Collection of human samples
All experiments were approved by the Ethics Committee of the National Institutes of Biomedical Innovation, Health, and Nutrition and were conducted in accordance with their guidelines (approval numbers: 177-08 and Kenei78-06). Informed consent was obtained from all participants.
For the discovery cohort, diabetic patients were recruited at Shinnanyo Hospital, Shunan City, Yamaguchi, Japan (Table S1); nondiabetic adult volunteers (control subjects; e.g., staff members of city offices and chamber of commerce) were recruited at health examination sites in surrounding communities (Table S1). From the 242 total participants possible, we excluded those who had received antibiotics within the previous 2 weeks (18 subjects), had traveled overseas within the previous month (two subjects), or had gastrointestinal disease (5 subjects), thus leaving a study population of 217 participants comprising 147 nondiabetic subjects, 45 patients with type 2 diabetes, and 25 patients with type 1 diabetes.
For a validation cohort, we recruited adult volunteers from among the city office staff of Minamiuonuma City, Niigata, Japan, at health examination sites (Table S8). From the 219 total participants possible, we excluded 24 who had received antibiotics within the previous 2 weeks (ten subjects), had traveled overseas within the previous month (six subjects), or had gastrointestinal disease (eight subjects), thus leaving a study population of 195 participants; all of these volunteers were nondiabetic.
Fecal samples were collected and placed in guanidine thiocyanate solution (TechnoSuruga Laboratory Co., Ltd, Shizuoka, Japan) as previously described95, which allows the sample to be stored at room temperature. The participants collected their stool sample at home without any restrictions, such as fasting, and submitted it to the hospital (diabetic patients) or health examination site (control subjects) within 5 days. Blood samples were collected at the hospital and health examination sites. Physical measurements, including body weight, height, blood glucose, and HbA1c, and disease information were obtained through health examinations and from medical records.
16S rRNA gene amplicon sequencing analysis
DNA was extracted from human fecal samples in guanidine thiocyanate solution by using the bead beating method and an automatic nucleic acid extraction system (Gene Prep Star PI-80X, Kurabo Industries, Ltd, Osaka, Japan) as previously described95. DNA was extracted from mouse fecal samples through a slight modification of this method. Briefly, a mouse fecal sample was placed in a 2-ml vial (Wakenbtech Co., Ltd, Tokyo, Japan) containing 0.5 ml of lysis buffer (No. 10, Kurabo Industries, Ltd) and 0.5 g of 0.1-mm glass beads. The mixture was mechanically disrupted by bead beating by using a Cell Destroyer PS1000 (Bio Medical Science, Tokyo, Japan) at 4260 rpm for 50 s at room temperature. After centrifugation at 13,000 × g for 5 min at room temperature, DNA was extracted from 0.2 ml of the supernatant by using a Gene Prep Star PI-80X device (Kurabo Industries, Ltd).
The 16S rRNA gene amplicon in human and mouse fecal DNA was sequenced as previously described95. The V3–V4 region of the 16S rRNA gene was amplified from the fecal DNA samples by using the following primers: forward, 5′- TCGTCGGCAGCGTCAGATGTGTATAAGCGACAGCCTACGGGNGGCWGCAG-3′; and reverse, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′96. A DNA library was prepared by using a Nextera kit Set A (Illumina, San Diego, California, USA), and 16S rRNA gene sequencing was performed by using MiSeq (Illumina) in accordance with the manufacturer’s instructions. The sequencing results were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) software package97 and QIIME Analysis Automating Script (Auto-q) (https://doi.org/10.5281/zenodo.1439555) as previously described98. Open-reference operational taxonomic unit (OTU) picking and taxonomy classification were performed based on sequence similarity (>97%) by using UCLUST software99 with the SILVA v128 reference sequence100.
Bacterial strains and culture
B. wexlerae (JCM 17041), B. vulgatus (JCM 5826), P. copri (JCM 13464), F. prausnitzii (JCM 31915), and B. faecihominis (JCM 31056) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. All bacterial strains were cultured anaerobically at 37 °C by using an anaerobic chamber (Bactron 300, Toei Kaisha, Ltd, Tokyo, Japan). For oral administration into mice, B. wexlerae was cultured anaerobically in reinforced clostridial medium (BD Difco, Franklin Lakes, NJ, USA) at 37 °C for 48 h until OD600 = 1.0–1.5. Cultures were stored as 0.5-ml aliquots at –80 °C until use for oral administration into mice. For measurement of starch, SCFAs, and metabolites and Raman spectroscopic analysis, B. wexlerae, B. vulgatus, P. copri, and F. prausnitzii were cultured in clostridial reinforced medium (BD Difco) at 37 °C for 48 h; at inoculation, OD600 was approximately 0.05 for all strains (B. wexlerae, 1.0; B. vulgatus, 1.1; P. copri, 0.8; and F. prausnitzii, 0.9). For the cross-feeding assay, B. faecihominis was cultured anaerobically at 37 °C in GAM broth (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) supplemented without or with B. wexlerae culture supernatant, succinate acid, sodium lactate, or sodium acetate; the pH of the medium was adjusted to 7.0.
Mice and high-fat diet-induced obesity and diabetes
Male C57BL/6 mice (age, 4 weeks) were purchased from Japan SLC (Shizuoka, Japan). Each group of five mice was maintained in a single cage in the specific pathogen-free animal facility and were fed a standard diet (Oriental Yeast, Tokyo, Japan; AIN-93M) for 2 weeks, followed by a high-fat diet composed of chemically defined materials (Oriental Yeast; AIN-93G) for 10 weeks; accordingly, this mouse model qualifies as an insulin-resistant and pre-diabetic model. During the duration of the experiments, mice had free access to food and water and received 0.5 ml of bacterial solution (i.e., 5 × 108 CFU) or fresh medium (as a vehicle control) by oral gavage three times each week, and the weights of each mouse and the total amount of diet consumed by each cage of mice were calculated every week. At 8 weeks, blood and fecal samples were collected for calculation of HOMA-IR and for measurement of the gut microbiome and SCFAs, respectively, and IPGTT was performed. At 9 weeks, a blood sample was collected for monitoring serum insulin concentration over time. At 10 weeks, mice were euthanized by cervical dislocation, after which tissues were collected by using surgical scissors. All experiments involving mice were approved by the Animal Care and Use Committee of the National Institutes of Biomedical Innovation, Health, and Nutrition (approval no. DS27-48R10) and were conducted in accordance with their guidelines.
Intraperitoneal glucose tolerance testing (IPGTT)
IPGTT was performed as described previously90 with modification. Briefly, mice were fasted overnight (16 h) and then intraperitoneally injected with d-(+)-glucose (2 g/kg body weight; 20% solution, Nacalai Tesque, Kyoto, Japan). To measure the blood glucose level, blood was obtained from the tail vein by cutting with a single-edged blade (Feather, Osaka, Japan) and analyzed on a ONE TOUCH Ultra Vue (LifeScan Japan, Tokyo, Japan) before and after glucose injection at the indicated time points. Blood insulin levels were measured by using an LBIS Mouse Insulin ELISA kit (Wako Pure Chemicals, Osaka, Japan) in accordance with the manufacturer’s instructions. Samples unsuitable for analysis, such as hemolyzed blood, were excluded.
Frozen tissue was analyzed histologically as described previously with minor modification101. Briefly, tissue samples were washed with PBS (Nacalai Tesque) on ice and frozen in Tissue-TeK OCT compound (Sakura Finetek, Tokyo, Japan) in liquid nitrogen. Frozen tissue sections (thickness, 6 µm) were prepared by using a cryostat (model CM3050 S, Leica, Wetzlar, Germany) and were fixed for 30 min at 4 °C in prechilled 95% ethanol (Nacalai Tesque) followed by 1 min at room temperature in prechilled 100% acetone (Nacalai Tesque).
For immunohistological analysis, tissue sections were washed with PBS for 10 min and then blocked in 2% (vol/vol) newborn calf serum in PBS for 30 min at room temperature in an incubation chamber (Cosmo Bio, Tokyo, Japan). Tissue sections were incubated with purified anti-F4/80 monoclonal antibody (1:100; catalog no. 123102, Biolegend, San Diego, CA, USA) and BODIPY493/503 (1:1000; catalog no. D3922, Molecular Probes, Eugene, Oregon, USA) in 2% (vol/vol) newborn calf serum in PBS for 16 h at 4 °C in the incubation chamber, washed once for 5 min each in 0.1% (vol/vol) Tween-20 (Nacalai Tesque) in PBS and in PBS only, and then stained with Cy3-anti-rat IgG (1:200; catalog no. 712-165-153, Jackson Immuno Research Laboratories, West Grove, Pennsylvania, USA) in 2% (vol/vol) newborn calf serum in PBS for 30 min at room temperature in the incubation chamber. To visualize nuclei, tissue sections then were washed twice (5 min each) with PBS and stained with DAPI (1 µM, AAT Bioquest, Sunnyvale, CA, USA) for 10 min at room temperature in the incubation chamber. Finally, tissue sections were washed twice with PBS, mounted in Fluoromount (Diagnostic BioSystems, Pleasanton, CA, USA), and examined under a fluorescence microscope (model BZ-X810, Keyence, Osaka, Japan); areas of fluorescence were calculated by using the software provided with the microscope (version 184.108.40.206, Keyence).
Cell preparation from epididymal adipose tissue
Epididymal adipose tissue (eAT) was minced into Krebs–Ringer bicarbonate HEPES (KRBH) buffer (120 mM NaCl, 4 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 10 mM NaHCO3, 30 mM HEPES, 20 µM adenosine, and 4% [wt/vol] BSA) by using scissors, centrifuged to remove blood cells, and then incubated in 2.67 mg/ml collagenase in KRBH buffer for 45 min at 37 °C with stirring. After the samples were centrifuged, we collected the stromal vascular fraction (i.e., the pellet, which contains immune cells) and mature adipocyte fraction (MAF; i.e., the supernatant) for flow cytometric analysis and RT-qPCR testing.
Flow cytometric analysis
Flow cytometry was performed as described previously, with slight modification91,102. Cells in the stromal vascular fraction of eAT were stained with an anti-CD16/32 monoclonal antibody (TruStain fcX; Biolegend) to avoid non-specific staining and 7-AAD (Biolegend) to detect dead cells. The cells were further stained with the fluorescently labeled antibodies BV421–anti-CD45 (Biolegend, clone 30-F11), PE–anti-I-Ab MHC class II (Biolegend, clone AF6-120.1), FITC-anti-CD206 (Biolegend, clone C068C2), PE-Cy7–anti-F4/80 (Biolegend, clone BM8), and APC-Cy7–anti-CD11b (Biolegend, clone M1/70). Samples were analyzed by using BD FACSAria III (BD Biosciences), and data were analyzed by using FlowJo 9.9 (Tree Star, Ashland, Oregon, USA). The CD45+CD11b+F4/80+ cells were defined as the macrophage population91. Among macrophages, the MHC II+/highCD206–/low cells and MHC II+CD206high cells were defined as the M1- and M2-like macrophage populations, respectively.
Reverse transcription and quantitative PCR (RT-qPCR) analysis
RT-qPCR analysis were performed as described previously101 with minor modification. Total RNA was isolated from purified or cultured cells by using Sepasol (Nacalai Tesque) and chloroform (Nacalai Tesque), precipitated by using 2-propanol (Nacalai Tesque), and washed with 75% (vol/vol) ethanol (Nacalai Tesque). RNA samples were incubated with DNase I (Invitrogen, Carlsbad, California, USA) to remove contaminating genomic DNA and then reverse-transcribed into cDNA (Superscript III reverse transcriptase, VIRO cDNA Synthesis Kit; Invitrogen).
Quantitative PCR analysis was performed by using a LightCycler 480 II (Roche, Basel, Switzerland) with FastStart Essential DNA Probes Master (Roche) or SYBR Green I Master reagents (Roche). Primer sequences were: Tnfα sense, 5′-CTGTAGCCCACGTCGTAGC-3′; Tnfα anti-sense, 5′-TTGAGATCCATGCCGTTG-3′; S100a8 sense, 5′-TCCTTGCGATGGTGATAAAA-3′; S100a8 anti-sense, 5′-GGCCAGAAGCTCTGCTACTC-3′; Pparγ sense, 5′-GAAAGACAACGGACAAATCACC-3′; Pparγ anti-sense, 5′-GGGGGTGATATGTTTGAACTTG-3′; Nrf1 sense, 5′-GCTCTCTGAGACGCTGCTTT-3′; Nrf1 anti-sense, 5′-GTGTTCAGTTTGGGTCACTCC-3′; Actb sense, 5′-AAGGCCAACCGTGAAAAGAT-3′; and Actb anti-sense, 5′-GTGGTACGACCAGAGGCATAC-3′.
The 3T3L1 cell line (JCRB 9014) was purchased from the Japanese Collection of Research Bioresources (JCRB) cell bank (Osaka, Japan). Differentiation of 3T3L1 adipocytes was performed as previously reported with modifications103. 3T3L1 cells were seeded at 3.5 × 104 cells per well in 12-well plates and incubated overnight (16 h) in DMEM (Nacalai Tesque) at 37 °C under 5% CO2 supplemented with 10% (vol/vol) newborn calf serum. The following day, the medium on the cells was changed to DMEM (Nacalai Tesque) supplemented with 10% (vol/vol) fetal bovine serum, and cells were cultured for 3 d; cells were then cultured in adipocyte differentiation medium (0.5 mM isobutyl-methylxanthine [Sigma-Aldrich, St. Louis, Missouri, USA], 1 µM dexamethasone [Sigma-Aldrich], 10 µg/ml insulin [Sigma-Aldrich] in DMEM [Nacalai Tesque] supplemented with 10% [vol/vol] fetal bovine serum) for 2 d and finally in DMEM (Nacalai Tesque) supplemented with 10% (vol/vol) fetal bovine serum for 6 d. During the terms, 1 or 10% B. wexlerae-cultured medium or uncultured fresh medium (reinforced clostridial medium [BD Difco]) was added to the 3T3L1 culture medium.
Mitochondrial mass analysis by flow cytometry using Mitogreen
For mitochondrial staining, 3T3L1 adipocytes were incubated with 100 nM Mitogreen (Takara Bio, Shiga, Japan) in PBS supplemented with 2% (vol/vol) newborn calf serum for 15 min at 37 °C under 5% CO2. Stained cells were washed with PBS, treated with trypsin, and then suspended in PBS supplemented with 2% (vol/vol) newborn calf serum. Samples were analyzed by using MACSQuant (Miltenyi Biotec, Bergisch Gladbach, Germany), and data were analyzed by using FlowJo 9.9 (Tree Star, Ashland, OR, USA).
The oxygen consumption rate and extracellular acidification rate were measured by using a flux analyzer (Seahorse Bioscience XF24 Extracellular Flux Analyzer, Agilent, Santa Clara, CA, USA) and XF Mito Stress Kit (Agilent). 3T3L1 cells were seed at 3.5 × 103 cells per well in 0.1% gelatin-coated Seahorse 24-well plates and differentiated into adipocytes as described above. After differentiation, B. wexlerae culture supernatant or uncultured fresh medium were added to the 3T3L1 adipocyte cultures to a final concentration of 10%; cultures were then incubated at 37 °C for 1 h. After incubation, the culture medium was changed to XF Base Medium Minimal DMEM (Agilent) supplemented with 10 mM glucose (Nacalai Tesque), 1 mM pyruvate (Nacalai Tesque), and 2 mM l-glutamine (Nacalai Tesque) for measurement. Compounds injected during the assay and their final concentrations were 1.5 µM oligomycin (inhibitor of ATP synthase), 1 µM FCCP (proton uncoupling agent), and 0.5 µM rotenone + 0.5 µM antimycin A (inhibitors of the mitochondrial respiration complex). XFe Wave software (Agilent) was used to analyze the results.
Oil red O staining
For measurement of lipid accumulation, 3T3L1 adipocytes were washed with PBS for 1 min, treated with 4% paraformaldehyde in PBS (Nacalai Tesque) at 37 °C for 30 min, washed with 60% (vol/vol) 2-propanol (Nacalai Tesque), and then dried at 37 °C for 15 min. The fixed cells were stained with 0.3% (wt/vol) oil red O (Sigma-Aldrich) in 60% (vol/vol) 2-propanol at 37 °C for 30 min, washed with distilled water three times, and then incubated with 100% 2-propanol at room temperature for 1 min. Absorbance at 490 nm was measured by using an iMark microplate reader (Bio-Rad, Hercules, CA, USA).
Hydrophilic metabolites were extracted as previously described with minor modification104,105. The eAT MAF (100 µl suspended in PBS), serum (50 µl), and bacteria-cultured medium (100 µl) were diluted with water (Wako Pure Chemicals) to a total volume of 200 µl, mixed with 400 µl of methanol (Wako Pure Chemicals) containing methionine sulfone (Wako Pure Chemicals) as an internal standard, and combined with 400 µl of chloroform (Nacalai Tesque). Samples of liver and gastrocnemius muscle were homogenized in methanol (400 µl) containing the internal standard (concentration, 100 mg/ml), and each suspension was mixed with 200 µl of water and 400 µl of chloroform.
Samples were centrifuged at 20,000 × g at 4 °C for 15 min, after which 200 µl of supernatant was centrifugally filtered through a 5-kDa cutoff filter (Human Metabolome Technologies, Inc., Tokyo, Japan). The filtrates were lyophilized, resuspended in ultrapure water (Wako Pure Chemicals), and analyzed by LC–MS/MS as previously described104 by using a Nexera system (Shimadzu, Kyoto, Japan) equipped with two LC-40D pumps, a DGU-405 degasser, an SIL-40C autosampler, a CTO-40C column oven, and a CBM-40 control module, coupled to an LCMS-8050 triple-quadrupole mass spectrometer (Shimadzu). A pentafluorophenylpropyl column (Discovery HS F5, 150 mm × 2.1 mm, 3 µm; Sigma-Aldrich) was used for the separation of metabolites. Instrument control and data analysis were performed by using the software LabSolutions LCMS with LC/MSMS Method Package for Primary Metabolites, ver. 2 (Shimadzu).
Raman spectroscopic analysis
Raman spectroscopic analysis was performed as described previously with minor modification106. Briefly, bacterial cells were cultured anaerobically for 48 h at 37 °C, collected by centrifugation at 10,000 × g for 10 min at 4 °C, and suspended in PBS. The cell suspension was analyzed under a confocal Raman microspectroscopic system, which was equipped with a 100×/1.4 N.A. objective lens. The spatial resolution of the system is 0.3 × 0.3 μm laterally and 2.6 μm in depth. The excitation wavelength was 532 nm, and the intensity was 5 mW at the sample point. The mapping scan measurements were performed in steps of 0.25 μm, with the exposure time of 1 s per point.
The data was preprocessed using IGOR Pro software (WaveMetrics, Inc., Lake Oswego, OR, USA) for wavenumber calibration and sensitivity correction. Subsequently, a multivariate curve resolution analysis was performed on the spectral data set obtained from all the mapping measurements107. In this analysis, the decomposed Raman spectra and their corresponding intensity images were calculated by alternating least-squares optimization, based on the initial value spectra created by concatenating the results of the singular value decomposition (SVD) and the reference spectra of glass and PBS.
Measurement of amylose and amylopectin
Bacterial cells were cultured anaerobically for 48 h at 37 °C, then collected by centrifugation at 10,000 × g for 10 min at 4 °C, and frozen at –80 °C, and lyophilized (EYELA FDU-2110, Tokyo Rikakikai Co., Ltd, Tokyo, Japan). Amylose and amylopectin contents were measured by using an Amylose/Amylopectin Assay kit (Megazyme, Bray, Ireland) in accordance with the manufacturer’s instructions.
SCFA measurement by HPLC
Murine fecal samples were mixed with 95% ethanol (Nacalai Tesque) to a concentration of 100 mg/ml and homogenized by using two 15-s pulses at 6500 rpm from a tissue homogenizer (Precellys 24, Bertin Instruments, Montigny-le-Bretonneux, France) with zirconia beads (Tomy Digital Biology Co., Ltd, Tokyo, Japan). The homogenate was centrifuged at 1600 × g for 10 min at 4 °C and the supernatant collected. The fecal supernatant or bacterial cultured medium were labeled using an FA Labeling kit (YMC Co., Ltd, Kyoto, Japan) in accordance with the manufacturer’s instructions. The labeled samples were analyzed on an HPLC system (Ultimate 3000, Thermo Fisher Scientific, Waltham, Massachusetts, USA) with a 6.0 × 250 mm YMC-Pack FA column (YMC), and the UV spectrum at 400 nm was measured.
Blautia-specific quantitative PCR analysis
Quantitative PCR analysis was performed by using a LightCycler 480 II (Roche) with Real-time PCR Detection Kit (Blautia: RI-0008) (TechnoSuruga Laboratory Co., Ltd) and TB Green Premix Ex Taq II (Takara Bio) in accordance with the manufacturer’s instructions for templates of human and mouse fecal DNA (25 ng).
Measurement of glucagon-like peptide-1 (GLP-1)
Serum was collected from mice at 10 weeks when they were euthanized for tissue sampling and stored at −80 °C until use. Serum GLP-1 was measured by using LBIS GLP-1 (Active) ELISA Kit (FUJIFILM Wako Shibayagi, Gunma, Japan) in accordance with the manufacturer’s instructions.
Measurement of energy excretion in mouse feces
Fecal samples were collected at 8 weeks, frozen at −80 °C, and lyophilized (EYELA FDU-2110). The lyophilized samples were completely combusted in a Calorimeter C5003 (IKA Japan K.K., Osaka, Japan), and the energy value (J) was calculated according to the amount of heat released during the combustion.
Measurement of spontaneous activity of mice
Physical spontaneous activity was measured as the number of infrared beams broken in both X and Y directions by using an activity monitoring system (ACTIMO-100, Shinfactory, Fukuoka, Japan). At 8 weeks, mice were placed in the individual cages, and movement was monitored for 24 h.
Statistics and reproducibility
The output of the QIIME pipeline in Biom table format was imported and analyzed in R (version 3.5.1) (https://www.R-project.org/). The alpha-diversity indices were calculated by using the estimate_richness function in the R package ‘phyloseq’108. The beta-diversity index, calculated according to the Bray–Curtis distance of genus-level data, was generated by using the vegdist function in the R package ‘vegan’109. Principal coordinate analysis (PCoA) was performed by using the dudi.pco function in the R package ‘ade4’110, and the PCoA figure was created by using the R package ‘ggplot2’111. Covariates of gut microbiome beta-diversity variation were identified by calculating the association between continuous or categorical phenotypes and the coordinates of genus-level communities with the envfit function in the ‘vegan’ R package. Multiple-regression analysis by forward selection and single regression analysis was used to identify the gut microbiota associated with BMI (i.e., lm function and step function in the R package ‘stats’). Multiple logistic regression analysis by forward selection and single logistic regression analysis were used to identify the gut microbiota associated with T2DM (i.e., glm function and step function in the R package ‘stats’). Heatmaps were created by using the R packages ‘corrplot’ (https://github.com/taiyun/corrplot) and ‘superheat’112. The area under the curve was calculated by using Prism 7 (GraphPad Software, La Jolla, California, USA). Statistical significance was evaluated through one-way ANOVA or two-way ANOVA for comparison of multiple groups and the Mann–Whitney U-test for two groups by using Prism 7 or R package. A P value less than 0.05 was considered to be significant.
No statistical method was used to predetermine sample size. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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