Paired T-test displays a significant reduction in caloric intake after accounting for calories provided directly by the FP (p<0.05). C: EAM mouse (data calculated from S4A) total caloric intake after accounting for calories provided by the microbiota after lactulose metabolism. Paired T-test displays a significant reduction in caloric intake after accounting for feeding by the microbiota (p<0.05). D: Total PYY concentration in portal vein plasma after lactulose or PBS treatment in EAM, GF, and SPF mice. Unpaired T-test revealed no significant differences between groups. E: Total GLP-1 concentration in portal vein plasma after lactulose or PBS treatment in EAM, GF, and SPF mice. Unpaired T-test revealed no significant differences between groups.

E. rectale is the principal hydrogen producer in the EAM.

A: Daily fluctuations of EAM strains in mouse feces. Feces were sampled over 2 days (n = 8). Shaded regions represent the dark cycle in the animal facility, from zt=12 (6PM) to zt=24 (6AM). B:Circadian food intake and hydrogen production of the EAM mouse over two days of measurement, where measurements are taken every 24 minutes (n = 16). Curve fit was calculated using formula y∼s(x; bs = “cs). SE is illustrated with grey shading surrounding line. Shaded regions represent the dark cycle in the animal facility, from zt=12 (6PM) to zt=24 (6AM). C: Circadian food intake and hydrogen production of the incomplete EAM (colonized with B. theta and E. coli) mouse (n = 4)

Lactulose disrupts the diurnal rhythm of microbial metabolism.

A:Hydrogen production temporarily increases after lactulose gavage, but not after PBS gavage in SPF mice (n = 11, Lactulose = 5, PBS = 6). Treatment is indicated by a dotted line at zt= 3.B: in vitro fermentation product production of EAM when grown on Lactulose. Measurements were taken at stationary phase of growth. C:Dry to wet feces ratio in EAM (n = 15, lactulose= 8, PBS = 7) and GF (n = 8, 4 in each group) mice. Unpaired T-test shows an increase (p<0.01) in the ratio in EAM mice after lactulose treatment. D: Total wet feces output after normalizing dry feces weight to mean dry/wet feces ratio in EAM (n = 8, 4 in each group) and GF (n = 8, 4 in each group) mice. Unpaired T-test shows an increase (p<0.05) in the total wet feces output in EAM mice after lactulose treatment. E: Dry to wet cecum content ratio in EAM (n= 17, lactulose= 10, PBS = 7).

Inducing microbial metabolism acutely alters circadian clock gene expression in small intestinal tissue.

A: Clock gene expression in gut tissue after EAM mouse treatment (n = 17, lactulose = 10, PBS = 7) measured via RT-PCR. Unpaired T-test revealed increases in gene expression in the colon and ileum after multiple testing correction (“*” = p<0.05, “**” = p<0.01). B: Clock gene expression in gut tissue after GF mouse treatment (n = 16, 8 in each group). No significant differences were found in gene expression. C: Clock gene expression in ileum tissue after SPF mouse treatment (n = 10, 5 in each group). Unpaired T-test revealed decrease in Cry1 expression after multiple testing correction (“*” = p<0.05).

Inducing microbial metabolism decreases host food intake in following active cycle.

A: EAM mouse (n = 15, lactulose = 8, PBS = 7) total food intake in dark phase prior to treatment (control) and following treatment (treatment). Paired T-test displays a reduction in food intake rate after lactulose treatment ( “**” = p<0.01). B: EAM mouse (n = 10, lactulose = 5, PBS = 5) food intake rate in dark phase prior to treatment (control) and two dark phases following treatment (treatment & treatment+1). Anova shows no significant change between the three days. C: GF mouse (n = 14, 7 in each group) total food intake. Paired T-test displays no difference in food intake rate after either treatment. D: SPF mouse (n = 8, lactulose = 4, PBS = 4) food intake rate in dark phase prior to treatment (control) and two dark phases following treatment (treatment & treatment+1). Anova shows no significant change between the three days.

Disruption of circadian food intake patterns is due to systemic effects of microbial metabolic products.

A: SPF mouse (n = 6, 3 in each group) food intake rate in dark phase prior to PBS or FP treatment (control) and following treatment (treatment). Paired T-test displays no change in food intake rate after FP treatment. B: Total ghrelin concentration in heart serum after lactulose or PBS treatment in EAM and GF mice. Unpaired T-test revealed no significant differences between groups in EAM mice, and an increase in ghrelin in GF mice (p<0.05). E: Total leptin concentration inheart serum after lactulose or PBS treatment in EAM and GF mice. Unpaired T-test revealed no significant differences between groups.