Supplemental Information. Colonic Pro-inflammatory Macrophages. Cause Insulin Resistance in an Intestinal. Ccl2/Ccr2-Dependent Manner

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1 Cell Metabolism, Volume Supplemental Information Colonic Pro-inflammatory Macrophages Cause Insulin Resistance in an Intestinal Ccl/Ccr-Dependent Manner Yoshinaga Kawano, Jun Nakae, Nobuyuki Watanabe, Tetsuhiro Kikuchi, Sanshiro Tateya, Yoshikazu Tamori, Mari Kaneko, Takaya Abe, Masafumi Onodera, and Hiroshi Itoh

2 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Generation of Tissue-specific Ccr Knockout Mice The mouse genomic DNA clone containing exons,, and of the Ccr gene locus was obtained from a BAC clone CH-O8 (BACPAC Resource) derived from the C7BL/6 mouse strain. The Ccr targeting construct consisted of 9. kb of the genomic sequence that was immediately of the first exon, followed by exon, intron, exon, a.-kb Neo-cassette, a frt-flanked Neo-resistance gene, and exon. A 76-bp of genomic sequence containing the open reading frame in exon of the Ccr gene was flanked by the loxp sequence. The SalI-linearized targeting vector was electroporated into TT embryonic stem (ES) cells (Yagi et al., 99) as described previously. G8-resistant clones were isolated and screened by PCR and Southern blotting. Seven out of 9 G8-resistant clones had undergone the desired homologous recombination. Positive clones were injected into CD- 8-cell stage embryos, and the chimeric male offspring were mated to C7BL/6 females, obtaining Ccr conditional knockout mice (Accession No. CDB6K: Primers for detection of the wild-type allele ( bp) and the loxp allele (6 bp) are: - GCCTATTCTCTTCTGTATCTC- and - TGGATGAACTGAGGTAACAT- (Figure SA-SD). Immunohistochemistry, Immunofluorescence and Histological Analysis For histological analysis, we removed the white adipose tissue and colon from mice, fixed the specimens in % 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., ) using anti-cd68 (Dako Denmark A/S), anti-claudin- (Invitrogen), or anti-asc (Santa-Cruz) antibodies. After washing with PBS, the sections were sequentially

3 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-8, 9, KEYENCE) by manually tracing at least more than adipocytes for each genotype. Isolation of Liver Macrophages The liver was removed and cut into small pieces with scissors, and then filtered through μm nylon mesh. The cells were collected in a new ml tube and incubated with mm EDTA and Collagenase B (Roche) with gentle shaking. After incubation with collagenase, the supernatants were centrifuged at 7 rpm and washed twice with PBS. The cell pellets were re-suspended in % Percoll, overlaid on 8% Percoll, and centrifuged at rpm for min 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 ml tube containing KRHAG buffer (M KCl, M CaCl, M KH PO, M MgSO, % BSA, mm HEPES (ph 7.8), mm glucose), and cut into small pieces. The pieces were incubated with Collagenase type Ⅰ (Wako) in KRHAG buffer (.mg/.ml) for min at 7 o C with gentle shaking. After filtering through μm nylon, the supernatant was centrifuged at rpm for min at 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

4 (Promega) according to the manufacturer s protocol. We performed reverse transcription using the PrimeScript TM 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., ). After centrifugation to remove insoluble material, the proteins in μg of lysate were separated using 8% SDS-PAGE for detection of p-akt and Akt (Cell signal) or % SDS-PAGE for detection of Claudin- and procaspase- p (sc-, Santa Cruz), and immunized using the indicated antibodies. H O Production The colon and epididymal fat were dissected from age-matched male C7BL/6J mice fed either a NCD or a HFD for or 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 μg/ml LPS for h with BFA added to the cell suspension for the final hours. Next, the cells were stained with surface marker antibodies, including F/8(BM8, APC, BioLegend), CDb (M/7, APC-Cy7, BioLegend), and CDc (N8, FITC, BioLegend), washed with PBS, and fixed in formaldehyde. The cells were permeabilized in PBS containing.% saponin and.% BSA (Sigma-Aldrich), stained with antibody for intracellular TNFα marker (MP-6XT, PerCP-Cy., BD Biosciences), and analyzed by flow cytometry.

5 Metagenomic analysis of 6S rrna To analyze the microbial population from NCD, HFD, M-CcrKO, Vil-CclKO mice in this study, metagenomics analysis using 6S 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.8..) Chimeric sequences were removed. We used a Cut-adapt (Ver.) to trim any over-read, and paired-end sequences from DNA sequencing reads. A total of, reads were generated per sample from these mice after filtering. Taxonomy is assigned to each OTU based on the 6S rdna database Greengenes (Ver.8) in the closed-reference manner. We excluded sequences that were not between and 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 (ToxinSensor TM Chromogenic LAL Endotoxin Assay kit) according to the manufacturer's protocol. Measurement of Cytokines in the Portal Vein Serum levels of IL8 and ILβ in the portal vein were measured using the mouse ELISA

6 kits (R&D SYSTEM for IL8 and mouse ELISA kit Quantikine, R&D SYSTEM for ILβ). Isolation of Intestinal Epithelial Cell for Real-time PCR The pieces of the colon and small intestine dissected from each mouse were incubated in mm DTT and.m EDTA in.% FBS-HANKS at 7 o C for minutes. The supernatant was then collected and filtered through 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 CDb magnetic beads (Miltenyi Biotec) for positive selection. The CDb + cells were cultured with ( 6 cells/ml) in HANKs Balanced Salt solution supplemented with.% FCS and % penicillin/streptomycin. Intestinal Permeability Fluorescein-isothiocyanate-conjugated dextran (FD: Sigma-Aldrich, Gillingham, UK) was administered to each mouse at. mg/g body weight by oral gavage. After h, blood was collected from the portal vein and diluted with an equal volume of PBS. The concentration of FD dextran was measured at 8 nm using a spectrophoto microplate reader. Co-culture of Caco- and CDb + Cell Caco- cells were cultured with DMEM supplemented with.% FBS and % penicillin/streptomycin for days. Differentiated Caco- cells at 8% confluence were seeded on the basal side of the Nunc TM CC insert -well plate (polycarbonate membrane, <.7 6 pores/cm ). CDb + cells sorted from the colons of mice fed a 6

7 NCD stimulated with a mixture of IL-β, IL-8, and LPS were added to polycarbonate membrane inserts. After 6 hours, Caco- 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. (). Loss of Pdk-Foxo signaling in myeloid cells predisposes to adipose tissue inflammation and insulin resistance. Diabetes 6, Nakae, J., Cao, Y., Hakuno, F., Takemori, H., Kawano, Y., Sekioka, R., Abe, T., Kiyonari, H., Tanaka, T., Sakai, J., et al. (). Novel repressor regulates insulin sensitivity through interaction with Foxo. EMBO J, 7-9. Sakaue, H., Ogawa, W., Matsumoto, M., Kuroda, S., Takata, M., Sugimoto, T., Spiegelman, B.M., and Kasuga, M. (998). Posttranscriptional control of adipocyte differentiation through activation of phosphoinositide -kinase. J Biol Chem 7, Yagi, T., Tokunaga, T., Furuta, Y., Nada, S., Yoshida, M., Tsukada, T., Saga, Y., Takeda, N., Ikawa, Y., and Aizawa, S. (99). A novel ES cell line, TT, with high germline-differentiating potency. Anal Biochem,

8 A Normal chow W High fat diet HFW HF6W HFW HF8W HFW NC B Length of small intestine NC HFW C Length (cm) p=. D Emr... Ly6c Cxcr Ccl... Tnf.. Ilb.. Il8.. Foxp.. E Epididymal fat F Mesenteric fat A Emr Emr Mesentery fat Ccl Ccl Tnf... Tnf.. Ilb.. Ilb... NC HFW HF8W HFW HF6W HFW G Liver Emr.. Ccl 6 Tnf.... Ilb.. Figure S

9 Figure S. Related to Figure. HFD analysis of small intestine. (A) The protocol for evaluating sequential change of intestinal immune state during HFD for weeks. (B)Representative image of the small intestine from NCD and HFD for weeks (C)The length of the small intestine from age-matched C7Bl6/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 C7Bl6/J mice fed with HFD compared with NCD. Data are normalized to β-actin expression and represent means + SEM from 8- mice in each group. P<. by one-way ANOVA. (E, F and G) Related to Figure. HFD-induced Pro-inflammatory Changes in Adipose Tissue and Liver. Normalized expression of inflammation-related genes (Emr, Ccl, Tnf, Ilb) in epididiymal fat (E), mesenteric fat (F) and liver (G) from age-matched C7Bl6/J mice fed with HFD compared with NCD. Data represent as the ratio to control in each gene. Data are means + SEM. P<. by one-way ANOVA.

10 A B Ly6c NC 8.% 87.7% 8.% Ly6c CXCR SiglecF HFW 98.9% %.68% Ly6c CXCR SiglecF 6 CXCR SiglecF(Eosinophil).. C D TNFα NC HFW SSC-A NC HFw.76%.7% CDb TNFα E F Number of F/8 + CDb + CDc - TNFα high (in living F/8 + cell cell) Number of F/8 + CDb + CDc - TNFα + cell TNFα ( pg/ml ) TNFα in cell culture supernatants NC CDb+ MACS cell HFW CDb+ MACS cell Figure S

11 Figure S. Related to Figure. HFD-induced Changes of Ly6c, Cxcr, and Siglecf Expression in Colon and FACS Analysis of F/8 + CDb + CDc - TNFα high from the Colon of NCD and HFD Mice. (A) Representative FACS analysis of Ly6c, CXCR, SiglecF among F/8 + CDb + CDc - cells in colonic LP from mice fed with NCD (the left panel) and HFD for weeks (the right panel). (B) Normalized expression of Ly6c, Cxcr, and Siglecf in colon from age-matched C7Bl6/J mice fed with HFD compared with NCD. Data represent as the ratio to control in each gene. Data are means + SEM. P<. by one-way ANOVA. (C and D) Representative FACS analysis of F/8 + CDb + CDc - TNFα high cell in colonic LP from mice fed with a NCD and with a HFD for weeks (C), and from mice fed with a NCD and a HFD for weeks (D). (E) Absolute number of F/8 + CDb + CDc - TNFα high cell in living F/8 positive cell. Data are means + SEM. P<. by one-way ANOVA. (F) Measurement of TNFα production by ELISA in culture supernatants of MACS sorted CD + cells from NCD and HFD for weeks. Data are expressed as mean + SEM. by one-way ANOVA.

12 A D g / BW Body weight (g)..... Body weight W W 6W 7W 8W 9W W W W W W Age (week) B Blood glucose (mg/dl) HF Control HF M-CcrKO E g/bw IPGTT 6 9 Time (min) Fat fat free free mass Control HF control C F M-CcrKO HF M-CcrKO % of Blood glucose ( min ) Insulin (ng/ml) 8 6 ITT 6 9 Time (min)... Insulin Time (min) cont HF control CcrLysM HF M-CcrKO Blood glucose ( mg/dl ) G OGTT HF control HF M-CcrKO 6 9 Time (min) Insulin (ng/ml) H I Insulin HF control HF M-CcrKO Time ( min ) Liver TG (mg/g tissue) Liver TG J Cell number of Epididymal fat F/8 + CDb + CD + CD6 - cell in epididymal fat 8 6 K Mesenteric fat Relative gene expression 8 6 NC HF Control HF M-CcrKO Emr Ccr Ccl Tnfa Ilb Il8 Ifng Il7 Foxp Il Il Siglecf L M-CcrKO MACS-sorted CDb + adipose tissue macrophage Relative gene expression... HF control HF M-CcrKO Emr Itgax Tnfa Ilb Il8 Arg Cd6 Il RelaBve gene expression M Skeletal Muscle control HF control CcrLysM HF M-CcrKO Figure S

13 Figure S. Related to Figure. Metabolic Phenotypes, FACS Analysis, and Gene Expression Analysis of M-CcrKO. (A) Body weight. (B) IPGTT. (C) ITT in M-CcrKO fed with a NCD. The black rhombus and green square indicate control and M-CcrKO, respectively. Data represent means + SEM of 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-CcrKO. 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-CcrKO mice fed with a HFD for weeks during IPGTT. Data are means + SEM of 8- mice in each genotype. (G) Oral glucose tolerance test (ogtt) of control and M-CcrKO mice fed with HFD for weeks (n=). Data are means + SEM. P<. by two-way ANOVA with Fisher's test. (H) Insulin secretion of control and M-CcrKO mice fed with HFD for weeks during ogtt. Data are means + SEM of 8- mice in each genotype. (I) Hepatic triglyceride content of control and M-CcrKO mice fed with HFD for weeks. (II) Data are means + SEM of 6-8 mice. by one-way ANOVA. (J) Absolute number of F/8 + CDb + CDc + CD6 - cell in whole epididymal fat. Data are means + SEM. by one-way ANOVA. (K) Normalized expression of inflammation-related genes in mesentery fat from NC control, HF control, and M-CcrKO mice fed with HFD for weeks. Data represent as the ratio to control in each gene and are means + SEM of 6 mice in each genotype. P<. by one-way ANOVA. (L) Normalized expression of inflammation-related genes in CDb + cell in stromal vascular fraction of epididymal fat from control and M-CcrKO mice fed with HFD for weeks. Data represent as the ratio to control in each gene and are means + SEM of mice in each genotype. P<. by one-way ANOVA. (M) Normalized expression of inflammation-related genes in skeletal muscle from control and M-CcrKO mice fed with HFD for weeks. Data represent as the ratio to control in each gene and are means + SEM of - mice in each genotype. P<. by one-way ANOVA.

14 Weight (g).. C NC HF control HF HF M-CcrKO M-CcrKO... Emr Tnf Tnfa Ccl Ilb Il8 Ifng Il7 Foxp Il Il Siglecf D HFD Control NCD Ccr HFD M-CcrKO Firmicutes Bacteroidetes Firmicutes Proteobacteria 8 6 Actinobacteria Deferribacteres Bacteroidetes..6 Small intestine B Cecum A Proteobacteria E F 8% 7% 6% % % % % % % G Bacteroidaceae 8 6 Deferribacteraceae Others Deferribacteraceae Dehalobacteriaceae Alcaligenaceae Peptostreptococcaceae Rhodothermaceae Odoribacteraceae Sphingobacteriaceae Prevotellaceae Verrucomicrobiaceae Flavobacteriaceae Lactobacillaceae Coriobacteriaceae Clostridiaceae Helicobacteraceae Erysipelotrichaceae Porphyromonadaceae Bacteroidaceae Desulfovibrionaceae Ruminococcaceae Lachnospiraceae 9% % H PC(.9%) PC(.8%) NCD HFD control HFD M-CcrKO Figure S

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

16 A 8W HF start B Ccl HFW Tamoxifen mg/day i.p. days HF6W Tamoxifen mg/day i.p. days HFW IPGTT, ITT analysis exon exon exon RFP loxp Functional C-C domain loxp C Control Control PCR Ccl-Ex-S - Ccl-Ex-AS recombination PCR Ccl-Ex-S - Ccl-6AS Vil-CclKO Recombination PCR bp Control PCR bp D g / BW..... NC control HF control HF Vil-Ccl HF Vil-CclKO g/bw E NC control HF control HF Vil-Ccl HF Vil-CclKO Fat free mass Insulin (ng/ml) F Insulin (IPGTT) control HF control. Cclvillin HF Vil-CclKO.. Time (min) G H I J OGTT Insulin(OGTT) Blood glucose (mg/dl) HF control Vil-CclKO HF Vil-CclKO 6 9 Time (min) Insulin (ng/ml).. HF control. HF Vil-CclKO Time min ) Liver TG (mg/g tissue) Weight (g).... Cecum Figure S

17 Figure S. Related to Figure. Generation and Analysis of Vil-CclKO (A) Protocol of HFD analysis of Vil-CclKO. (B and C) Recombination of conditional Ccl-targeted allele (Ccl-RFP flox/flox ) by intestine-specific tamoxifen-inducible Cre transgenic mice (Vil-Cre-ER T ). (B) Construct of Ccl-targeted allele and primers for control and recombination PCR. (C) Representative data for recombination PCR on genomic DNA isolated from various tissues of Vil-CclKO 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-CclKO. Data are expressed as mean + SEM. P<. 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-CclKO and are expressed as mean + SEM. (F) Insulin secretion of control and Vil-CclKO mice during IPGTT. Data represent means + SEM of 8 mice in each genotype. (G) OGTT of control and Vil-CclKO mice fed with HFD for weeks (n=). Data are means + SEM. P<. by two-way ANOVA with Fisher's test. (H) Insulin secretion of control and Vil-CclKO mice fed with HFD for weeks during ogtt. Data are means + SEM of 8- mice in each genotype. (I) Hepatic triglyceride content of NC control, HF control, and Vil-CclKO mice fed with HFD for weeks. Data are means + SEM of 8 mice. P<. by one-way ANOVA. (J) The weight of the cecum from NC control, HF control and Vil-Ccl KO mice fed with HFD for weeks. Data represent as the ratio to control in each gene and are means + SEM of mice in each genotype. P <. by one-way ANOVA.

18 A... Small intestine NC HF control HF Vil-CclKO B Colon.. Arg Mr Cd6 NC HF control HF Vil-CclKO C F 8 6 Liver Emr Ccr Ccl Tnfa Ilb Il8 Ifng Il7 Foxp Il Il Siglecf Bacteroidetes Firmicutes Proteobacteria Deferribacteres D Concentration In portal vein (pg/ml) Tenericutes ILβ Synergistetes E HFD Control HFD Vil-CclKO Firmicutes Bacteroidetes Proteobacter G % 8% 6% % % % H Desulfovibrionaceae Helicobacteraceae Others Deferribacteraceae Dehalobacteriaceae Alcaligenaceae Peptostreptococcaceae Rhodothermaceae Odoribacteraceae Sphingobacteriaceae Prevotellaceae Verrucomicrobiaceae Flavobacteriaceae Lactobacillaceae Coriobacteriaceae Clostridiaceae Helicobacteraceae Erysipelotrichaceae Porphyromonadaceae Bacteroidaceae Desulfovibrionaceae Ruminococcaceae Lachnospiraceae Deferribacteraceae I PC((8.%) PC(.%) NCD HFD control HFD Vil-CclKO Figure S6

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

20 A B Adipocyte number Number of F/8 + CDb + CDc + CD6 - cell ( / SVF total cell) Adipocyte size (µm) Adipocyte size (μmm ) C Serum Ccl Serum Ccl (pg/ml) 8 6 D Ccr Blood HF control.% E Blood Ly6c + Ccr + cell F Blood Ly6c + Ccr + cell P Ccr Blood HF Vil-CclKO.6% Ly6c % of P Number (/ living blood cells) Ly6c Figure S7

21 Figure S7. Related to Figure 6. Adipocyte Size, FACS Analysis, and Serum Ccl Concentration of Vil-CclKO (A) Histogram of adipocyte size and number of control (blue bar) and Vil-CclKO (yellow bar) fed with HFD for weeks. (B) Absolute number of F/8 + CDb + CDc + CD6 - cell among 6 SVF total cell from control and Vil-CclKO fed with HFD for weeks. Data are means + SEM. P<. by one-way ANOVA. (C) Serum Ccl concentration of control and Vil-CclKO fed with HFD for weeks. by one-way ANOVA. (D) Representative FACS analysis of blood monocyte, expressed as Ly6c + Ccr + cell in blood from mice fed with control (the upper panel) and Vil-CclKO (the bottom panel) fed with HFD. (E and F) Percentage (E) and Absolute number (/ living blood cell) (F) of blood monocyte from mice fed with control and Vil-CclKO fed with HFD

22 Supplemental table. Related to Figure,, and. The composition of HFD used in this study HFD-6 (Oriental, g / g) Milk Casein L-cystein Corn starch Maltodextrin Sucrose Soybean oil Powdered cellulose AIN-9G Mineral mixture AIN-9 Vitamin mixture Choline bitartrate Butterfat Tert-butylhydroquinone Total calorie ( kcal/g)