ody weight (g) ody weight (g) 34 3 Male 3 27 Female 26 24 22 18 7 9 11 13 15 17 19 21 23 21 18 15 7 9 11 13 15 17 19 21 23 Age (weeks) Age (weeks) Supplementary Figure 1. Lean phenotypes in mice regardless of gender. ody weights from ages 7 to 23 weeks in male (left) and female (right) and mice fed normal chow diet.
ody weight (g) 24 22 2 18 16 Supplementary Figure 2. Lean phenotypes of mice were also detected in mice given the high-fat diet (HFD). The body weight of and mice (n = 4) were monitored following feeding with HFD for 3 weeks. Data shown are at age 12 weeks and are mean ± s.e.m. Statistical analyses were done with two-tailed paired t-test. P<.5.
Relative mrna expression Relative mrna expression pg / ml of serum A 8 6 4 2 1.5 1. F4/8 2. 1.5 TNFα 1..5.5 C Small Intestine Large Intestine Supplementary Figure 3. Lean phenotypes of 24-week-old mice were not associated with inflammation. (A) Levels of proinflammatory cytokines in serum of and mice (total n = 7) by cytometric bead array mouse-inflammatory kit (D iosciences). () mrna expression levels of F4/8 (left) and TNFα (right) in adipose tissue by real-time PCR. (C) Hematoxylin-eosin staining of small and large intestines. Scale bar = 1 μm. Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 3 to 5 mice per experiment. Statistical analyses were done with two-way ANOVA with onferroni post-hoc test (A) and two-tailed paired t- test (). P<.5;, not significant.
Orthogonal component 1 Modeled correlation A 6 1. 4 2 Lactic acid. -2-4 Acetic acid utyric acid C -6-6 -4-2 2 4 6 Acetate 15 12 9 6 3 Predictive component utyrate 4 3 2 1 4 3 2 1 Propionate 1 8 6 4 2 Lactate Propionic acid -1. -.8 -.4..4.8 Covariance Supplementary Figure 4. Orthogonal partial least squares discriminate analysis (OPLS-DA) of fecal metabolome data of and mice. (A) Cross-validated score plots from OPLS-DA of 1 H-nuclear magnetic resonance (NMR) data in feces of and mice (total n = 7). () S-plots for predictive component from OPLS-DA of 1 H-NMR data of in feces of and mice (total n = 7). (C) Quantification of shortchain fatty acids (i.e., acetate, butyrate, and propionate) and lactate in feces of and mice (total n = 7) by gas chromatography-mass spectrometry. Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 3 to 4 mice per experiment. Statistical analyses were done with two-tailed paired t-test. P<.5, P<.1.
ody weight (g) 32 3 (S) (CH) (CH) (S) 28 26 24 15 16 17 18 19 2 21 Age (weeks) Supplementary Figure 5. Compeatory body weight of mice in co-housing (CH) cages and shared fecal microbes. ody weights of (n = 5) and (n = 4) mice in CH cages. ody weights of and mice housed separately (S) are shown by linear graphs of filled gray and blue circles, respectively.
EF62759_s DQ815942_s DQ815871_g_uc EF62759_f_uc_s acteroides acidifacie acteroides sartorii 4P363_s EF64598_s EF46456_s Parabacteroides distasonis A21165_s EF96_s EF63769_s DQ815871_s EU622763_s EU791194_s A66322_s AY239469_g_uc acteroidales_uc_s EF46459_s DQ815748_s EF97615_s EF64981_s EF4686_s EF6376_s HM12428_g_uc A66319_s acteroides_uc EF6319_s EF46536_s EF62759_g_uc EF46817_s EU457676_s acteroides uniformis EU456683_s EF4683_s Prevotellaceae_uc_s EF9757_s HM123997_s FJ511984_s acteroides coprocola EF63121_s EF63835_s EF46481_s EF46712_s EF46368_s Parabacteroides_uc EF63734_s A66279_s EU643_s FJ88499_s DQ815599_s EU6213_s A66254_s EF46417_s EF46766_s FJ879877_s EF63798_s EF63662_s HQ74248_s JQ8513_s DQ815311_s EU5541_s EU791177_s EF64622_s Pseudoflavonifractor_uc A626943_s EF6461_s Oscillibacter_uc GQ451281_s Lachnospiraceae_uc_s HM123978_s A66283_s DQ815599_g_uc DQ815781_s Lactobacillus brevis Ruminococcaceae_uc_s EU51538_s EU6321_s Coprobacillus_f_uc_s Faecalibacterium prausnitzii FJ881243_s EF64623_s A626922_s EF6288_s 4P1451_s JQ84524_s DQ81597_s AJ38395_s HM124141_s Helicobacter mastomyrinus Parasutterella excrementihominis A2741_s 4P3191_s EF46813_s Mycoplasmataceae_f1_uc_s AJ4239_s DQ7779_s AJ4239_g_uc Mucispirillum schaedleri ETC Supplementary Figure 6. Color legend of pyrosequencing data for species levels in feces of mice shown in Fig. 3C.
Relative abundance (%) 6 5 4 3 2 1 5 Supplementary Figure 7. iological assignment of acteroides contigs in DNA metagenomes. The comparison of relative abundance of contigs number in feces of and mice (total n = 6). Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 3 mice per experiment. Statistical analyses were done with two-way ANOVA with onferroni post-hoc test. P<.5; P<.1;, not significant.
Colony-Forming Units A 15 1 -CH -CH 5 - CH - CH 216 bp 15 15 Supplementary Figure 8. Expanded were determined in feces of mice co-housed with mice. (A) Colony-forming units (CFUs) of. acidifacie on acteroides ile Esculin (E) agar with feces of and mice (n = 3) in co-housing (CH) cage for 23 weeks. () Representative gel images of PCR analysis using -specific primer (F : 5 -CTGCCTCATACTCGGGGATA-3, R : 5 - CGTAGGAGTTTGGACCGTGT- 3 ; product size : 216 bp). The 15 colonies were randomly picked per plate for DNA templates. Data shown are the mean values ± SEM from individual mice. Statistical analyses were done with two-tailed paired t-test. P<.5.
A Nil Day 1 after feeding DAPI. acidifacie number (x1 7 / g) 25 2 15 1 5 Nil 1 2 3 4 5 Days after administration Supplementary Figure 9. Orally administered. acidifacie () can temporarily reside in colon. Colon tissues and feces were obtained at Nil and on days 1 5 after oral administration of (5 1 9 CFU / 1 μl ; total n = 6) and stained with -specific FISH (fluorescence in situ hybridization) probes. (A) Representative confocal images of (white arrows) in the colon tissues. () Quantification of in the feces at indicated time points. were counted in 2 regio per slide. Data shown are the mean values ± SEM from individual mice from 3 independent experiments with 2 mice per experiment. Statistical analyses were done with two-way ANOVA with onferroni post-hoc test. P<.1;, not detected.
Energy expenditure (kcal kg -1 hr -1 ) Total activity (x1 4 ) (beam breaks per day) RER (VCO 2 / VO 2 ) lood glucose (mg dl -1 ) lood glucose (mg dl -1 ) (mm 2 ) (μm 2 ) ody weight change Food uptake (g / mouse / day) A C NCD (%) 18 16 14 12 1 8 1 2 3 4 5 6 7 8 9 1 (Weeks) Area 4 35 3 25 2 D 4 3 2 1 8 6 4 2 Area E 4 3 2 GTT 3 2 ITT 1 1 F 26 22 3 6 9 Time (min) 12 6 4 3 6 9 Time (min) 12 1. 18 14 2.75 Light Dark 1 2 4 6 8 1 12 14 16 18 2 22 Light Dark.5 Light Dark 2 4 6 8 1 12 14 16 18 2 22 Supplementary Figure 1. Effective functio of. acidifacie () in regulating body weight and fat mass in normal chow diet (NCD)-fed 6 mice given or. (A) Representative photos and body weight over 1 weeks (left and right panels, respectively). was administered orally (5 1 9 CFU / 1 μl) daily (total n = 1). () Oral food intake with or. (C) Magnetic resonance imaging analysis (total n = 6). (D) Histological changes of adipose tissues (left panel) and size of adipocytes (right panel) of - and -fed mice during NCD (total n = 6). (E) Glucose tolerance test (GTT) (left panel, n = 9) and iulin tolerance test (ITT) (right panel, total n = 12) results by time point after intraperitoneal injection of glucose or iulin. (F) Energy expenditure, total activity, and respiratory exchange ratio (RER) of - or -fed mice (total n = 1). Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 3 to 6 mice per experiment. Statistical analyses were done with two-tailed paired t-test (-D) and with two-way ANOVA with onferroni post-hoc test (A and E-F). P<.5, P<.1, P <.1;, not significant.
ody weight change Food uptake (g / mouse / day) A (%) 26 22 18 NCD+ NCD+S HFD+ HFD+S 4 3 2 NCD + NCD + S HFD + HFD + S 14 1 1 1 2 3 4 5 6 7 8 9 1 Weeks post administration Supplementary Figure 11. Mice given. sartorii (S) and normal-chow diet (NCD) or highfat diet (HFD) had similar body weight and food intake. (A) ody weight over 1 weeks following oral administration of S (total n = 1) (5 1 9 CFU / 1 μl). () Oral food intake with or S (total n = 1). Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 5 mice per experiment. Statistical analyses were done with two-way ANOVA with onferroni post-hoc test (A) and two-tailed paired t-test ()., not significant.
mg/kg/min mg/kg/min Glucose infusion rate (GIR) 75 6 Hepatic glucose production (HGP) 3 15 Heat-inactivated 45 3 15-15 -3 Supplementary Figure 12.. acidifacie () administration improves both hepatic and peripheral iulin seitivity. Hyperiulinemic-euglycemic clamp studies of -, heat-inactivated -fed mice for 6 weeks and their control mice (total n = 6) fed a normal chow diet. Infusion dose of iulin during the clamp study were determined 3mU based on preliminary experiments. Whole-body glucose uptake (peripheral iulin seitivity, A) and iulin-mediated suppression of hepatic glucose production rates (hepatic iulin seitivity, ) are significantly increased in -fed mice compared to heat inactivated -fed group. Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 3 mice per experiment. Statistical analyses were done with two-tailed paired t-test. P<.5;, not significant.
A Acetate utyrate Propionate Lactate ( P =.5742 ) ( P =.28 ) ( P =.693 ) ( P =.934) 12 25 25 5 1 2 2 4 8 15 15 3 6 4 1 1 2 2 5 5 1 Acetate ( P =.8652 ) 2 15 1 5 utyrate ( P =.596 ) 2. 1.5 1..5 Propionate ( P =.215 ) 6. 4.5 3. 1.5 Lactate ( P =.9693) 1..8.6.4.2 Supplementary Figure 13. Levels of short-chain fatty acids (SCFAs) and lactate in feces after oral. acidifacie () administration for 1 weeks. Levels of acetate, butyrate, propionate, and lactate in feces measured by gas chromatographymass spectrometry in mice given normal chow diet (A; total n = 6) or high-fat diet (; total n = 6). Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 3 mice per experiment. Statistical analyses were done with two-tailed paired t-test. P<.1;, not significant.
Relative mrna expression Relative mrna expression A Fatty acid synthesis β-oxidation Thermogenesis 2.5 2. 1.5 1..5 2. Fatty acid synthesis β-oxidation Thermogenesis 1.5 1..5 Supplementary Figure 14. Similar expression levels of lipid oxidation in livers and small intestines of and mice. Expression level of mrna genes related to fatty acid synthesis (FasN, HSL, PEPCK, SCD1, and PPARγ), β-oxidation (PPARα), and thermogenesis (PRDM16, PGC1a, Cidea, and GLUT4) were determined by real-time PCR using livers (A) and small intestines () of and mice (total n = 8). Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 3 to 5 mice per experiment. Statistical analyses were done with two-way ANOVA with onferroni post-hoc test.
Area (x 1 3, μm 2 ) A 2 15 1 5 Supplementary Figure 15.. acidifacie () administration does not induce β-cell overstimulation. Pancreas tissues were obtained from mice (total n = 6) administrated with (5 1 9 CFU / 1 μl) for 1 weeks. (A) Representative confocal images of islets (red color for α-cell and green color for β-cell). Scale bar = 5 μm. The sectio were sequentially reacted with mouse anti-glucagon immunoglobulin G (IgG) Ab (K79b1; Sigma-Aldrich, St. Louis, MO) and rabbit polyclonal anti-iulin Ab (Santa Cruz iotechnology, Santa Cruz, CA), and then PE-conjugated anti-mouse IgG (eioscience, San Diego, CA) and FITC-conjugated anti-rabbit IgG (eioscience, San Diego, CA), respectively. () The size of β-cells region was quantified using ImageJ software program. The islet were randomly selected in 1 regio per slide. Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 3 mice per experiment. Statistical analyses were done with two-tailed paired t-test. P<.1;, not significant.
Triglyceride (mg dl -1 ) Total cholesterol (mg dl -1 ) Triglyceride (mg dl -1 ) Total cholesterol (mg dl -1 ) Triglyceride (mg dl -1 ) Total cholesterol (mg dl -1 ) Triglyceride (mg dl -1 ) Total cholesterol (mg dl -1 ) A 5 12 4 3 2 1 9 6 3 15 1 8 6 6 6 6 6 4 5 2 C 8 6 4 2 15 1 5 1 8 6 4 2 3 2 1 NCD HFD Supplementary Figure 16. Triglyceride and cholesterol levels were similar in serum of, fecal microbiota traplantation (FMT), and. acidifacie ()-fed mice. Concentratio of serum triglycerides and total cholesterol were analyzed using enzymatic assay kits in mice (A; total n = 5), FMT mice (; total n = 5), and -fed mice (C; total n = 5). Data shown are the mean values ± SEM from individual mice from 2 independent experiments with 2 to 3 mice per experiment. Statistical analyses were done with two-tailed paired t-test., not significant. NCD, normal chow diet; HFD, high-fat diet.
(nmol / g feces) (nmol / g feces) (nmol / g feces) (nmol / g feces) (nmol / g feces) 1 85 7 3 2 1 1 5 A (2) 3 2 1 1 5 (3) 15 5 6 C (2) 4 15 1 8 D (1) E-F (5) 5 4 3 2 3 4 1 2 15 1 5 3 G (25) 8 6 4 2 1 H (1) 2 1 5 12 I (1) 7 L-M (12) 9 5 6 4 3 2 2 1 25 N (31) 8 O (9) 15 5 3 6 4 2 1 2 1 5
(nmol / g feces) (nmol / g feces) (nmol / g feces) (nmol / g feces) 3 P (26) 3 U-X (13) 2 1 2 1 2 45 1 3 15 R-S (19) 7 5 3 1 6 4 2 4 3 2 1 4 3 2 1 T (2) 3 1'- & 2'- (23) 2 1 12 9 6 3 2 3'- & 4'- (21) 4 3 2 1 3 5'- & 6'- & 7'- (15) 1 2 1 Supplementary Figure 17. The profiles of 292 metabolites in feces of and mice. The metabolites in feces of and mice (n = 7) were measured by capillary electrophoresis timeof-flight mass spectrometry (CE-TOF-MS). All metabolites are arranged in alphabetical order. Data shown are the mean values ± SEM from individual mice. Statistical analyses were done with two-way ANOVA with onferroni post-hoc test. P<.5, P<.1 and P<.1;, not detected.
Colony-Forming Units (Log 1 ) 7 6 5 4 3 2 1 6 24 Time for co-culture (hours) Supplementary Figure 18.. acidifacie () can be regulated by autophagy machinery of CD11c + cells. Numbers of in bone marrow-derived CD11c + cells were determined on EG agar plate at 6 and 24 hours after co-cultured with (MOI=1). one marrow were obtained from or mice (total n = 6). Data shown are the mean values ± SEM from individual mice from 3 independent experiments with 2 mice per experiment. Statistical analyses were done with two-tailed paired t-test. P<.5;, not detected.