PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING METABOLIC DISEASES, COMPRISING BACTEROIDES ACIDIFACIENS AS ACTIVE INGREDIENT

Abstract

The present disclosure relates to a composition for preventing or treating metabolic diseases, in which the composition includes Bacteroides acidifaciens as an active ingredient. In addition, the present disclosure relates to a composition for oxidizing fat or inhibiting DPP-4, in which the composition includes Bacteroides acidifaciens as an active ingredient. In addition, the present disclosure relates to a transformant expressing a lean phenotype, in which an Atg7 gene is deleted in dendritic cells.

Claims

1. A method for preventing or treating diabetes, the method comprising administering Bacteroides acidifaciens to a subject in need of prevention or treatment of diabetes.

2. A method for preventing or treating diabetes according to claim 1, wherein the Bacteroides acidifaciens has a higher ratio of intestinal total bacteria in a lean phenotype as compared to obesity or a standard phenotype.

3. A method for preventing or treating diabetes according to claim 1, wherein the Bacteroides acidifaciens activates fat oxidation in adipose tissue, inhibits the activity of intestinal DPP-4 (dipeptidal peptidase-4), and increases GLP-1.

Description

DESCRIPTION OF DRAWINGS

[0072] FIGS. 1A-1F are diagrams collectively showing a lean phenotype in an Atg7.sup.?CD11c mouse.

[0073] FIG. 1A The body weight change of Atg7.sup.f/f and Atg7.sup.?CD11c mice were monitored for 23 weeks (left panel). Body weight (middle panel) and fat mass (right panel) of 24-week-old male Atg7.sup.f/f and Atg7.sup.?CD11c mice on a normal chaw diet (NCD). (n=8).

[0074] FIG. 1B Photographs of 24-week-old Atg7.sup.f/f and Atg7.sup.?CD11c mice.

[0075] FIG. 1C Mill of abdominal adipose tissue of 24-week-old male Atg7.sup.f/f and Atg7.sup.?CD11c mice on a NCD.

[0076] FIG. 1D Histological changes (left panel) and adipocyte size (right panel) in adipose tissue of Atg7.sup.f/f and Atg7.sup.?CD11c mice. Scale bars=50 ?m.

[0077] FIG. 1E Levels of glucose (right panel) and insulin (left panel) in the serum of Atg7.sup.f/f and Atg7.sup.?CD11c mice on a NCD under non-fasting conditions.

[0078] FIG. 1F GTT (Glucose tolerance test; left panel) and ITT (insulin tolerance test; right panel) results for male Atg7.sup.f/f and Atg7.sup.?CD11c mice.

[0079] All data are shown as mean?s.e.m. *P<0.05, **P<0.01, and ***P<0.001.

[0080] FIGS. 2A-2E are diagrams showing that a lean phenotype is originated from visceral symbiotic bacteria.

[0081] FIG. 2A Photographs of co-housing (CH; middle) and separated (left and right ends) 24-week-old male Atg7.sup.f/f and Atg7.sup.?CD11c mice.

[0082] FIG. 2B Body weight (left panel) and fat mass (right panel) of 24-week-old male Atg7.sup.f/f and Atg7.sup.?CD11c mice in a co-housing and a separated cage. (n=3 or 4).

[0083] FIG. 2C Results of monitoring the body weight of each mouse after co-housing (CH) for an additional 10 weeks (n=3 to 9).

[0084] FIG. 2D Body weight (left panel) and fat mass (right panel) of untreated B6 mice after 18 weeks of movement of feces in an Atg7.sup.f/f or Atg7.sup.?CD11c mouse (n=5).

[0085] FIG. 2E Levels of glucose (right panel) and insulin (left panel) in the serum after movement of fecal extract of Atg7.sup.f/f or Atg7.sup.?CD11c mouse under non-fasting conditions (n=5).

[0086] All data are mean?s.e.m values. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

[0087] FIGS. 3A-3D are diagrams collectively showing that B. acidifaciens (BA) is expanded in the feces of an Atg7.sup.?CD11c mouse in visceral symbiotic bacteria.

[0088] FIG. 3A Phylum level detected by pyrosequencing.

[0089] FIG. 3B Pyrosequencing data (n=6) from the class to the genus (left to right).

[0090] FIG. 3C Representative graphs of species representing the distribution of BA in the feces detected by pyrosequencing. Red arrow=BA.

[0091] FIG. 3D IECs (intestinal epithelial cells) of Atg7.sup.f/f and Atg7.sup.?CD11c mice determined by BA-specific FISH (fluorescence in situ hybridization) probe and the number of BA increased in lumens of colon (n=3). Scale bar=100 ?m. All data are mean?s.e.m values. *P<0.05; ns, not significant.

[0092] FIGS. 4A-4F are diagrams collectively showing that B. acidifaciens (BA) regulates the body weight and fat mass of diet-induced B6 mouse obesity.

[0093] FIG. 4A Photographs of high-fat diet (HFD; left panel) and PBS- and BA-fed mice. The body weight of each group was monitored for 10 weeks (right panel). BA was orally administered (5?10.sup.9 CFU/100 ?l) (n=5).

[0094] FIG. 4B Oral dietary intake with PBS or BA (n=5).

[0095] FIG. 4C MRI of PBS- and BA-fed mice.

[0096] FIG. 4D Histological changes in adipose tissue (left panel) and adipocyte size (right panel) of PBS- and BA-fed mice in HFD.

[0097] All data are shown as mean?s.e.m of ?2 independent experiments.

[0098] FIG. 4E Results of GTT (glucose tolerance test; left panel, n=8 or 9) and ITT (insulin tolerance test; right panel, n=7-12) using the serum of PBS- and BA-fed mice measured at a certain point after intraperitoneal injection of glucose or insulin.

[0099] FIG. 4F Energy consumption (top left panel), total activity (top right panel), and RER (respiratory exchange ratio; bottom left panel) (n=5) of PBS- or BA-fed mice. All data are expressed as mean?s.e.m. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

[0100] FIGS. 5A-5E are diagrams collectively showing that B. acidifaciens(BA) induces fat oxidation in adipose tissue through PPAR? activity.

[0101] FIG. 5A At the end of each experiment, the expression levels of mRNAs of genes related to fatty acid synthesis (FasN, HSL, PEPCK, SCD1, and PPAR?), ?-oxidation (PPAR?), thermogenesis (PRDM16, PGC1a, Cidea, and GLUT4) were determined through real-time PCR using the epididymis adipose tissues of Atg7.sup.f/f and Atg7.sup.?CD11c mice (n=5).

[0102] FIG. 5B The expression levels of mRNAs of genes related to fatty acid synthesis (FasN, HSL, PEPCK, SCD1, and PPAR?), ?-oxidation (PPAR?), thermogenesis (PRDM16, PGC1a, Cidea, and GLUT4) were determined through real-time PCR using the epididymis adipose tissues of mice transplanted with fecal microorganisms (n=5).

[0103] FIG. 5C The expression levels of mRNAs of genes related to fatty acid synthesis (FasN, HSL, PEPCK, SCD1, and PPAR?), ?-oxidation (PPAR?), thermogenesis (PRDM16, PGC1a, Cidea, and GLUT4) were determined through real-time PCR using the epididymis adipose tissues of BA-fed mice (n=5).

[0104] FIG. 5D The PPAR? expression level in adipose tissue after 1, 7 and 14 days of daily BA administration were analyzed through RT-PCR.

[0105] FIG. 5E The TGR5 expression level in adipose tissue after 1, 7 and 14 days of daily BA administration were analyzed through RT-PCR.

[0106] All data are mean?s.e.m of ?2 independent experiments. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

[0107] FIGS. 6A-6D are diagrams collectively showing that B. acidifaciens (BA) regulates DPP-4 (dipeptidal peptidase-4) secretion to induce GLP-1 production.

[0108] FIG. 6A Levels of glucose (right panel) and insulin (left panel) in the serum of PBS- and BA-fed mice (normal chow diet, NCD; high-fat diet, HFD).

[0109] FIG. 6B Levels of activated GLP-1 in the serum of PBS- and BA-fed mice (normal chow diet, NCD; high-fat diet, HFD; n=5).

[0110] FIG. 6C One hour after administration of BA or BA culture supernatant or medium alone to untreated B6 mice, the level of DPP-4 in the small intestine was confirmed by light emission analysis.

[0111] FIG. 6D Quantification of cholates (left panel) and taurine (right panel) in the feces of PBS- and BA-fed mice (n=5) using CE-MS (Capillary Electrophoresis Mass Spectrometry).

[0112] All data are mean?s.e.m of ?2 independent experiments. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

[0113] FIG. 7 is a diagram showing a mechanism by which B. acidifaciens (BA) can prevent or treat insulin sensitivity and obesity.

[0114] Specific visceral symbiotic bacteria (i.e., BA) expanded in a lean phenotype Atg7.sup.?CD11c mouse were identified. Administration of BA results in the activation of fat oxidation through the bile acid-TGR5-PPAR? axis in adipose tissue, resulting in high energy consumption. At the same time, BA activates visceral DPP-4, followed by the increase in GLP-1, thereby contributing to glucose homeostasis. Bile acids, cholate and taurine also contribute to GLP-1 activity through a TGR5 receptor and improve insulin sensitivity.

[0115] PPAR?, peroxisome proliferator-activated receptor ?; SCFAs, short-chain fatty acids.

[0116] FIG. 8 is a diagram showing that an Atg7.sup.?CD11c mouse shows a lean phenotype regardless of gender.

[0117] Body weight of male (FIG. 8 left panel) and female (FIG. 8 right panel) Atg7.sup.f/f and Atg7.sup.?CD11c mice between 7 and 23 weeks of age on a NCD.

[0118] FIGS. 9A-9C are diagrams showing that the lean phenotype of 24-week-old Atg7.sup.?CD11c mouse is not associated with inflammation.

[0119] FIG. 9A Levels of pre-inflammatory cytokines in the serum of Atg7.sup.f/f and Atg7.sup.?CD11c mice (n=7) measured using a cytometric bead array mouse-inflammatory kit (BD Biosciences).

[0120] FIG. 9B F4/80 (left panel) and TNF? (right panel) mRNA expression levels through real-time PCR.

[0121] FIG. 9C Results of hematoxylin eosin staining of small intestine (top panels) and colon (bottom panels). Scale bar=100 ?m.

[0122] All data are mean?s.e.m in 2 independent experiments. *P<0.05; ns, not significant.

[0123] FIGS. 10A-10B are diagrams showing OPLS-DA (Orthogonal partial least squares discriminate analysis) of fecal metabolites of Atg7.sup.f/f and Atg7.sup.?CD11c mice.

[0124] FIG. 10A Cross-validated score plots from OPLS-DA of 1H-NMR (nuclear magnetic resonance) in the feces of Atg7.sup.f/f and Atg7.sup.?CD11c mice (n=7).

[0125] FIG. 10B S-plot for the predicted components from OPLS-DA of 1H-NMR data in the feces of Atg7.sup.f/f and Atg7.sup.?CD11c mice (n=7).

[0126] FIG. 10C Quantification of short chain fatty acids in the feces of Atg7.sup.f/f and Atg7.sup.?CD11c mice (n=5) using gas chromatography-mass spectrometry (e.g., left to right: acetates, butyrates and propinate) and lactate (far right panel). All data are mean?s.e.m. *P<0.05, **P<0.01.

[0127] FIG. 11 is a diagram showing compensated body weight changes of a mouse of the same kind in co-housing (CH) cage and fecal microorganisms.

[0128] The body weight of Atg7.sup.f/f (n=5) and Atg7.sup.?CD11c (n=4) mice in a CH cage and the body weight of Atg7.sup.f/f and Atg7.sup.?CD11c mice (S) raised separately are shown by a line graph based on gray and blue circles, respectively.

[0129] FIG. 12 is a diagram showing that pyrosequencing data at the species level in the feces of an Atg7.sup.?CD11c mouse shown in FIG. 3C show a color legend.

[0130] FIG. 13 is a diagram showing the biological arrangement of Bacteroides contig in the DNA metagenome.

[0131] The relative abundance of the number of contigs in the feces of Atg7.sup.f/f and Atg7.sup.?CD11c mice are compared. All data are shown as mean?s.e.m. * P<0.05; ***P<0.001; ns, not significant.

[0132] FIGS. 14A-14B are diagrams collectively showing that B. acidifaciens (BA) that are orally administered remains in the colon temporarily.

[0133] Colons and feces were obtained at nil and after 1-5 days after oral administration of BA (5?10.sup.9 CFU/100 ?l), and stained with BA-specific FISH (fluorescence in situ hybridization) probes.

[0134] FIG. 14A The confocal image of the BA (yellow arrow).

[0135] FIG. 14B Quantification of BA in the feces at a specific point of time. The number was counted at 20 sites per slide. All data are shown as mean?s.e.m of three independent experiments. ***P<0.001; N.D., not detected.

[0136] FIGS. 15A-15F are diagrams collectively showing the effective function of the body weight and fat mass of B. acidifaciens (BA) in NCD-fed B6 mice provided with BA or PBS.

[0137] FIG. 15A Photographs and body weight after 10 weeks (left and right panels, respectively). BA was orally administered daily (5?10.sup.9 CFU/100 ?l).

[0138] FIG. 15B Oral dietary intake with PBS or BA.

[0139] FIG. 15C MRI analysis.

[0140] FIG. 15D Histological changes of adipose tissues (left panel) and changes in adipocyte size (right panel).

[0141] FIG. 15E Results of GTT (glucose tolerance test) (left panel, n=8 or 9) and ITT (insulin tolerance test) (right panel, n=7-12) at a specific point of time after intraperitoneal injection of glucose or insulin.

[0142] FIG. 15F Energy consumption (left panel), total activity (middle panel), and RER (respiratory exchange ratio; right panel) of PBS- or BA-fed mice (n=5).

[0143] All data are shown as mean?s.e.m. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

[0144] FIGS. 16A-16B are diagrams showing that mice fed with NCD or HFD and B. sartorii (BS) have similar body weight and dietary intake.

[0145] FIG. 16A Body weight for 10 weeks after oral administration of BS (n=5) (5?10.sup.9 CFU/100 ?l).

[0146] FIG. 16B Dietary intake. Data are mean?s.e.m. of two independent experiments. ns, not significant.

[0147] FIG. 17 is a diagram showing that the administration of B. acidifaciens (BA) improves liver and peripheral insulin sensitivity.

[0148] It is the result of performing hyperinsulinemic-euglycemic clamp for BA-, heat-inactivated BA-fed mice and NCD-fed control mice (n=3) for 6 weeks. During the clamp experiment, the amount of insulin solution was determined to be 3 mU based on the first experiment. The inhibition of systemic glucose uptake (peripheral insulin sensitivity, FIG. 17 left panel) and insulin-mediated liver glucose production (liver insulin sensitivity, FIG. 17 right panel) were significantly increased in BA-fed mice as compared to heat inactivated BA-fed group. All data are shown as mean?s.e.m. *P<0.05; ns, not significant.

[0149] FIGS. 18A-18B are diagrams showing the levels of SCFAs (short-chain fatty acids) and lactate levels in the feces after oral administration of B. acidifaciens (BA) for 10 weeks.

[0150] FIG. 18A Levels of acetate, butyrate, propionate, and lactate in the feces of mice with NCD (panels from left to right; n=5) were measured through gas chromatography-mass spectrometry. All data are mean?s.e.m. *P<0.01; ns, not significant.

[0151] FIG. 18B Levels of acetate, butyrate, propionate, and lactate in the feces of mice with HFD (panels from left to right; n=6) were measured through gas chromatography-mass spectrometry. All data in FIGS. 18A-18B are mean?s.e.m. *P<0.01; ns, not significant.

[0152] FIGS. 19A-19B are diagrams showing the similar levels of lipid metabolism in liver and small intestine in Atg7.sup.f/f and Atg7.sup.?CD11c mice.

[0153] FIG. 19A The expression levels of mRNAs of genes related to fatty acid synthesis (FasN, HSL, PEPCK, SCD1, and PPAR?), ?-oxidation (PPAR?), thermogenesis (PRDM16, PGC1a, Cidea, and GLUT4) were determined through real-time PCR using liver of Atg7.sup.f/f and Atg7.sup.?CD11c mice (FIG. 19A; n=5). All data are mean?s.e.m.

[0154] FIG. 19B The expression levels of mRNAs of genes related to fatty acid synthesis (FasN, HSL, PEPCK, SCD1, and PPAR?), ?-oxidation (PPAR?), thermogenesis (PRDM16, PGC1a, Cidea, and GLUT4) were determined through real-time PCR using small intestine of Atg7.sup.f/f and Atg7.sup.?CD11c mice. All data are mean?s.e.m.

[0155] FIGS. 20A-20B are diagrams showing that B. acidifaciens (BA) does not induce ?-cell hyperpolarization.

[0156] Pancreatic tissues were obtained from mice (n=5) administered with (5?10.sup.9 CFU/100 ?l) for 10 weeks.

[0157] FIG. 20A The confocal image of the pancreatic islet (?-cells are red, ?-cells are green). Scale bar=50 ?m. Sections were continuously reacted with mouse anti-glucagon IgG Ab (K79bB10; Sigma-Aldrich, St. Louis, Mo.) and rabbit polyclonal anti-insulin Ab (Santa Cruz Biotechnology, Santa Cruz, Calif.), they were reacted with PE-conjugated anti-mouse IgG (eBioscience, San Diego, Calif.) and FITC-conjugated anti-rabbit IgG (eBioscience, San Diego, Calif.), respectively.

[0158] FIG. 20B The size of a ?-cell region was quantified using an ImageJ software program. The pancreatic islets were randomly selected as 10 sites per slide. All data are mean?s.e.m. ***P<0.001; ns, not significant.

[0159] FIGS. 21A-21C are graphs showing triglyceride and cholesterol levels in plasma of Atg7.sup.?CD11c, fecal microbiota transplantation (FMT), and B. acidifaciens (BA)-fed mice.

[0160] FIG. 21A The concentrations of plasma triglycerides and total cholesterol were analyzed by using an enzyme assay kit in Atg7.sup.?CD11c mice (n=3). All data are mean?s.e.m. ns, not significant.

[0161] FIG. 21B The concentrations of plasma triglycerides and total cholesterol were analyzed by using an enzyme assay kit in FMT mice (n=5). All data are mean?s.e.m. ns, not significant.

[0162] FIG. 21C The concentrations of plasma triglycerides and total cholesterol were analyzed by using an enzyme assay kit in BA-fed mouse (n=5). All data are mean?s.e.m. ns, not significant. NCD, normal chow diet; HFD, high-fat diet.

[0163] FIG. 22 is a diagram showing that B. acidifaciens (BA) can regulate self-digestion of CD11c.sup.+ cells.

[0164] The number of BA in bone marrow-derived CD11c.sup.+ cells after 6 and 24 hours of co-incubation with BA were determined on EG agar plates (MOI=10). Bone marrow was obtained from Atg7.sup.f/f and Atg7.sup.?CD11c mice. All data are mean?s.e.m of three independent experiments. * P<0.05; N.D., not detected.

MODES OF THE INVENTION

[0165] Hereinafter, the present disclosure will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present disclosure is not limited to these examples.

Example 1: Animal Experiment

[0166] All animal experiments were approved by the Asian Animal Experimental Ethics Committee (permit number: PN 2014-13-069). All experiments were performed under anesthesia with ketamine (100 mg/kg) and xylazine (20 mg/kg).

Example 2: Mice and Bacteria Strain

[0167] C57BL/6 (B6), and CD11c-Cre, Villine-Cre, and LysM-Cre mice were purchased from Charles River Laboratories (Orient Bio Inc., Sungnam, Korea) and Jackson Laboratory (Bar Harbor, Me.). ATG7.sup.flox/flox mice were provided by Masaaki Komatsu (Tokyo Metropolitan Institute of Medical Science, Japan). Atg7.sup.?CD11c mice were prepared by crossbreeding CD11c.sup.cre mice and ATG7.sup.f/f mice in the animal laboratory of Seoul Asian Medical Center. All mice were fed with sterile feed and drinking water under non-pathogenic conditions. B. acififaciens (JCM10556) and B. sartorii (JCM17136) were purchased from Japan Collection of Microorganisms (JCM) of RIKEN BioResource Center.

Example 3: 454 Pyrosequencing Analysis

[0168] cDNA was extracted from the feces using QIAamp DNA stool mini kits (Qiagen, Valencia, Calif.). PCR amplification was performed using primers targeting the V1 to V3 sites of 16S rRNA gene. For the amplification of bacteria, primer 9F

TABLE-US-00001 SEQIDNO:1 (5-CCTATCCCCTGTGTGCCTTGGCAGTC-TCAG-AC- AGAGTTTGATCMTGGCTCAG-3;
the underlined sequence means primer at the target site) to which a barcode is attached and 541R

TABLE-US-00002 SEQIDNO:2 (5-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-X-AC- ATTACCGCGGCTGCTGG-3;
X means a specific barcode of each object) (http://oklbb_ezbiocloud_net/content/1001) were used.

[0169] Amplification was performed under the following conditions: initiation of denaturation at 95? C. for 5 minutes, 30 cycles denaturation at 95? C. for 30 seconds, primer annealing at 55? C. for 30 seconds, and amplification at 72? C. for 30 seconds, and final extension at 72? C. for 5 minutes. The same concentrations of the presumed products were pooled together and short pieces (non-target objects) were removed using an AMPure bead kit (Agencourt Bioscience, Beverly, Mass.). The quality and size of the products were measured on Bioanalyzer 2100 (Agilent, Palo Alto, Calif.) using DNA 75001 chip. Sequencing of the mixed amplifications was performed via emulsion PCR, and then was placed on a picotiter plate. Sequencing was performed on Chunlab (Seoul, Korea) on the GS Junior Sequencing System (Roche, Branford, Conn.). Pyrosequencing data analysis was performed according to the prior art (Lim Y. W. et al.).

Example 4: Measurement of CE-TOF-MS (Capillary Electrophoresis (CE) Time-of-Flight Mass Spectrometry)

[0170] Quantitative analysis of the charged metabolites using CE-TOF-MS was performed as follows. 10 mg of lyophilized fecal samples were milled using 3-mm zirconia-silica beads (BioSpec Products, Bartlesville, Okla.), and as an internal standard, they were homogenized using 400 ?l of MeOH containing 20 ?M each of methionine sulfone (Wako, Osaka, Japan) as a cation, IVIES (Dojindo, Kumamoto, Japan) as an anion, and CSA (D-Camphol-10-sulfonic acid; Wako). Subsequently, 200 ?l of de-ionized water and 500 ?l of chloroform were added. By using a Shakemaster neo (Bio Medical Science, Tokyo, Japan), they were stirred in 1,500 r.p.m. for 10 minutes, the solution was centrifuged at 4,600 g for 15 minutes at 4? C., and the protein was removed by filtering using a Millipore 5,000-Da cut-off filter (Millipore, Billerica, Mass.). The filtrate was lyophilized and dissolved in 25 ?l water containing 200 ?M each of 3-aminopyrrolidine (Sigma-Aldrich) and trimesate (Wako) as reference compounds. All CE-TOF-MS experiments were conducted using Agilent Technologies equipment: CE capillary electrophoresis system, G3250AA LC/MSD TOF system, 1100 series binary HPLC pump, G1603A CE-MS adapter, and G1607A CE-ESI-MS sprayer kit. Data were treated (MasterHands) using internal software (Sugimoto M et al.) to determine peak annotation and quantification.

Example 5: Gas Chromatography Mass Spectrometry (GC-MS) Measurement

[0171] The organic concentration in the feces was determined by gas chromatography-mass spectrometry. A partial sample (80 ml) of the ether extract of the feces was mixed with N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide. The vial was sealed, heated in boiling water at 80? C. for 20 minutes, and then placed at room temperature for 48 hours for derivatization. The derivatized samples were treated with a 6890N Network GC System (Agilent Technologies) equipped with an HP-5MS column (0.25 mm?30 m?0.25 mm) and a 5973 Network Mass Selective Detector (Agilent Technologies).

[0172] Pure helium (99.9999%) was used as carrier gas and was delivered at a rate of 1.2 ml min.sup.?1.

[0173] The outlet pressure was set at 97 kPa divided by 20:1. The inlet and travel line temperatures were 250 and 260? C., respectively. The temperature program was used as follows: 60? C. (3 minutes), 60-120? C. (5? C./min), 120-300? C. (20? C./min). Subsequently, 1 ?l of each sample was injected for a reaction time of 30 minutes. The organic acid concentration was quantified by comparing the peak area with the standard.

Example 6: Measurement of GLP-1 (Glucagon-Like Peptide-1)

[0174] Blood samples were obtained from a control group and BA-fed mice, and were centrifuged at 1800 g at 4? C. for 30 minutes. DPP-4 (dipeptidyl peptidase-4) inhibitor was added and GLP-1 concentration was determined using GLP-1 ELISA kit (Shibayagi).

Example 7: Measurement of DPP-4

[0175] DPP-4 levels were measured. After 6 hours of fasting in wild-type B6 mice, BA (5?10.sup.9 CFU/100 ?l) or its culture supernatant (100 ?l/head) or culture medium alone was administered with DPP-4 inhibitor sitagliptin (40 mg/mouse; Merck Sharp Dohme and Chibret Laboratories, Rahway, N.J.) followed by glucose for 30 minutes. After 15 minutes, intestinal epithelial cells of the ileum were recovered from pretreated mice and washed with PBS to remove luminal materials. Mucus was scraped off, epithelium was cut into 1-2 mm in length, and placed in 1 ml PBS. The sliced tissues was spun down to centrifugation (6,000 g, 4? C., 5 min), and 50 ?l of supernatant was incubated using DPP-4 Glo protease assay (Promega, Madison, Wis.) at 37? C. for 2 hours together with kit reagents. DPP-4 activity was calculated as the value of a control sample in the absence of sitagliptin.

Example 8: Statistics

[0176] GraphPad Prism software (GraphPad, La Jolla, Calif.) was used for statistical analysis. Significant differences between the two groups were analyzed by two-tailed paired t-test or Mann-Whitneyt t-test. A plurality of groups were analyzed using two-way ANOVA followed by Bonferroni post-hoc test (*, P<0.05; **, P<0.01; ***, P<0.001).

Example 9: Identification of Reduced Body Weight and Fat Mass in Atg7.SUP.?CD11c .Mice

[0177] In order to identify the role of immune cell self-digestion action in the occurrence of metabolic diseases, the body weight and action were observed in dendritic cells (Atg7.sup.?CD11c), alimentary canal epithelial cells (Atg7.sup.?villin), and macrophages (Atg7?LysM) of Atg7 conditional knockout mice. The mice were fed with NCD (normal chow diet). Thereafter, it was confirmed that the difference in body weight between the Atg7.sup.?CD11c mice and their litter control group mice (Atg7.sup.flox/flox (f/f)) was increased (FIG. 1A).

[0178] Importantly, it was confirmed that 24-week-old Atg7.sup.?CD11c mice had very low body weight and fat mass as compared to Atg7.sup.f/f mice (FIGS. 1A and 1B). A lean phenotype (Atg7.sup.?CD11c; FIG. 8) is shown both in females and males.

[0179] In MRI (Magnetic Resonance Imaging) analysis, abdominal adipose tissues that were remarkably reduced in Atg7.sup.?CD11c mice were confirmed in both axial and coronal directions as compared to their litter Atg7.sup.f/f mice (FIG. 1C). In addition, the single adipocyte size of visceral adipose tissue obtained from Atg7.sup.?CD11c mice was significantly small as compared to Atg7.sup.f/f mice (FIG. 1D).

[0180] In order to confirm the involvement of systemic or mucosal inflammation in Atg7.sup.?CD11c mice of a lean phenotype, the level of proinflammatory cytokine in serum and mRNA expression of F4/80 and TNF? in visceral adipose tissue were confirmed, and the tissues of the small intestine and colon were analyzed. It was confirmed that Atg7.sup.?CD11c mice showed similar or decreased levels of multiple markers of systemic and mucosal inflammation indicating that the lean phenotype of Atg7.sup.?CD11c mice was not related to inflammation (FIGS. 9A and 9B).

[0181] Importantly, higher insulin and subsequent low glucose levels than Atg7.sup.f/f mice under non-fasting conditions were identified in the serum of Atg7.sup.?CD11c mice (FIG. 1E). It was confirmed that the insulin resistance determined by GTT (glucose tolerance test) and ITT (insulin tolerance test) in Atg7.sup.?CD11c mice as compared to the litter Atg7.sup.f/f was improved in Atg7.sup.?CD11c mice as compared to the litter Atg7.sup.f/f (FIG. 1F). To sum up, these data indicate that Atg7.sup.?CD11c mice have reduced fat mass and improved glucose homeostasis.

Example 10: Identification of Low SCFAs Levels in the Feces of Atg7.SUP.?CD11c .Mice

[0182] Since aged Atg7.sup.?CD11c mice have low body weight and fat mass, the relevance between a lean phenotype and energy use was confirmed using CE-TOF-MS (capillary electrophoresis time-of-flight mass spectrometry) of Example 4 and GC-MS (gas chromatography mass spectrometry) of Example 5 in the feces.

[0183] Individual plots of Atg7.sup.?CD11c mice in OPLS-DA (orthogonal partial least squares discriminant analysis) are clearly distinct from Atg7.sup.f/f mice (FIG. 10A). Furthermore, some SCFAs such as acetate, butyrate, propionate and lactate are located at the remote spot from the axis (FIG. 10B), which indicates that these factors contribute to distinguish Atg7.sup.f/f and Atg7.sup.?CD11c mice. In reality, the amount of acetate, butyrate, and propionate in Atg7.sup.?CD11c mice was remarkably reduced as compared to Atg7.sup.f/f mice, whereas the amount of lactate was higher (FIG. 10C).

Example 11: Identification that Symbiotic Bacteria are Related to a Lean Phenotype of Aged Atg7.SUP.?CD11c .Mice

[0184] In order to confirm whether a lean phenotype of Atg7.sup.?CD11c mice is related to symbiotic bacteria, co-housing (CH) and FMT (fecal microbiota transplantation) experiments were performed. From birth, Atg7.sup.?CD11c and Atg7.sup.f/f mice shared a cage and exposed feces.

[0185] As a result, Atg7.sup.f/f mice that shared the cage with Atg7.sup.?CD11c, lost more both body weight and fat as compared to Atg7.sup.f/f mice (FIGS. 2A and 2B; FIG. 11). In addition, Atg7.sup.?CD11c mice that shared the cage with Atg7.sup.f/f mice increased body weight and fat compared to Atg7.sup.?CD11c mice (FIGS. 2A, and 2B; FIG. 11). In order to confirm whether the phenotype of CH mice was due to symbiotic microorganisms, mice were transferred to their respective cages after co-housing experiments. As shown in FIG. 2C, Atg7.sup.?CD11c mice lost body weight when they were raised alone, whereas Atg7.sup.f/f mice did not. In addition, the oral administration of the fecal extract of Atg7.sup.?CD11c mice to wild-type B6 mice for 12 weeks resulted in significantly lower body weight and fat mass than the feces of wild type B6 or Atg7.sup.f/f mice (FIG. 2D). In addition, importantly, wild-type B6 mice to which the fecal extract of Atg7.sup.?CD11c mice is administered showed higher insulin levels and subsequent low serum glucose levels as compared to the mice to which the extract of Atg7.sup.f/f is administered (FIG. 2E).

[0186] To sum up, these results indicate that they play an essential role in the lean phenotype of Atg7.sup.?CD11c mice.

Example 12: Expansion of Bacteroides acidifaciens (BA) in the Feces of Atg7.SUP.?CD11c .Mice

[0187] Metagenomics analysis was used to confirm the diversity and composition of intestinal symbiotic bacteria. In pyrosequencing analysis, it was confirmed that the faces of Atg7.sup.f/f and Atg7.sup.?CD11c mice were mutually similar in the primary distribution of intestinal microorganisms at the Bacteroidetes, Firmicutes, and Proteobacteria ratios, and phylum level (FIG. 3A). There was no similar or significant difference in the distribution of Bacteroidia (class), Bacteroidales (order), Bacteroidaceae (family), and Bacteroides (genus) (FIG. 3B). However, at the species level, it was confirmed that the ratio of BA was significantly extended in the feces of Atg7.sup.?CD11c mice as compared to Atg7.sup.f/f mice (5.48?1.76% vs. 0.77?0.18%) (FIG. 3C, red arrow; FIGS. 12 and 13).

[0188] On the other hand, there was no difference in the ratio of the other Bacteroides species including B. sartorii in the feces of Atg7.sup.?CD11c or Atg7.sup.f/f mice (FIG. 13).

[0189] In alpha diversity, the species abundance of the fecal microorganisms of Atg7.sup.?CD11c mice (Chao 1 index) was remarkably reduced, whereas the biodiversity (Shannon/Simpson index) was similar to the fecal microorganism of Atg7.sup.f/f mice (Table 1 below).

TABLE-US-00003 TABLE Chao1 Shannon Simpson Reciprocal Atg7.sup.f/f 932.12 ? 109.72 4.39 ? 0.43 0.04 ? 0.02 Atg7.sup.?CD11c 604.41 ? 203.83* 4.28 ? 0.28 0.04 ? 0.03

[0190] FISH (fluorescence in situ hybridization) analysis was performed to confirm the expansion of BA in Atg7.sup.?CD11c mice of a lean phenotype. As shown in FIG. 3D, the number of increased BA was detected in the lumen of the colon, and it was confirmed that a small amount of BA was internalized into the colon epithelial cells of Atg7.sup.?CD11c mice.

[0191] To sum up, these results indicate that, in symbiotic bacteria, BA has been expanded in lean phenotype intestines.

Example 13: Identification that the Oral Administration of BA to High Fat Diet (HFD)-Provided B6 Mice Induces a Lean Phenotype

[0192] In order to confirm whether extended BA regulates lipid metabolism, BA (JCM10556) was obtained and cultured to obtain large amounts of microorganisms, which were provided to untreated B6 mice.

[0193] In order to determine the optimal conditions of administration, colon tissues and BA in mice fed with BA (5?10.sup.9 CFU/100 ?l) were quantitated by FISH analysis. One day after oral administration, a large number of BA were detected in the lumen of colon epithelial cells (FIG. 14A).

[0194] After 2 days of oral administration, the number of BA in the peak feces subsequently disappeared and recovered rapidly (FIG. 14B). It was confirmed that the oral administration of BA reduced body weight and fat mass of wild-type B6 mice provided with NCD and HFD without dietary effects (FIGS. 4A-4C; FIGS. 15A-15C). In contrast, no body weight loss was observed in B. sartorii-provided mice used as control groups (FIG. 16A). In addition, the size of single adipocytes in adipose tissue of the epididymal was significantly lower in BA- and HFD-provided B6 mice as compared to PBS- and HFD-provided mice (FIG. 4D). In addition, the insulin resistance determined by GTT and ITT improved remarkably in BA- and HFD-provided mice as compared to PBS- and HFD-provided mice (FIG. 4E).

[0195] A hyperinsulinemic-euglycemic clamp technique using heat-inactivated BA as a control group was used to confirm the effect of BA feeding on liver and peripheral insulin sensitivity.

[0196] Interestingly, the BA feeding improved liver and peripheral insulin sensitivity (FIG. 17). It was confirmed that the oral administration of BA showed a decrease in butylate in the feces of NCD-fed mice, but it was confirmed that levels of acetate, propionate, and lactate were not changed (FIG. 18A). A similar trend was confirmed in the HFD-fed group (FIG. 18B). In order to measure energy consumption, activity and substrate utilization, the mice provided with BA were monitored after they were individually raised in a CLAMS (a comprehensive laboratory animal monitoring system) cage for 5 days. It was confirmed that the group of PBS or BA-provided mice showed a similar locomotor activity and respiratory exchange ratio, whereas BA- and HFD-fed mice consumed more energy as compared to PBS- and HFD-fed mice (FIG. 4F). The NCD-fed mice to which oral BA is administered showed a similar effect (FIG. 15F).

[0197] To sum up, the long-term administration of BA induces energy consumption, and accordingly, causes a lean phenotype of dominance in diet-induced obese mice.

Example 14: Identification that a Lean Phenotype Mouse Shows an Increase in PPAR? (Peroxisomeproliferator-Activated Receptor ?) Expression in Adipose Tissues

[0198] Expression levels of genes related to lipid metabolism in adipose tissue, liver, and small intestine were analyzed based on the detection of decreased body weight and fat mass in Atg7.sup.?CD11c, FMT B6, and BA-fed B6 mice. Importantly the expression of genes related to lipid ?-oxidation, particularly PPAR?, increased only in adipose tissue of the epididymis of Atg7.sup.?CD11c mice (FIG. 5A). No significant difference could be identified in the small intestine and liver (FIGS. 19A and 19B). To be consistent with these results, PPAR? expression was significantly up-regulated in adipose tissue of the epididymis of B6 mice fed with the fecal extract of Atg7.sup.?CD11c and mice fed with HFD and BA for 10 weeks (FIGS. 5B and 5C).

[0199] The level of PPAR? expression in B6 mice by BA administration by the time-dependent method was measured to confirm whether the enhanced ?-oxidation level was activated by the bacteria alone or was not activated by the product of a lean phenotype.

[0200] Interestingly, the level of mRNA of PPAR? in epididymis adipose tissue of B6 mice was significantly increased after 2 weeks of BA administration (FIG. 5D). In addition, the expression levels of TGR5, GPBAR1 (G-protein-coupled bile acid receptor), which can stimulate energy consumption through PPAR? activity, were measured.

[0201] As a result, it was confirmed that BA administration increased the level of TGR5 expression in adipose tissue (FIG. 5E). These results indicate that a lean phenotype by BA initiates fat oxidation in adipose tissue according to TGR5-PPAR? activity.

Example 15: Identification that BA Mediates Production of GLP-1 (Glucagon-Like Peptide-1) by Regulation of DPP4 (Dipeptidylpeptidase-4) and Production of Bile Acids

[0202] The role of BA in glucose homeostasis was confirmed. As expected, BA-fed B6 mice showed higher insulin and lower glucose levels in serum than PBS-fed B6 mice (FIG. 6A).

[0203] In order to confirm whether this increase in plasma insulin levels was due to over-stimulation of ?-cells, ? and ? cells were stained in pancreatic tissue 10 weeks after BA feeding.

[0204] As a result, it was confirmed that the BA feeding did not induce hypersensitivity of ?-cells (FIGS. 20A and 20B).

[0205] In order to confirm the mechanism of high levels of insulin secretion in BA-fed lean mice, levels of GLP-1 stimulating insulin release into the blood were measured.

[0206] GLP-1 levels in serum were remarkably increased in NCD and HFD-fed mice (FIG. 6B).

[0207] It was confirmed that the level of DPP-4, a well-known enzyme with inhibiting activity of GLP-1, decreased in the small intestine and ileum after oral administration of BA or its culture supernatant (FIG. 6C).

[0208] In addition, DPP-4 activity was measured, reflecting the amount of protein. Previous studies have shown that bile juice plays a key role in glucose homeostasis through stimulation of GLP-1 secretion through TGR5 activity.

[0209] As a result, it was confirmed a significantly increased level of deconjugated cholate, salt of cholic acid, and taurine from primary bile acid in the feces of B6 mice fed with BA for 10 weeks, but significant loss of cholesterol could not be confirmed (FIG. 6D; FIG. 21C). These results indicate that BA or its metabolites lower the activity of the DPP-4 enzyme, and accordingly, causing GLP-1 activity, thereby improving insulin sensitivity and glucose resistance.

[0210] To sum up, the present inventors have confirmed that the specific intestinal symbiotic bacteria (i.e., BA) are extended in the lean phenotype Atg7.sup.?CD11c mice. The administration of BA results in the activation of fat oxidation through the bile acid-TGR5-PPAR? axis in adipose tissue, resulting in high energy consumption.

[0211] At the same time, BA activates visceral DPP-4, followed by the increase in GLP-1, thereby contributing to glucose homeostasis. Bile acids, cholate and taurine contributed to GLP-1 activity through a TGR5 receptor and improved insulin sensitivity.

[0212] Accordingly, it can be understood that BA plays an important role in the prevention or treatment of metabolic diseases such as diabetes and obesity (FIG. 7).