Cell Metabolism, Volume 24

Supplemental Information

Colonic Pro-inflammatory Macrophages

Cause Insulin Resistance in an Intestinal

Ccl2/Ccr2-Dependent Manner

Yoshinaga Kawano, Jun Nakae, Nobuyuki Watanabe, Tetsuhiro Kikuchi, SanshiroTateya, Yoshikazu Tamori, Mari Kaneko, Takaya Abe, Masafumi Onodera, and HiroshiItoh

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SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Generation of Tissue-specific Ccr2 Knockout Mice

The mouse genomic DNA clone containing exons 1, 2, and 3 of the Ccr2 gene locus

was obtained from a BAC clone CH25-320O8 (BACPAC Resource) derived from the

C57BL/6 mouse strain. The Ccr2 targeting construct consisted of 9.1 kb of the genomic

sequence that was immediately 5’ of the first exon, followed by exon 1, intron 1, exon 2,

a 2.3-kb Neo-cassette, a frt-flanked Neo-resistance gene, and exon 3. A 1763-bp of

genomic sequence containing the open reading frame in exon 3 of the Ccr2 gene was

flanked by the loxP sequence. The SalI-linearized targeting vector was electroporated

into TT2 embryonic stem (ES) cells (Yagi et al., 1993) as described previously.

G418-resistant clones were isolated and screened by PCR and Southern blotting.

Seven out of 192 G418-resistant clones had undergone the desired homologous

recombination. Positive clones were injected into CD-1 8-cell stage embryos, and the

chimeric male offspring were mated to C57BL/6 females, obtaining Ccr2 conditional

knockout mice (Accession No. CDB0610K:

http://www2.clst.riken.jp/arg/mutant%20mice%20list.html). Primers for detection of the

wild-type allele (440 bp) and the loxP allele (600 bp) are: 5’-

GCCTATTCTCTTCTGTATCTC-3’ and 5’- TGGATGAACTGAGGTAACAT-3’ (Figure

S4A-S4D).

Immunohistochemistry, Immunofluorescence and Histological Analysis

For histological analysis, we removed the white adipose tissue and colon from mice,

fixed the specimens in 4% paraformaldehyde and embedded them in paraffin. After

rehydration and permeabilization, we stained the specimens with hematoxylin and eosin.

Immunofluorescence was performed as described previously (Kawano et al., 2012)

using anti-CD68 (Dako Denmark A/S), anti-Claudin-1 (Invitrogen), or anti-ASC

(Santa-Cruz) antibodies. After washing with PBS, the sections were sequentially

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incubated with secondary antibody and visualized using the Liquid DAB Substrate

Chromogen System (DakoCytomation) for CD68 single-staining in white adipose tissue

and colon. Crypt depth, the number of goblet cells in the colon, and the size and number

of adipocytes in white adipose tissue were determined using a fluorescence microscope

(BZ-8000, 9000, KEYENCE) by manually tracing at least more than 300 adipocytes for

each genotype.

Isolation of Liver Macrophages

The liver was removed and cut into small pieces with scissors, and then filtered

through 100μm nylon mesh. The cells were collected in a new 15 ml tube and incubated

with 10mM EDTA and Collagenase B (Roche) with gentle shaking. After incubation with

collagenase, the supernatants were centrifuged at 1700 rpm and washed twice with

PBS. The cell pellets were re-suspended in 40% Percoll, overlaid on 80% Percoll, and

centrifuged at 2000 rpm for 20min at room temperature. The white interphase was

collected in a new tube, and centrifuged, and analyzed by FACS immediately.

Isolation of the Stromal Vascular Fraction

The epididymal fat was removed, transferred to a 50ml tube containing KRHAG buffer

(1M KCl, 1M CaCl2, 1M KH2PO4, 1M MgSO4, 5% BSA, 200 mM HEPES (pH 7.8), 200

mM glucose), and cut into small pieces. The pieces were incubated with Collagenase

type Ⅰ (Wako) in KRHAG buffer (3.0mg/1.5ml) for 45 min at 37oC with gentle shaking.

After filtering through 100μm nylon, the supernatant was centrifuged at 1500rpm for

5min at 4℃ and washed twice in Pharm Lyse (BD Bioscience) buffer. After hemolytic

incubation with lysing solution (BD Bioscience), the cells were washed twice again and

analyzed by FACS immediately.

RNA Isolation and Real-time PCR

Isolation of total RNA was performed using the SV Total RNA Isolation System

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(Promega) according to the manufacturer’s protocol. We performed reverse

transcription using the PrimeScriptTM RT Reagent Kit, and real-time PCR using the

SYBR GREEN detection protocol by STRATAGENE (An Agilent Technologies division,

Germany). All primer sequences are available upon request.

Western Blotting

For Western blotting, we homogenized tissues as described previously (Nakae et al.,

2012). After centrifugation to remove insoluble material, the proteins in 150 μg of lysate

were separated using 8% SDS-PAGE for detection of p-Akt and Akt (Cell signal) or 14%

SDS-PAGE for detection of Claudin-1 and procaspase-1 p10 (sc-514, Santa Cruz), and

immunized using the indicated antibodies.

H2O2 Production

The colon and epididymal fat were dissected from age-matched male C57BL/6J mice

fed either a NCD or a HFD for 4 or 24 weeks. We measured peroxidase activity in the

colon and epididymal fat using with the Amplex Red Hydrogen Peroxide/Peroxidase

Assay Kit (Invitrogen) according to the manufacturer's protocol.

Intracellular Staining of TNF-α

Intracellular staining of TNF-α was performed using the Fix and Permeabilization Kit

(eBioscience). First, cells were stimulated with 1μg/ml LPS for 4 h with BFA added to

the cell suspension for the final 2 hours. Next, the cells were stained with surface

marker antibodies, including F4/80(BM8, APC, BioLegend), CD11b (M1/70, APC-Cy7,

BioLegend), and CD11c (N418, FITC, BioLegend), washed with PBS, and fixed in

formaldehyde. The cells were permeabilized in PBS containing 0.5% saponin and 0.5%

BSA (Sigma-Aldrich), stained with antibody for intracellular TNFα marker (MP-6XT22,

PerCP-Cy5.5, BD Biosciences), and analyzed by flow cytometry.

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Metagenomic analysis of 16S rRNA

To analyze the microbial population from NCD, HFD, M-Ccr2KO, Vil-Ccl2KO mice in this

study, metagenomics analysis using 16S rRNA sequencing was performed. DNA sample

for assessment of the microbial community was extracted from lyophilized cecal content

using the QIAamp DNA Stool Mini Kit (Qiagen). Illumina Miseq was used to sequence

complex microbial populations. Sequence data was processed and filtered by the

quantitative insights into microbial ecology (QIIME, Ver 1.8.0.) Chimeric sequences were

removed. We used a Cut-adapt (Ver 1.1) to trim any over-read, and paired-end

sequences from DNA sequencing reads. A total of 110,000 reads were generated per

sample from these mice after filtering. Taxonomy is assigned to each OTU based on the

16S rDNA database Greengenes (Ver 13.8) in the closed-reference manner. We excluded

sequences that were not between 200 and 1000 nucleotides in length and clustered into

Operational Taxonomic Units (OTUs). OTU analyses were performed by clustering at the

similarity 97% level with UCLUST. Beta diversity was measured using the weighted/

unweighted UniFrac analysis. UniFrac distance matrics, which was were used to compute

average distances within and between various groups of samples, were generated using by

QIIME’s BaseSpace. Weighted UniFrac PCoA plots were used to visualize the similarities

or dissimilarities of variables. Hierarchical clustering method was used to produce UPGMA

(Unweighted Pair Group Method with Arithmetic Mean) tree in genus level.

Endotoxin Measurement

The concentration of endotoxin in the portal vein was measured using the endotoxin

assay kit (ToxinSensorTM Chromogenic LAL Endotoxin Assay kit) according to the

manufacturer's protocol.

Measurement of Cytokines in the Portal Vein

Serum levels of IL18 and IL1β in the portal vein were measured using the mouse ELISA

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kits (R&D SYSTEM for IL18 and mouse ELISA kit Quantikine, R&D SYSTEM for IL1β).

Isolation of Intestinal Epithelial Cell for Real-time PCR

The pieces of the colon and small intestine dissected from each mouse were incubated

in 10 mM DTT and 0.5M EDTA in 1.5% FBS-HANKS at 37oC for 30 minutes. The

supernatant was then collected and filtered through 100 μm nylon mesh and centrifuged

to pellet. The pellets were washed four times and resuspended in the RNA lysis buffer of

the SV Total RNA Isolation System (Promega)

Magnetic Activated Cell Sorting

Mononuclear cells were isolated from the colon and incubated with CD11b magnetic

beads (Miltenyi Biotec) for positive selection. The CD11b+ cells were cultured with (2×

106 cells/ml) in HANKs’ Balanced Salt solution supplemented with 1.5% FCS and 1%

penicillin/streptomycin.

Intestinal Permeability

Fluorescein-isothiocyanate-conjugated dextran (FD4: Sigma-Aldrich, Gillingham, UK)

was administered to each mouse at 0.4 mg/g body weight by oral gavage. After 4 h,

blood was collected from the portal vein and diluted with an equal volume of PBS. The

concentration of FD4 dextran was measured at 485 nm using a spectrophoto microplate

reader.

Co-culture of Caco-2 and CD11b+ Cell

Caco-2 cells were cultured with DMEM supplemented with 1.5% FBS and 1%

penicillin/streptomycin for 20 days. Differentiated Caco-2 cells at 80% confluence were

seeded on the basal side of the NuncTM CC insert 12-well plate (polycarbonate

membrane, < 1.7×106 pores/cm2 ). CD11b+ cells sorted from the colons of mice fed a

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NCD stimulated with a mixture of IL-1β, IL-18, and LPS were added to polycarbonate

membrane inserts. After 36 hours, Caco-2 cells were harvested and centrifuged and the

cell pellets suspended in RIPA lysis buffer.

SUPPLEMENTAL REFERENCES

Kawano, Y., Nakae, J., Watanabe, N., Fujisaka, S., Iskandar, K., Sekioka, R., Hayashi,

Y., Tobe, K., Kasuga, M., Noda, T., et al. (2012). Loss of Pdk1-Foxo1 signaling in

myeloid cells predisposes to adipose tissue inflammation and insulin resistance.

Diabetes 61, 1935-1948.

Nakae, J., Cao, Y., Hakuno, F., Takemori, H., Kawano, Y., Sekioka, R., Abe, T.,

Kiyonari, H., Tanaka, T., Sakai, J., et al. (2012). Novel repressor regulates insulin

sensitivity through interaction with Foxo1. EMBO J 31, 2275-2295.

Sakaue, H., Ogawa, W., Matsumoto, M., Kuroda, S., Takata, M., Sugimoto, T.,

Spiegelman, B.M., and Kasuga, M. (1998). Posttranscriptional control of adipocyte

differentiation through activation of phosphoinositide 3-kinase. J Biol Chem 273,

28945-28952.

Yagi, T., Tokunaga, T., Furuta, Y., Nada, S., Yoshida, M., Tsukada, T., Saga, Y.,

Takeda, N., Ikawa, Y., and Aizawa, S. (1993). A novel ES cell line, TT2, with high

germline-differentiating potency. Anal Biochem 214, 70-76.

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Figure S1. Related to Figure 1. HFD analysis of small intestine. (A) The protocol for evaluating sequential change of intestinal immune state during HFD for 24 weeks. (B)Representative image of the small intestine from NCD and HFD for 4 weeks (C)The length of the small intestine from age-matched C57Bl6/J mice (n=6) fed with HFD compared with NCD. Data are expressed as mean + SEM. P-value by one-way ANOVA.(D) Normalized gene expression of inflammatory related genes in the small intestine of age-matched C57Bl6/J mice fed with HFD compared with NCD. Data are normalized to β-actin expression and represent means + SEM from 8-10 mice in each group. *P<0.05 by one-way ANOVA. (E, F and G) Related to Figure 1. HFD-induced Pro-inflammatory Changes in Adipose Tissue and Liver. Normalized expression of inflammation-related genes (Emr1, Ccl2, Tnf, Il1b) in epididiymal fat (E), mesenteric fat (F) and liver (G) from age-matched C57Bl6/J mice fed with HFD compared with NCD. Data represent as the ratio to control in each gene. Data are means + SEM. *P<0.05 by one-way ANOVA.

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Figure S2. Related to Figure 2. HFD-induced Changes of Ly6c, Cx3cr1, and Siglecf Expression in Colon and FACS Analysis of F4/80+CD11b+CD11c-TNFαhigh from the Colon of NCD and HFD Mice.(A) Representative FACS analysis of Ly6c, CX3CR1, SiglecF among F4/80+CD11b+CD11c- cells in colonic LP from mice fed with NCD (the left panel) and HFD for 4 weeks (the right panel). (B) Normalized expression of Ly6c, Cx3cr1, and Siglecf in colon from age-matched C57Bl6/J mice fed with HFD compared with NCD. Data represent as the ratio to control in each gene. Data are means + SEM. *P<0.05 by one-way ANOVA.(C and D) Representative FACS analysis of F4/80+CD11b+CD11c-TNFαhigh cell in colonic LP from mice fed with a NCD and with a HFD for 4 weeks (C), and from mice fed with a NCD and a HFD for 12 weeks (D).(E) Absolute number of F4/80+CD11b+CD11c-TNFαhigh cell in living 105 F4/80 positive cell. Data are means + SEM. *P<0.05 by one-way ANOVA.(F) Measurement of TNFα production by ELISA in culture supernatants of MACS sorted CD11+ cells from NCD and HFD for 4 weeks. Data are expressed as mean + SEM. n.s. by one-way ANOVA.

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Figure S3. Related to Figure 3. Metabolic Phenotypes, FACS Analysis, and Gene Expression Analysis of M-Ccr2KO.(A) Body weight. (B) IPGTT. (C) ITT in M-Ccr2KO fed with a NCD. The black rhombus and green square indicate control and M-Ccr2KO, respectively. Data represent means + SEM of 3 mice in each genotype.(D) Fat composition of epididymal fat, subcutaneous fat, mesentery fat, perirenal fat and brown adipose tissue from NC, HF control and M-Ccr2KO. Data are % of body weight and expressed as mean + SEM. (E) Fat free mass. Data are defined as total body mass except for epididymal fat, subcutaneous fat, mesentery fat, perirenal fat and brown adipose tissue and are expressed as mean + SEM. (F) Insulin secretion of control and M-Ccr2KO mice fed with a HFD for 10 weeks during IPGTT. Data are means + SEM of 8-10 mice in each genotype. (G) Oral glucose tolerance test (oGTT) of control and M-Ccr2KO mice fed with HFD for 10 weeks (n=10). Data are means + SEM. *P<0.05 by two-way ANOVA with Fisher's test.(H) Insulin secretion of control and M-Ccr2KO mice fed with HFD for 10 weeks during oGTT. Data are means + SEM of 8-10 mice in each genotype. (I)  Hepatic triglyceride content of control and M-Ccr2KO mice fed with HFD for 10 weeks. (II)  Data are means + SEM of 6-8 mice. n.s. by one-way ANOVA.(J) Absolute number of F4/80+CD11b+CD11c+CD206- cell in whole epididymal fat. Data are means + SEM. n.s. by one-way ANOVA.(K) Normalized expression of inflammation-related genes in mesentery fat from NC control, HF control, and M-Ccr2KO mice fed with HFD for 10 weeks. Data represent as the ratio to control in each gene and are means + SEM of 6 mice in each genotype. *P<0.05 by one-way ANOVA.(L) Normalized expression of inflammation-related genes in CD11b+ cell in stromal vascular fraction of epididymal fat from control and M-Ccr2KO mice fed with HFD for 10 weeks. Data represent as the ratio to control in each gene and are means + SEM of 4 mice in each genotype. *P<0.05 by one-way ANOVA. (M) Normalized expression of inflammation-related genes in skeletal muscle from control and M-Ccr2KO mice fed with HFD for 10 weeks. Data represent as the ratio to control in each gene and are means + SEM of 3-5 mice in each genotype. *P<0.05 by one-way ANOVA.

Small intestine

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Figure S4. Related to Figure 4. Analysis of Cecum and Small Intestine of M-Ccr2KO(A) The weight of the cecum from NC control, HF control, and M-Ccr2KO mice fed with a HFD for 10 weeks. Data represent as the ratio to control in each gene and are means + SEM of 4 mice in each genotype. *P < 0.05 by one-way ANOVA.(B) Normalized expression of inflammation-related genes in the small intestine from NCcontrol, HF control, and M-Ccr2KO mice fed with HFD for 10 weeks. Data represent as the ratio to control in each gene and are means + SEM of 6 mice in each genotype. *P<0.05 by one-way ANOVA.(C, D, E and F) The 16S rRNA Sequencing Analysis of Microbiota at the Phylum and Family Level in M-Ccr2KO(C) Pie charts showing relative abundances of bacterial phyla in the cecum microbiota of control mice fed a NCD (n=4), fed a HFD (n=4), and M-Ccr2KO fed a HFD (n=4). (D) The 16S rRNA sequencing analysis of the cecum microbiota at the phylum level with Illumina MiSeq in Paired-End mode (150×2 bp) of cecal DNA product from control fed with a NCD, HFD, M-Ccr2KO fed with a HFD. These data were expressed as a percentage of total DNA sequence from each Phylum of microbiota and mean + SEM. *P<0.05 by one-way ANOVA. (E) Bar plot analysis on family taxon. (F) The 16S rRNA sequencing analysis of the cecum microbiota at the family level. These data were expressed as a percentage of total DNA sequence from each Family of microbiota and mean + SEM. *P<0.05 by one-way ANOVA. (G) Principal Coordinate Analysis (PCoA) of weighted UniFrac distances from the cecum microbiota of control mice fed a NCD (n=4), fed a HFD (n=8), and M-Ccr2KO fed a HFD (n=4) (H) Hierarchical clustering dendrogram of the cecum microbiota based on genus-level classifications.

Figure S5

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1mg/day i.p.5days

A

500bp

200bp

Control Vil-Ccl2KO

Control PCR

RecombinationPCR

C

Control PCR 　 Ccl2-Ex1-S1 - Ccl2-Ex1-AS1recombination PCR　 Ccl2-Ex1-S2 - Ccl2-6AS

Ccl2

exon1 exon2 exon3 RFPloxP loxP

Functional C-C domain

B

00.20.40.60.8

1

Fat free mass

g/B

W

NCcontrol HFVil-Ccl2 KO

E

0

0.5

1

1.5

2

2.5

0 15 30

Insu

lin (n

g/m

l)

Time (min)

controlCcl2villin

0

100

200

300

400

0 30 60 90 120Blo

od g

luco

se (m

g/dl

)

Time (min)

OGTT HF controlVil-Ccl2KO

＊ ＊

00.5

11.5

22.5

3

0 15 30

Insu

lin (n

g/m

l)

Time（min )

Insulin(OGTT)

HF controlHF Vil-Ccl2KO

Insulin (IPGTT)

00.005

0.010.015

0.020.025

g / B

W

NCcontrol HFVil-Ccl2 KO

*

* ***

*

* n.s.

HF Vil-Ccl2KOHF control

HF controlHF Vil-Ccl2KO

HF Vil-Ccl2KO02468

101214

Live

r TG

(mg/

g tis

sue)

HF Vil-Ccl2KO

HF Vil-Ccl2KOHF control

00.10.20.30.40.50.6

Wei

ght (

g)

Cecum*

n.s.

D F

G H I J

Figure S5. Related to Figure 5. Generation and Analysis of Vil-Ccl2KO (A) Protocol of HFD analysis of Vil-Ccl2KO. (B and C) Recombination of conditional Ccl2-targeted allele (Ccl2-RFPflox/flox) by intestine-specific tamoxifen-inducible Cre transgenic mice (Vil-Cre-ERT2). (B) Construct of Ccl2-targeted allele and primers for control and recombination PCR. (C) Representative data for recombination PCR on genomic DNA isolated from various tissues of Vil-Ccl2KO compared with control mice.(D) Analysis of fat composition of epididymal fat, subcutaneous fat, mesentery fat, perirenal fat and brown adipose tissue from NC control, HF control, and Vil-Ccl2KO. Data are expressed as mean + SEM. *P<0.05 by one-way ANOVA.(E) Fat free mass. Data are defined as total body mass except for epididymal fat, subcutaneous fat, mesentery fat, perirenal fat and brown adipose tissue from NC, HF control and Vil-Ccl2KO and are expressed as mean + SEM.(F) Insulin secretion of control and Vil-Ccl2KO mice during IPGTT. Data represent means + SEM of 8 mice in each genotype. (G) OGTT of control and Vil-Ccl2KO mice fed with HFD for 10 weeks (n=10). Data are means + SEM. *P<0.05 by two-way ANOVA with Fisher's test.(H) Insulin secretion of control and Vil-Ccl2KO mice fed with HFD for 10 weeks during oGTT. Data are means + SEM of 8-10 mice in each genotype.(I) Hepatic triglyceride content of NC control, HF control, and Vil-Ccl2KO mice fed with HFD for 12 weeks. Data are means + SEM of 8 mice. *P<0.05 by one-way ANOVA.(J) The weight of the cecum from NC control, HF control and Vil-Ccl2 KO mice fed with HFD for 10 weeks. Data represent as the ratio to control in each gene and are means + SEM of 4 mice in each genotype. *P < 0.05 by one-way ANOVA.

Figure S6

Small intestineA

C

01020304050607080

Con

cent

ratio

n In

por

tal v

ein

(pg/

ml)

D

00.5

11.5

22.5

3

Rel

ativ

e ge

ne e

xpre

ssio

n

NCHF control HF Vil-Ccl2KO

** *

0

2

4

6

8

10

Emr1 Ccr2 Ccl2 Tnfa Il1b Il18 Ifng Il17 Foxp3 Il10 Il4 SiglecfRel

ativ

e ge

ne e

xpre

ssio

n

Liver

** *

* *

IL1β

B

n.s.

0

0.5

1

1.5

2

Arg1 Mr Cd163

Rel

ativ

e ge

ne e

xpre

ssio

n

NCHF controlHF Vil-Ccl2KOHF Vil-Ccl2KO

Colon

HFD Control

HFD Vil-Ccl2KO

F

05

101520253035

Bacteroidetes

01020304050607080

Firmicutes

05

10152025303540

Proteobacteria

00.10.20.30.40.50.60.70.8

Deferribacteres

00.10.20.30.40.50.60.70.8

Tenericutes

00.05

0.10.15

0.20.25

0.30.35

Synergistetes

**

* ** * * *n.s.

n.s.

% o

f tot

al

% o

f tot

al

% o

f tot

al

% o

f tot

al

% o

f tot

al

% o

f tot

al

0%20%40%60%80%

100%OthersDeferribacteraceaeDehalobacteriaceaeAlcaligenaceaePeptostreptococcaceaeRhodothermaceaeOdoribacteraceaeSphingobacteriaceaePrevotellaceaeVerrucomicrobiaceaeFlavobacteriaceaeLactobacillaceaeCoriobacteriaceaeClostridiaceaeHelicobacteraceaeErysipelotrichaceaePorphyromonadaceaeBacteroidaceaeDesulfovibrionaceaeRuminococcaceaeLachnospiraceae

0

5

10

15

20

25

Desulfovibrionaceae

**

% o

f tot

al

02468

101214

Helicobacteraceae

*

*

% o

f tot

al

00.10.20.30.40.50.60.70.8

Deferribacteraceae

* *

% o

f tot

al

HF Vil-Ccl2KO

-0.4-0.3-0.2-0.1

00.10.20.30.40.50.6

-0.4 -0.2 0 0.2 0.4 0.6 0.8

NCDHFD controlHFD Vil-Ccl2KO

PC1(51.0%)

PC2(

(38.

0%)

E

G

H

I

Firmicutes BacteroidetesProteobacter

Figure S6. Related to Figure 6. Analysis of Inflammation Related Gene Expression of Intestine and Liver in Vil-Ccl2KO(A) Normalized expression of inflammation-related genes in the small intestine from NC.Control, HF control, and Vil-Ccl2KO mice fed with HFD for 10 weeks. Data represent as the ratio to control in each gene and are means + SEM of 6 mice in each genotype. *P<0.05 by one-way ANOVA.(B) Normalized expression of anti-inflammatory macrophage-related genes in the colon from NC control, HF control, and Vil-Ccl2KO mice fed with HFD for 10 weeks. Data represent as the ratio to control in each gene and are means + SEM of 4-6 mice in each genotype. (C) Normalized expression of inflammation-related genes in liver from NC control, HF control, and Vil-Ccl2KO fed with HFD for 12 weeks. Data are normalized to b-actin expression level and represent as the ratio to control in each gene and are means + SEM of 6 mice in each genotype. *P<0.05 by one-way ANOVA.(D) The concentration of IL1b in the portal vein from control and Vil-Ccl2KO fed with HFD for 12 weeks. Data are means + SEM of 4-6 mice in each genotype. n.s by one-way ANOVA.(E) The 16S rRNA Sequencing Analysis of Microbiota at the Phylum and Family Level in Vil-Ccl2KO. Pie charts showing relative abundances of bacterial phyla in the cecum microbiota of control (n=4) and Vil-Ccl2KO fed a HFD (n=4). (F) The 16S rRNA sequencing analysis of the cecum microbiota at the phylum level with Illumina MiSeq in Paired-End mode (150×2 bp) of cecal DNA product from control fed with a NCD (n=4), HFD (n=4), Vil-Ccl2KO fed with a HFD (n=4). These data were expressed as a percentage of total DNA sequence from each Phylum of microbiota and mean + SEM. *P<0.05 by one-way ANOVA.(G) Bar plot analysis on family taxon.(H) The 16S rRNA sequencing analysis of the cecum microbiota at the family level. These data were expressed as a percentage of total DNA sequence from each Family of microbiota and mean + SEM. *P<0.05 by one-way ANOVA. (I)Principal Coordinate Analysis (PCoA) of weighted UniFrac distances from the cecum microbiota of control mice fed a NCD (n=4), fed a HFD (n=4), and Vil-Ccl2KO fed a HFD (n=4).

Figure S7

A

020406080

100120140160

Adi

pocy

te n

umbe

r

Adipocyte size (μmm2)

0

500

1000

1500

2000

2500

3000

Num

ber o

f F4

/80+

CD

11b+

CD

11c+

CD

206-

cell

( / 1

05 S

VF to

tal c

ell)

*

B

020406080

100120140

Seru

m C

cl2

(pg/

ml)

Serum Ccl2C

BloodHF Vil-Ccl2KO

D BloodHF control

Ly6c

Ccr2

Ly6c

Ccr2

1.42%

1.46%

P1

00.20.40.60.8

11.21.41.61.8

% o

f P1

n.s.

n.s.

EBlood

Ly6c+Ccr2+ cell

0200400600800

10001200140016001800

Num

ber　

(/ 10

5 liv

ing

bloo

d ce

lls)

n.s.

FBlood

Ly6c+Ccr2+ cell

Adipocyte size (µm2)

Figure S7. Related to Figure 6. Adipocyte Size, FACS Analysis, and Serum Ccl2 Concentration of Vil-Ccl2KO(A) Histogram of adipocyte size and number of control (blue bar) and Vil-Ccl2KO (yellow bar) fed with HFD for 12 weeks. (B) Absolute number of F4/80+CD11b+CD11c+CD206- cell among 106 SVF total cell from control and Vil-Ccl2KO fed with HFD for 12 weeks. Data are means + SEM. *P<0.05 by one-way ANOVA.(C) Serum Ccl2 concentration of control and Vil-Ccl2KO fed with HFD for 12 weeks. n.s. by one-way ANOVA.(D) Representative FACS analysis of blood monocyte, expressed as Ly6c+Ccr2+ cell in blood from mice fed with control (the upper panel) and Vil-Ccl2KO (the bottom panel) fed with HFD.(E and F) Percentage (E) and Absolute number (/105 living blood cell) (F) of blood monocyte from mice fed with control and Vil-Ccl2KO fed with HFD

Milk Casein

L-cystein

Corn starch

Maltodextrin

Sucrose

Soybean oil

Powdered cellulose

AIN-93G Mineral mixture

AIN-93 Vitamin mixture

Choline bitartrate

Butterfat

Tert-butylhydroquinone

Total calorie ( kcal/100g)

HFD-60 (Oriental, g / 100g)

19.82

0.3

3.7458

9.98

34.0

1.0

5.0

3.5

1.0

0.25

20.0

0.0042

529

Supplemental table 1. Related to Figure 1, 3, and 5.The composition of HFD used in this study