Male

18

22

26

30

34

Bo

dy w

eig

ht (g

)

Age (weeks)

9 11 13 15 17 19 21 23 7

Female

15

18

21

24

27

30

Age (weeks)

9 11 13 15 17 19 21 23 7 B

od

y w

eig

ht (g

)

Atg7 f/f

Atg7 ΔCD11c

Supplementary Figure 1. Lean phenotypes in Atg7ΔCD11c mice regardless of gender.

Body weights from ages 7 to 23 weeks in male (left) and female (right) Atg7f/f and Atg7ΔCD11c

mice fed normal chow diet.

16

18

20

22

24

*

Atg7 f/f

Atg7 ΔCD11c

Body w

eig

ht

(g)

Supplementary Figure 2. Lean phenotypes of Atg7ΔCD11c mice were also detected in mice

given the high-fat diet (HFD).

The body weight of Atg7f/f and Atg7ΔCD11c 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<0.05.

A

pg

/ m

l o

f se

rum

0

20

40

60

80

*

Atg7 f/f

Atg7 ΔCD11c

B Atg7 f/f

Atg7 ΔCD11c

Rela

tive

mR

NA

e

xp

ressio

n

ns

0

0.5

1.0

1.5

2.0

TNFα

Re

lative m

RN

A

exp

ressio

n

0

0.5

1.0

1.5

F4/80

ns

C Atg7 f/f Atg7 ΔCD11c

Small

Intestine

Large

Intestine

Supplementary Figure 3. Lean phenotypes of 24-week-old Atg7ΔCD11c mice were not associated

with inflammation.

(A) Levels of proinflammatory cytokines in serum of Atg7f/f and Atg7ΔCD11c mice (total n = 7) by

cytometric bead array mouse-inflammatory kit (BD Biosciences). (B) mRNA expression levels of

F4/80 (left) and TNFα (right) in adipose tissue by real-time PCR. (C) Hematoxylin-eosin staining of

small and large intestines. Scale bar = 100 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 Bonferroni post-hoc test (A) and two-tailed paired t-test (B). *P<0.05; ns, not significant.

B A

C Atg7 f/f

Atg7 ΔCD11c

0

10

20

30

40

**

(μm

ol g

-1)

Propionate

0

2

4

6

8

10

(μm

ol g

-1)

*

Lactate

(μm

ol g

-1)

**

0

30

60

90

120

150

Acetate

0

10

20

30

40

(μm

ol g

-1)

**

Butyrate

Predictive component

Ort

ho

go

na

l co

mp

on

en

t 1

-200 0 -400 -600 200 400 600

200

400

600

-200

-400

-600

0

Atg7 f/f

Atg7 ΔCD11c

Covariance

Mo

de

led

co

rre

latio

n

1.0

0.0

-1.0

-0.8 -0.4 0.0 0.4 0.8

Acetic acid

Butyric acid

Propionic acid

Lactic acid

Supplementary Figure 4. Orthogonal partial least squares discriminate analysis (OPLS-DA) of fecal

metabolome data of Atg7f/f and Atg7ΔCD11c mice.

(A) Cross-validated score plots from OPLS-DA of 1H-nuclear magnetic resonance (NMR) data in

feces of Atg7f/f and Atg7ΔCD11c mice (total n = 7). (B) S-plots for predictive component from OPLS-DA

of 1H-NMR data of in feces of Atg7f/f and Atg7ΔCD11c mice (total n = 7). (C) Quantification of short-

chain fatty acids (i.e., acetate, butyrate, and propionate) and lactate in feces of Atg7f/f and Atg7ΔCD11c

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<0.05, **P<0.01.

24

26

28

30

32

Age (weeks)

15 16 17 18 19 20 21

Bo

dy w

eig

ht (g

)

Atg7 f/f (CH)

Atg7 ΔCD11c (CH)

Atg7 f/f (S)

Atg7 ΔCD11c (S)

Supplementary Figure 5. Compensatory body weight of mice in co-housing (CH) cages and

shared fecal microbes.

Body weights of Atg7f/f (n = 5) and Atg7ΔCD11c (n = 4) mice in CH cages. Body weights of

Atg7f/f and Atg7ΔCD11c mice housed separately (S) are shown by linear graphs of filled gray

and blue circles, respectively.

EF602759_s DQ815942_s DQ815871_g_uc EF602759_f_uc_s Bacteroides acidifaciens Bacteroides sartorii 4P003630_s EF604598_s EF406456_s Parabacteroides distasonis AB021165_s EF096000_s EF603769_s DQ815871_s EU622763_s EU791194_s AB606322_s AY239469_g_uc Bacteroidales_uc_s EF406459_s DQ815748_s EF097615_s EF604981_s EF406806_s EF603706_s HM124280_g_uc AB606319_s Bacteroides_uc EF603109_s

EF406536_s EF602759_g_uc EF406817_s EU457676_s Bacteroides uniformis EU456683_s EF406830_s Prevotellaceae_uc_s EF097057_s HM123997_s FJ511984_s Bacteroides coprocola EF603121_s EF603835_s EF406481_s EF406712_s EF406368_s Parabacteroides_uc EF603734_s AB606279_s EU006430_s FJ880499_s DQ815599_s EU006213_s AB606254_s EF406417_s EF406766_s FJ879877_s EF603798_s EF603662_s

HQ740248_s JQ085130_s DQ815311_s EU505041_s EU791177_s EF604622_s Pseudoflavonifractor_uc AB626943_s EF604610_s Oscillibacter_uc GQ451281_s Lachnospiraceae_uc_s HM123978_s AB606283_s DQ815599_g_uc DQ815781_s Lactobacillus brevis Ruminococcaceae_uc_s EU510538_s EU006321_s Coprobacillus_f_uc_s Faecalibacterium prausnitzii FJ881243_s EF604623_s AB626922_s EF602808_s 4P001451_s JQ084524_s DQ815907_s AJ308395_s

HM124141_s Helicobacter mastomyrinus Parasutterella excrementihominis AB270041_s 4P003191_s EF406813_s Mycoplasmataceae_f1_uc_s AJ400239_s DQ777900_s AJ400239_g_uc Mucispirillum schaedleri ETC

Supplementary Figure 6. Color legend of pyrosequencing data for species levels in feces of

Atg7ΔCD11c mice shown in Fig. 3C.

Supplementary Figure 7. Biological assignment of Bacteroides contigs in DNA

metagenomes.

The comparison of relative abundance of contigs number in feces of Atg7f/f and Atg7ΔCD11c

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 Bonferroni post-hoc test. *P<0.05; ***P<0.001; ns, not significant.

0

5

10

20

30

40

50

60

Rela

tive a

bundance (

%)

ns

ns ns

ns

*** Atg7 f/f

Atg7 ΔCD11c

ns ns ns ns ns

ns

*

Atg7 f/f-CH Atg7 ΔCD11c-CH

Supplementary Figure 8. Expanded BA were determined in feces of Atg7f/f mice co-housed with

Atg7ΔCD11c mice.

(A) Colony-forming units (CFUs) of B. acidifaciens on Bacteroides Bile Esculin (BBE) agar with feces of

Atg7f/f and Atg7ΔCD11c mice (n = 3) in co-housing (CH) cage for 23 weeks. (B) Representative gel images

of PCR analysis using BA-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<0.05.

A

B

0 15

BA BA

0 15

0

500

1000

1500

Colo

ny-F

orm

ing

Un

its Atg7 f/f-CH

Atg7 ΔCD11c-CH

216 bp

Atg7 f/f

*

A

B

BA

nu

mb

er

(x1

07

/ g

)

Days after administration

0

5

10

15

20

25

Nil 1 2 3 4 5

***

***

N.D. N.D. N.D.

Day 1 after BA feeding D

AP

I B. a

cid

ifacie

ns

Nil

Supplementary Figure 9. Orally administered B. acidifaciens (BA) can temporarily reside in

colon.

Colon tissues and feces were obtained at Nil and on days 1–5 after oral administration of BA

(5 × 109 CFU / 100 μl ; total n = 6) and stained with BA-specific FISH (fluorescence in situ

hybridization) probes. (A) Representative confocal images of BA (white arrows) in the colon

tissues. (B) Quantification of BA in the feces at indicated time points. BA were counted in ≥

20 regions 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 Bonferroni post-hoc test. ***P<0.001; N.D., not detected.

B

0

1

2

3

4 ns

Fo

od

up

take

(g

/ m

ou

se

/ d

ay)

D C

F

0 2 4 6 8 10 12 14 16 18 20 22 10

14

18

22

26

Light Dark

PBS

BA

En

erg

y e

xp

en

ditu

re

(kca

l kg

-1 h

r-1)

0.50

0.75

1.00

PBS

BA

RE

R (

VC

O2

/ V

O2)

0 2 4 6 8 10 12 14 16 18 20 22

Light Dark

To

tal a

ctivity (

x1

04)

(be

am

bre

aks p

er

da

y)

0

2

4

6

Light Dark

PBS

BA

ns

ns

E PBS

BA

0 Blo

od

glu

co

se

(m

g d

l-1)

0 30 60 90

Time (min)

120

GTT

*

100

200

300

400 ITT

0 30 60 90

Time (min)

120

Blo

od

glu

co

se

(m

g d

l-1)

0

100

200

300 B

A

PB

S

Area

(μm

2)

0

2000

4000

6000

8000

***

NCD

Bo

dy w

eig

ht ch

an

ge

0 1 2 3 4 5 6 7 8 9 10

(%)

** ** ** **

80

100

120

140

160

180

*

200

250

300

350

400

Area

(mm

2) ns

Supplementary Figure 10. Effective functions of B. acidifaciens (BA) in regulating body weight and fat mass

in normal chow diet (NCD)-fed B6 mice given BA or PBS.

(A) Representative photos and body weight over 10 weeks (left and right panels, respectively). BA was

administered orally (5 × 109 CFU / 100 μl) daily (total n = 10). (B) Oral food intake with PBS or BA. (C)

Magnetic resonance imaging analysis (total n = 6). (D) Histological changes of adipose tissues (left panel)

and size of adipocytes (right panel) of PBS- and BA-fed mice during NCD (total n = 6). (E) Glucose tolerance

test (GTT) (left panel, n = 9) and insulin tolerance test (ITT) (right panel, total n = 12) results by time point

after intraperitoneal injection of glucose or insulin. (F) Energy expenditure, total activity, and respiratory

exchange ratio (RER) of PBS- or BA-fed mice (total n = 10). 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 (B-D) and with two-way ANOVA with Bonferroni post-hoc test (A and E-F).

*P<0.05, **P<0.01, ***P <0.001; ns, not significant.

(Weeks)

PB

S

BA

A

Bo

dy w

eig

ht ch

an

ge

100

140

180

220

260

0 1 2 3 4 5 6 7 8 9 10

HFD+PBS

NCD+BS

NCD+PBS

HFD+BS

(%)

Weeks post administration

A B

HFD + PBS

NCD + BS

NCD + PBS

HFD + BS

Fo

od

up

take

(g / m

ou

se

/ d

ay)

0

1

2

3

4

ns

ns

Supplementary Figure 11. Mice given B. sartorii (BS) and normal-chow diet (NCD) or high-

fat diet (HFD) had similar body weight and food intake.

(A) Body weight over 10 weeks following oral administration of BS (total n = 10) (5 × 109 CFU

/ 100 μl). (B) Oral food intake with PBS or BS (total n = 10). 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 Bonferroni post-hoc test (A) and

two-tailed paired t-test (B). ns, not significant.

0

15

30

45

60

-30

-15

0

15

30

mg/k

g/m

in

Glucose infusion rate (GIR) Hepatic glucose production (HGP)

mg/k

g/m

in

*

*

ns

ns

BA

Heat-inactivated BA

PBS

75 ns

Supplementary Figure 12. B. acidifaciens (BA) administration improves both hepatic and peripheral

insulin sensitivity.

Hyperinsulinemic-euglycemic clamp studies of BA-, heat-inactivated BA-fed mice for 6 weeks and their

control mice (total n = 6) fed a normal chow diet. Infusion dose of insulin during the clamp study were

determined 3mU based on preliminary experiments. Whole-body glucose uptake (peripheral insulin

sensitivity, A) and insulin-mediated suppression of hepatic glucose production rates (hepatic insulin

sensitivity, B) are significantly increased in BA-fed mice compared to heat inactivated BA-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<0.05; ns, not

significant.

A

B

0

5

10

15

20

(μm

ol g

-1)

Acetate

(μm

ol g

-1)

Butyrate

0

1.5

3.0

4.5

6.0

(μm

ol g

-1)

Propionate

0

0.2

0.4

0.6

0.8

1.0

(μm

ol g

-1)

Lactate

PBS

BA ns

ns

ns

ns

0

0.5

1.0

1.5

2.0

( P = 0.9693) ( P = 0.2150 ) ( P = 0.0596 ) ( P = 0.8652 )

0

20

40

60

80

100

120

(μm

ol g

-1)

(μm

ol g

-1)

0

5

10

15

20

25

(μm

ol g

-1)

0

1

2

3

4

5

0

5

10

15

20

25

(μm

ol g

-1)

**

PBS

BA ns ns

ns

Acetate Butyrate Propionate Lactate

( P = 0.0934) ( P = 0.0693 ) ( P = 0.0028 ) ( P = 0.5742 )

Supplementary Figure 13. Levels of short-chain fatty acids (SCFAs) and lactate in feces after oral

B. acidifaciens (BA) administration for 10 weeks.

Levels of acetate, butyrate, propionate, and lactate in feces measured by gas chromatography-

mass spectrometry in mice given normal chow diet (A; total n = 6) or high-fat diet (B; 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<0.01; ns, not

significant.

A

B

Rela

tive

mR

NA

exp

ressio

n

Atg7 f/f

Atg7 ΔCD11c

Fatty acid synthesis β-Oxidation Thermogenesis

0

0.5

1.0

1.5

2.0

Rela

tive

mR

NA

exp

ressio

n

0

0.5

1.0

1.5

2.0

2.5 Atg7 f/f

Atg7 ΔCD11c

Fatty acid synthesis β-Oxidation Thermogenesis

Supplementary Figure 14. Similar expression levels of lipid oxidation in livers and small

intestines of Atg7f/f and Atg7ΔCD11c 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 (B) of Atg7f/f

and Atg7ΔCD11c 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 Bonferroni post-hoc test.

PBS BA

Supplementary Figure 15. B. acidifaciens (BA) administration does not induce β-cell overstimulation.

Pancreas tissues were obtained from mice (total n = 6) administrated with BA (5 × 109 CFU / 100 μl)

for 10 weeks. (A) Representative confocal images of islets (red color for α-cell and green color for β-cell).

Scale bar = 50 μm. The sections were sequentially reacted with mouse anti-glucagon immunoglobulin G

(IgG) Ab (K79bB10; Sigma-Aldrich, St. Louis, MO) and rabbit polyclonal anti-insulin Ab (Santa Cruz

Biotechnology, Santa Cruz, CA), and then PE-conjugated anti-mouse IgG (eBioscience, San Diego, CA)

and FITC-conjugated anti-rabbit IgG (eBioscience, San Diego, CA), respectively. (B) The size of β-cells’

region was quantified using ImageJ software program. The islet were randomly selected in 10 regions

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<0.001; ns,

not significant.

A B

0

5

10

15

20

Are

a (

x 1

03

, μm

2)

ns

BA PBS

A

B

0

50

100

150

0

20

40

60

80

Tri

gly

ce

rid

e (

mg

dl-1

)

To

tal ch

ole

ste

rol (m

g d

l-1)

Atg7 f/fB6

Atg7 ΔCD11cB6

B6B6

ns ns ns ns

C

0

20

40

60

80

0

50

100

150

Tri

gly

ce

rid

e (

mg

dl-1

)

ns

To

tal ch

ole

ste

rol (m

g d

l-1)

ns

0

20

40

60

80

100

ns

Tri

gly

ce

rid

e (

mg

dl-1

)

0

100

200

300 ns

To

tal ch

ole

ste

rol (m

g d

l-1)

NCD HFD

0

10

20

30

40

50

0

30

60

90

120

Tri

gly

ce

rid

e (m

g d

l-1)

To

tal ch

ole

ste

rol (m

g d

l-1)

ns ns

Supplementary Figure 16. Triglyceride and cholesterol levels were similar in serum of

Atg7ΔCD11c, fecal microbiota transplantation (FMT), and B. acidifaciens (BA)-fed mice.

Concentrations of serum triglycerides and total cholesterol were analyzed using enzymatic

assay kits in Atg7ΔCD11c mice (A; total n = 5), FMT mice (B; total n = 5), and BA-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. ns, not significant. NCD, normal chow diet; HFD, high-fat diet.

Atg7 f/f

Atg7 ΔCD11c

G (25)

0

1000

2000

3000 5000

10000

15000

20000

I (10)

0

200

400

600

900

1200

(nm

ol /

g f

eces)

B (3)

0

500

1000

10000

20000

30000

***

D (10) E-F (5)

A (20)

0

500

1000 1000

2000

3000

7000

10000

8500

N.D

.

C (20)

H (10)

0

500

1000 2000

4000

6000

8000

N.D

. L-M (12)

0

1000

2000 3000

5000

7000

N.D

.

N (31)

0

500

100

1500

200

2500

300

N.D

.

O (9)

0

50

100 200

400

600

800

N.D

.

(nm

ol /

g f

eces)

(nm

ol /

g f

eces)

(nm

ol /

g f

eces)

0

40

80 100

150

400

0

100

200

300

400

500

***

0

30

60

500

1500

N.D

.

(nm

ol /

g f

eces)

U-X (13)

0

150

300

450

1000

2000

3000

Supplementary Figure 17. The profiles of 292 metabolites in feces of Atg7f/f and Atg7ΔCD11c mice.

The metabolites in feces of Atg7f/f and Atg7ΔCD11c mice (n = 7) were measured by capillary electrophoresis time-

of-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

Bonferroni post-hoc test. *P<0.05, **P<0.01 and ***P<0.001; N.D., not detected.

1'- & 2'- (23)

0

100

200

300

**

3'- & 4'- (21)

0

100

200 300

600

900

1200 *

P (26)

0

1000

2000

10000

20000

30000

***

N.D

.

N.D

.

N.D

.

R-S (19)

0

200

400

600 1000

3000

5000

7000 ***

N.D

.

T (20)

5'- & 6'- & 7'- (15)

0

100

200

300

1000

2000

3000

4000 N

.D.

(nm

ol /

g f

eces)

(nm

ol /

g f

eces)

(nm

ol /

g f

eces)

(nm

ol /

g f

eces)

0

100

200

300

400

1000

2000

3000

4000

*

N.D

.

Supplementary Figure 18. B. acidifaciens (BA) can be regulated by autophagy machinery of

CD11c+ cells.

Numbers of BA in bone marrow-derived CD11c+ cells were determined on EG agar plate at 6

and 24 hours after co-cultured with BA (MOI=10). Bone marrow were obtained from Atg7f/f or

Atg7ΔCD11c 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<0.05; N.D., not detected.

Atg7 f/f

Atg7 ΔCD11c

Time for co-culture (hours)

0

1

2

3

4

5

6

7 *

ns

0 6 24

N.D.

Colo

ny-F

orm

ing

Un

its (

Lo

g1

0)