Supplemental Table 1. Gene expression in gastroc muscles of germ-free mice when compared to sedentary SPF control values.
Gene SED SPF CON Fold Change (2^-∆∆CT)
SED GF Fold Change (2^-
∆∆CT)
VWR GF Fold Change (2^-
∆∆CT)
Group p value
PGC1α 1.12±0.18 3.52±0.50* 3.51±0.79* 0.002
MCT1 1.02±0.06 0.62±0.04* 0.64±0.05* <0.001
LC3 1.03±0.08 1.41±0.23 1.87±0.22* 0.02
SDH 1.02±0.08 1.13±0.05 1.19±0.07 0.263
GLUT4 1.01±0.05 0.51±0.08* 0.44±0.07* <0.001
MCT4 1.04±0.09 1.03±0.12 0.81±0.17 0.410
PPARγ 2.18±1.25 62.65±10.18* 51.05±6.12* <0.001
Values are means ± standard error. *represents a significant (p<0.05) difference from respective sedentary SPF group. Note: these samples were obtained immediately after an acute exhaustive bout of exercise due to mitigate the potential for microbial
contamination making it difficult to interpret VWR training effect. N = 9, 10, and 8 for SPF SED, SED GF, and VWR GF, respectively.
Supplemental Figure 1. Food (a) and water (b) disappearance in SPF mice.
Supplemental Figure 1. Food (a) and water (b) disappearance in SPF mice. Food and water disappearance was measured ~weekly during the course of the experiment in SPF mice. For food disappearance (a), *signifies that CON was significantly lower than both VWR and VWR/ABX groups. For water disappearance (b), *signifies that CON and VWR were significantly lower than ABX and ABX/VWR, while **signifies that CON was significantly lower than all other groups.
Supplemental Figure 2. Effects of wheel running and broad spectrum antibiotic treatment on body weight change over time.
Supplemental Figure 2. Effects of wheel running and broad-spectrum antibiotic treatment on body weight over time in males (a) and females (b). There were significant time main effects for both sexes indicating that both gained weight during the experiment.
In males (2a), there was a significant time x VWR interaction (F18,270 = 10.7; p = 0.000) indicating that VWR mice had lower body weights over time. In females (2b), there were significant time x VWR (F18,270 = 2.25; p = 0.003) and time x ABX (F18,270 = 2.95; p = 0.000) interactions indicating that VWR mice gained weight in the latter stages of the experiment and ABX treatment also lead to weight gain late in the experiment. Data are mean + sem for n = 4-5 per group.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
21 22 23 24 25 26 27 28 29 30 31
CON
VWR ABX
VWR/ABX Males
Time Points
Body Weight (g)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
17 18 19 20 21 22 23 24 25
CON VWR ABX
VWR/ABX Females
Time Points
B o d y W e ig h t (g )
Supplemental Figure 3. Body weight changes over time for male (a) and female (b) SPF mice
CON
VW
R ABX
VWR ABX GF
GF VWR
0 1 2 3 4 5
6 a
a,b a
b
c c
a) Males
Group
B o d y W e ig h t C h a n g e ( g )
CON
VW
R ABX
VWR ABX GF
GF VWR
0 1 2 3 4 5 6
* *
b) Females
Group
B o d y W e ig h t C h a n g e ( g )
Supplemental Figure 3. Body weight changes over time for male (a) and female (b) SPF mice. For males (a), there were
significant (<0.001) time and time x exercise effects (driven largely by lower body weights in VWR), and for females (b), there were significant (<0.001) time, time x exercise, and time x ABX effects.
Supplemental Figure 4. Total area under the curve (AUC) for 30, 60, and 90 minute timepoints during intraperitoneal glucose tolerance test
Supplemental Figure 4. Total area under the curve (AUC) for 30, 60, and 90 minute timepoints during intraperitoneal glucose tolerance test. When the 0 minute (baseline) timepoint is omitted, ABX did not affect total AUC, but Germ free status significantly increased it (p<0.001). This was unaffected by VWR in all groups. Data are mean + sem for n = 9, 10, 9, 9, 10, and 9 for CON, VWR, ABX, ABX VWR, GF, and GF VWR respectively.
Supplemental Figure 5. Effects of ABX or germ-free status on diurnal wheel running behavior
Supplemental Figure 5. Effects of ABX or germ-free status on diurnal wheel running behavior. There were significant (p < 0.05) time and time x group effects, but no main effect of group. asignifies a difference between VWR and GF VWR, crepresents difference between GF VWR and both other groups. Data are mean + sem for n = 9, 9, 10, and 8 for VWR, VWR/ABX, and GF VWR,
respectively.