Metagenomics analysis in the Swedish IGT cohort
The rarefied abundance levels of metagenomics species (CAG00017), annotate as Intestinimonas butyriciproducens, and 33 KEGG orthologies (KOs) involved in the fructoselysine metabolism were obtained from a previous study aiming to characterize the gut microbial changes in prediabetes and diabetes based on the Swedish IGT cohort (n=1,011)[16]. The relative differences of IB were then compared across individuals with distinct glucose intolerance levels versus the healthy control group. To examine the potential importance of fructoselysine metabolism to glucose intolerance, the relative abundances of each KO and/or the whole pathway (based on aggregated sum values of all KOs) were associated with 12 common clinical variables, such as the levels of fasting glucose, insulin, HbA1c, and triglycerides, indicative of the T2D status, respectively. The study was approved by the IRB of Sahlgrenska Hospital, Gothenburg University and all subjects provided written informed consent.
Isolation of Intestinimonas from human stool
Fresh fecal samples were collected in 15 ml falcon tubes containing anaerobic phosphate buffer (pH7) and later stored in 25% glycerol in 5 ml anaerobic bottles kept at -80 oC freezer. 0.5 ml of these fecal slurries was transferred to 10 ml anaerobic bicarbonate-buffered mineral salt medium (CP medium) containing 40mM lysine as energy and carbon source to enrich lysine-fermenting bacteria. The headspace was filled with CO2/N2 (1:4) at 1.5 atm and incubation was at 37oC. Subsequently, the enrichment cultures were transferred two more times in the same medium before being plated on YCFA agar medium containing 40 mM lysine as substrate (YCFA_L). Single colonies were picked and plated at least 3 times on the same medium which resulted in an axenic culture. The purity of the strains, designated as strain GL3, AS-BT and IY4 was confirmed by 16S rRNA gene sequencing and microscopy. The strains were routinely maintained in YCFA_L medium at 37 °C. 16S gene sequences of three isolates and strain AF211 were aligned with the multiple sequence aligner SINA [60] and merged with the Silva SSU Ref database (release 111). A phylogenetic tree of three isolates and Intestinimonas AF211 and closely related strains was constructed in the ARB software package (v. 6) by an algorithm [61].
Enrichment medium was done in anaerobic bicarbonate-buffered mineral salt medium (CP medium) [62] consisting of (l −1): 0.53 g Na2HPO4 . 2H2O, 0.41 g KH2PO4, 0.3 g NH4Cl, 0.11 g CaCl2 . 2H2O, 0.10 g MgCl2 . 6H2O, 0.3 g NaCl, 4.0 g NaHCO3 and 0.48 g Na2S . 9H2O as well as alkaline and acid trace elements (each 1 ml l −1) and vitamins (0.2 ml l −1) [62]. The alkaline trace element solution contained the following (mM): 0.1 Na2SeO3, 0.1 Na2WO4, 0.1 Na2MoO4 and 10 NaOH. The acid trace element solution was composed of the following (mM): 7.5 FeCl2, 1 H3BO4, 0.5 ZnCl2, 0.1 CuCl2, 0.5 MnCl2, 0.5 CoCl2, 0.1 NiCl2 and 50 HCl. The vitamin solution had the following composition (g l −1): 0.02 biotin, 0.2 niacin, 0.5 pyridoxine, 0.1 riboflavin, 0.2 thiamine, 0.1 cyanocobalamin, 0.1 p-aminobenzoic acid and 0.1 pantothenic acid.
YCFA medium (l −1): 10 g soy peptone, 10 g yeast extract, 4 g NaHCO3, 2.7g sodium acetate, 4.5g K2HPO4, 0.7g KH2PO4, 0.9 g NH4Cl, 0.9 g NaCl, 0.09 MgSO4, 0.09 CaCl2, 1 ml vitamin solution (1mg biotin, 1mg cobalamin, 3mg PABA, 5mg folic acid, 15mg pyridoxamine in 100 ml H2O), 1ml resazurine (0.5 g/l), 0.5g L-cysteine. In case of agar medium, 10g noble agar (DIFCO) was added to YCFA liquid before autoclave. The agar medium was then brought inside an anaerobic chamber and poured on agar plates. Those plates were then left slightly open for 30min till the agar got dried. The plates were kept for a maximum a week in the chamber before use. All streaking and plating were performed in the anaerobic chamber while the plate incubation was done in anaerobic jars filled with N2/CO2 in the gas phase by a gas exchange machine.
Genome sequencing and fructoselysine pathway gene analysis
Strain GL3, IY4 and AS-BT were cultivated in 50ml YCFA_L liquid medium for an overnight at 37oC. The bacterial cells were harvested at the late exponential phase by centrifuging at 4700 rpm at 4oC. The cell pellets were used for DNA extraction using MasterPure™ Gram Positive DNA Purification Kit (Epicentre) according to the manufacturer’s instructions. After checking the quality on a Nanodrop, 30µl of high-quality DNA solution were send in dry ice to GATC for draft genomes using Illumina sequencing technology. Draft genome assemblies were constructed using the MyPro assembly pipeline [63]. Raw reads were quality checked using FastQC [64]. Reads were trimmed and subsampled to a total coverage of 100X (50X for forward reads, 50X for reverse reads), then assembled using 4 different assembly tools: VelvetOptimiser [65], Edena [66], SOAPdenovo [67] and SPAdes [68]. The resulting contigs were ordered with r2cat [69] using the Intestinimonas butyriciproducens AF211 genome (CP011307) as a reference and overlapping contigs merged resulting in the final genome assembly. Genome assemblies were then annotated using RAST [70]. The annotation was done by RAST server [70]. Functional prediction of proteins was verified manually by BLASTing the amino acid sequences in Pfam [71], Brenda, Interpro [72] and Uniprot databases. In addition, the screening of antibiotic resistance genes was done using ABRICATE against the NCBI, ARG-ANNOT, ResFinder and VFDB databases.
Fructoselysine growth experiment
Conversion of fructoselysine was tested in CP medium containing 5 mM fructoselysine (provided by TRC, North York, Canada). The inoculum was 2.5% from active cultures of strain GL3, IY4, AS-BT and AF211 for the growth test. All strains were pre-cultured in YCFA containing 20mM lysine. The bacterial cultures were sampled during the growth up to 48 hours for substrate and end metabolite measurements as described below.
Antibiotic resistance profile
The E-test was done to identify minimal inhibitory concentrations (MICs) according to the manufacturer's protocol (bioMérieux, France). Both strains were pre-grown in RCM broth (overnight cultures) and 50 µl was spread on RCM agar plates (1.5% w/v agar) until the agar surface was dry and the liquid was absorbed by the agar. Two E-test strips were used per antibiotic and considered as duplicates. Antibiotics tested included ciprofloxacin, cefotaxime, erythromycin, oxacillin, teicoplanin, tetracycline, tobramycin, vancomycin and sulfamethoxazole. The concentration range was 0.016–256 µg/ml for chloramphenicol, oxacillin, tetracycline, tobramycin and vancomycin, and 0.016–32 µg/ml for ciprofloxacin, cefotaxime, erythromycin, teicoplanin and sulfamethoxazole. MIC values were recorded directly from the strips after 24 h, 48 h and rechecked after 4 days.
As tetW gene was detected in the genomes of most Intestinimonas strains and some of Intestinimonas strains were found to be resistant in erythromycin from the Etest, tetracycline and erythromycin were selected to perform the MIC test in liquid according to EFSA guideline. The test was done in 10ml YCFA medium containing lysine as substrate in anaerobic bottles filled with CO2/N2 (1:4) at 1.5 atm. The concentration of tetracycline was 2-fold reduction in each bottle from 256µg/ml to 1µg/ml. The 256 ug/ml tetracycline bottle was prepared in 20 ml growth medium by adding 1ml of tetracycline filter sterilized stock solution (5.12mg/ml) to 19 ml complete medium. This medium was then serial diluted two-fold to get concentrations of 128, 64, 32, 16, 8, 4, 2 and 1 ug/ml. All these bottles were inoculated 2% with an overnight culture. The growth was monitored by OD measurement at 24h, 48h, 72h and 96h. Bottles without tetracycline were used as a positive control. MIC test for erythromycin in liquid was done in the same way by replacing tetracycline by erythromycin.
Animal studies
All animal studies were approved by the Institute Ethical Committee. C57BL6/J male mice were purchased from Charles River at the age of 4 weeks, fed a regular chow diet and kept under regular 12h/12h light/dark cycles. To determine the impact of IB on whole-body metabolism, male mice were randomized in 4 groups (N=12) receiving 3 times/week 2x109 CFU Intestinimonas butyriciproducens GL3 or placebo solution (anaerobic PBS) by oral gavage and fed ad libitum a low-fat diet (LFD, 10%kcal from fat, Research Diets, D12450Ji) or high-fat diet (HFD, 60%Kcal from fat, Research Diets, D12492i). Mice were fed LFD/HFD for 13 weeks and IB/placebo supplementation started one week before switching to special diets. To avoid cage-effects on the microbiota composition, 3 mice were housed in one cage. Body weight and food consumption were monitored once a week. At the end of the study, insulin-tolerance test (ITT) was performed in obese mice on HFD: after 6 hour-fasting, mice received an intraperitoneal injection of insulin (0.5U/kg); blood glucose levels were assessed by tail prick using glucometer strips at 0, 15, 30, 60 minutes post-injection. To avoid unnecessary discomfort and suffering due to potential hypoglycemic events, lean mice fed a LFD were not subjected to ITT. Mice were sacrificed under anesthesia (5% isoflurane, O2 flow of 2 L/minute), blood was collected by cardiac puncture, harvested organs were stored in formalin (for later paraffin-embedding) and snap-frozen in liquid-nitrogen. White-adipose tissues were weighed immediately after collection.
Protein bound fructoselysine in high fat diet and low fat diet
Protein bound fructoselysine in the two diet was indirectly quantified through furosine concentration according to the method of Troise et al. [73] by using a Nexera U-HPLC system coupled with a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan). In brief, a 0.5 g aliquot of each chow was added to 4 mL of HCl (7.4 M) and then incubated at 110 °C, for 20 h. After filtering, 400 µL of the hydrolysate suspension was dried under vacuum using a rotary evaporator and reconstituted in 400 µL of 80% aqueous acetonitrile along with d4-furosine as internal standard (final concentration 200 µg/l). For separation of furosine and its internal standard, a core-shell Kinetex HILIC column (2.6 µm, 2.1 mm × 100 mm, Phenomenex) thermostated at 30 °C, with a flow rate of 0.4 mL/min was used. The mobile phases consisted of 0.1% formic acid (solvent A), 0.1% formic acid in acetonitrile (solvent B), and 50 mmol/L ammonium formate (solvent C). The gradient was as follows (t in [min]/[%B]): (0.0/80), (3.5/40), (6.5/40), with 4.5 min for equilibration, while solvent C was kept at 10%. Positive ionization multiple reaction monitoring (MRM) mode was used; the spray voltage was 4.0 kV and the collision energies (CE) were the following (in bold quantifier ions for furosine): furosine (m/z 255.3 à130, 84, CE: 12 and 18), furosine-d4 (m/z 259 à 134, 88 CE: 12 and 15). Profile data were acquired and analyzed through LabSolutions (Shimadzu Corporation).
Targeted liquid chromatography tandem mass spectrometry of mouse cecum and plasma samples
Fructoselysine, lysine and SCFAs in CP medium, mice caecum and mice plasma were analysed by liquid chromatography high resolution tandem mass spectrometry (LC-MS/MS) by means of a Vanquish Core LC system interfaced to an Exploris 120, hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Because of target analytes concentration and matrix effect, SCFA in mice plasma underwent a different procedure including 3-nitrophenylhydrazine (3-NPH) derivatization. For fructoselysine and lysine quantification in CP medium, samples were diluted in a solution acetonitrile/water (50:50, v/v) according to the linearity range used for calibration curve. For fructoselysine quantification in caecum and plasma, analytical protocol was adapted from Wolf et al. [7], with minor modifications. Briefly, 20 µL of plasma or 20 µL of caecum supernatants were diluted in ice cold methanol (ratio 1:3). Suspensions were centrifuged at 12700 rpm for 10 min, 4°C and 50 µL were dried under vacuum in a centrifugal evaporator (Savant, Thermo Fisher Scientific). Dried samples were resuspended in a solution consisting of 50% acetonitrile in water. Lysine and its Amadori compound were separated at 35°C through a zwitterionic sulfobetaine column (Atlantis Premier BEH, Z-HILIC, 100 x 2.1, 1.7 µm, Waters, Etten-Leur, the Netherlands) with the following gradient of solvent B (minutes/%B): (0/5), (1/5), (2/50), (6/50). Mobile phases consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B) and the flow rate was 0.2 mL/min. Heated electrospray (H-ESI) interface parameters were as follows: static spray voltage 3.3 kV, ion transfer tube and vaporizer temperature were both at 280 °C; sheath gas flow and auxiliary gas flow were 30 and 5 arbitrary units. The analyzer resolution was set at 60000 (FWHM at m/z 200), fructoselysine and lysine were identified and quantified in product ion scan positive mode screening the precursor ions (C12H24N2O7 [M+H]+ 309.1656, for fructoselysine and C6H14N2O2 [M+H]+ 147.1128 for lysine) with an increasing normalized collision energies set at 25, 50 and 60% to improve fragmentation pattern and screening the product ions, monitoring for both fructoselysine and lysine the characteristic fragment ion at m/z 84.0808. For product ion scan mode, Orbitrap resolution was set at 15000 (FWHM at m/z 200) and the quadrupole resolution was set at 1. A linear calibration curve was built in the range 100-10000 nM and concentration reported in mM was measured through standard addition technique by using CP medium, caecum or plasma as blank samples.
For acetate, butyrate and isobutyrate separation in CP growth experiments, supernatants were directly diluted in o-phosphoric acid (0.5 % final concentration in water, 1:10, v:v), while for mice caecum content, samples were centrifuged at 4 °C, 12000 rpm for 15 min and supernatants diluted 1:10 v/v in 0.5% o-phosphoric acid. Samples were centrifuged at 12000 rpm before transferring clear supernatants to glass vial. Analytes were separated through a graphite column thermostated at 40°C (Hypercarb, 100 x 1.0, 1.7 µm, Thermo Fisher Scientific) with the following gradient of solvent B (minutes/%B): (0/0), (2/0), (6/75), (8/75). Mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) and the flow rate was 0.1 mL/min. H-ESI parameters were as follows: static spray voltage 3.2 kV, ion transfer tube and vaporizer temperature were both at 280 °C; sheath gas flow and auxiliary gas flow were 35 and 7 arbitrary units. The analyzer resolution was set at 60000 (FWHM at m/z 200), working in the scan range 50-350. Acetate, butyrate and isobutyrate were preliminary identified in full MS to evaluate effective separation of the two C4:0 isomers. Acetate and butyrate were quantified in full MS scan positive ion mode screening the two precursor ions (C2H4O2 [M+H]+ 61.0284 and C4H8O2 [M+H]+ 89.0597) with a mass accuracy below 3 ppm. A linear calibration curve was built in the range 0.5-10 mM by using acetate and butyrate as internal standard and concentration reported in mM.
For fructoselysine and SCFA analytical procedures in product ion scan mode and in full MS acquisition mode, profile data were collected using Xcalibur 4.5 (Thermo Fisher Scientific, Waltham, MA) and fragmentation spectra were recorded by using Free Style software (v. 1.8, Thermo Fisher Scientific, Waltham, MA). EASY-IC with fluoranthene in positive ion mode (m/z 202.0777 [M]+) was used to improve mass accuracy in both full scan and product ion scan mode. Analytical performance robustness, sensitivity, reproducibility, repeatability, linearity, accuracy, carry over and matrix effects were evaluated by following the procedures previously reported by Troise and coworkers through authentic analytical standard according to an in-house procedure developed in Trace Finder environment (v. 5.1, Thermo Fisher Scientific, Waltham, MA) encompassing identification of isotopic distribution, elemental composition, mass accuracy below 3 ppm for precursors and product ions and number of scan points higher than 8.
SCFA concentration in mouse plasma samples were quantified according to the procedure detailed by Garcia-Rivera et al [74] with minor modifications. Briefly, 10 µL of plasma were spiked with 1 µL of SCFA internal standard mix including 13C2-acetate, 13C3-propionate and 13C4-butyrate (final concentration 0.1 mM for each carbon labelled compound). Upon protein precipitation with the addition of 60 µL of 75:25 methanol: water (v/v) solution, samples were mixed with 60 µL of 3-NPH (200 mM) and 10 µL of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, 120 mM in 6% pyridine). Samples were incubated at room temperature (22°C) for 45 min under gentle shaking in an orbital shaker. Derivatization reaction was stopped upon the addition of 10 µL quinic acid (200 mM) and incubation under shaking at room temperature for 15 min. Samples were centrifuged at 15000 rpm for 5 min at 4°C and supernatants diluted up to 1 mL with 10:90 methanol:water solution (v/v). Before transferring to glass vial, samples were centrifuged again at 4°C, 5 min, 15000 rpm. Hydrazone derivative quantitation was achieved by a U-HPLC system (Ultimate 3000 RS, Thermo Fisher Scientific) interfaced to a linear ion trap hybrid Orbitrap high resolution mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scinetific). Chromatographic separation included a reversed phase C18 column thermostated at 40°C (Kinetex C18 PS, 100 x 2.1 mm, 2.6 µm, Phenomenex, Torrance, CA) with the following gradient of solvent B (minutes/%B): (0/5), (5/5), (12.3/35), (13.3/85), (14/99), (16/99). Mobile phases consisted of water (solvent A) and acetonitrile (solvent B) and the flow rate was 0.2 mL/min. LC stream was interfaced to an electrospray ion source (ESI) working in negative ion mode scanning the ion in the m/z range 100-400; resolution was set at 30000 (FWHM at m/z 200), capillary temperature was 300°C, while sheath and auxiliary gases were set at 25 and 15 arbitrary units. Analytes profile data in full MS mode were collected using Xcalibur 2.1 (Thermo Fisher Scientific). Calibration curve was performed by internal standard technique in the linearity range 1-1000 µM by using the same procedure detailed above for plasma samples. Analytical performances for the three procedures are detailed in Supplementary Table 5.
RNA isolation
RNA was isolated from iWAT tissues, which were stored at -80˚C until analysis, using standard RNA isolation protocol. In short, biopsies were mixed with 1ml TriPure (Roche) and homogenized using a ceramic beads homogenizer. After adding 0.2ml chloroform to 1ml Tripure solution, samples were centrifuged (15 min, 12000 x g, 4˚C). The aqueous phase was transferred and mixed with 0.5ml isopropanol and centrifuged (15 min, 12000 x g, 4˚C). afterwards the pellets were resuspended in 1ml of 70% ethanol and centrifuged (15 min, 7500 x g, 4˚C). RNA was eluted in 20 µl RNAse free water. RNA concentrations were measured using the NanoDrop 1000 (Thermo Scientific).
Real Time quantitative polymerase chain reaction (RT-qPCR)
1mg of RNA was converted to cDNA with SensiFAST cDNA synthesis kit (Meridian Bioscience) according to the manufacturer’s instructions. qPCR was performed on a CFX Opus 384 PCR machine (BioRad) using SensiFAST SYBR No-ROX Green (Meridian Bioscience).Gene expression was normalized with the expression of the housekeeping gene Hprt; relative gene expression was calculated with the “delta Ct” method and shown as 2ˆ- delta Ct. All primers were manufactured by Sigma-Aldrich; primer sequences are provided in Supplementary Table 6.
Western blotting
iWAT tissues were lysated in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (cOmplete™ Protease inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails, Sigma) using a ceramic beads homogenizer. After centrifugation, the lipid layer was removed and protein concentration was determined by BCA assay (Thermo Fisher Scientific). b-mercaptoethanol was added as a reducing agent to sample lysates. Samples were loaded on 4-12% NuPage Bis-Tris polyacrylamide gels (Invitrogen). Proteins were transferred to PVDF membranes (BioRad), which were blocked in 5% milk in TBS-T (Tris Buffered Saline – Tween-20). Membranes were incubated overnight a 4˚C with primary antibodies: rabbit antibodies anti-IRS2 (insulin receptor substrate 2, L1326, Cell Signaling, 1:1000) and mouse anti-beta-actin (Genetex, 1:10000). Horseradish peroxidase (HRP)-conjugated secondary antibodies (polyclonal goat anti-rabbit IgG or monoclonal goat anti-mouse IgG, Dako, 1:3000) were incubated for 1 hour at room temperature. HRP activity was visualized with peroxidase substrate for enhanced chemiluminescence and imaged with ChemiDoc MP Imaging System (BioRad). Densitometric quantification analysis was performed using the Image J software. All protein levels were normalized to the loading control (b-actin).
ELISA
Sections of frozen iWAT tissues were cut and homogenized in 400 µl RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (cOmplete™ Protease inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails, Sigma) using a ceramic beads homogenizer. Protein concentration was determined with BCA assay (Thermo Fisher Scientific). The tissue concentrations of TNF-α, IL-6 and IL-1β cytokines were quantified using respectively the mouse TNFalpha/IL-6 DuoSet ELISA (R&D Systems) and ELISA MAX™ Deluxe Set Mouse IL-1β (BioLegend) according to the manufacturers’ protocol. The absorbance was measured at an optical density (OD) of 450 nm and 570 nm using the Tunable Microplate Reader VersaMax (Molecular Devices, USA). The cytokine levels were normalized for protein concentrations.
Histology and Immunohistochemical staining
Tissues were fixed in 10% buffered formalin overnight, dehydrated in 70% ethanol and embedded in paraffin. Sections of 4 µm were cut and stained for macrophage marker F4/80. Formalin-fixed paraffin-embedded (FFPE) sections were deparaffinized in 100% xylene and rehydrated in ethanol (100%, 96% and 70%) and H2O, followed by block of endogenous peroxidase in 3% H2O2 methanol for 20 minutes and heat-induced epitope retrieval (HIER) in citrate buffer pH 6.0 at 100 °C for 10 min. iWAT sections were incubated with the following antibodies: FITC anti-mouse F480 (BioLegend) diluted 1:5000 for 2 hours at room temperature, rabbit anti-FITC (Bio-Rad) diluted 1:1000 for 1 hour at room temperature, finally BrightVision Poly-HRP-conjugates goat anti-rabbit IgG (ImmunoLogic) diluted 1:2 for 30 minutes at room temperature. Staining was visualized with 3,3’Diaminobenzidine (DAB) kit (Sigma Aldrich) and counterstaining was performed using hematoxylin. Per sections, ten pictures were captured at random using a Leica MC170 HD stand-alone microscope camera (Danaher Corporation, USA). Subsequently, analysis of the digital images was conducted using Image-J software.
Statistical analysis of the in vivo data
Statistical differences between the placebo and IB-treated groups were assessed using Mann Whitney test, and the differences were considered statistically significant with P values < 0.05.