Animal studies

Donor mice: C57BL/6J (B6) or lean mice and leptin receptor knock-out (db/db) mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA) and were acclimatised for 2 weeks in our vivarium by maintaining under 12 hours light-dark cycle before the start of experiments or collection of stools. At the age of 4 weeks, the mice (n=5) were divided into four groups- (1) B6-NC (lean) fed with normal chow, (2) B6-LFD-fed with a LFD (10% kcal fat, Research Diets Inc, New Brunswick, USA, NJ), (3) db/db- fed with normal chow and (4) DIO- B6 mice fed with HFD (60% kcal fat, Research Diets, New Brunswick, New Jersey, USA). When mice were 16 weeks old, faecal samples were collected from each group for microbiome, metabolite, FMTs, CMTs and FCM studies. For FMT and CMT studies, B6 mice (age of 8 weeks) were divided into three groups (n=5–8 in each group): (1) lean-FMT/CMT, (2) db/db-FMT/CMT and (3) DIO-FMT/CMT), and, after gut cleansing using antibiotics and polyethylene glycol (as published and described earlier by us and others41–43) recipient mice were administered lean, db/db and DIO faecal/cecal slurry, respectively, for 7 days. Daily body weight, food and water intake data were collected, and stool collection and gut permeability, MTT, and ITT assays were done during/after treatments. For ethanolamine administration studies, 8–10 week old B6/lean mice (n=5–8 per group) were treated daily with ethanolamine (1 gm/kg body weight) for up to 7 days by oral gavage; data and samples were collected at the indicated time points. For the MSD feeding study, we fed 8–10 weeks-old mice (n=6–9) with MSD compared with an isocaloric control diet (online supplemental table 8a,b) for 2 weeks and collected data and samples. For lentivirus transfection studies in mice, the lentiviral particle carrying mimetic-miR-101a-3p was administered through enema twice a week in B6 mice and compared with scrambled miRNA lentivirus ingested controls (n=5 in each group). Healthy B6 mice were anaesthetised with 3.75% isofluorane and were administered with Lentivirus carrying miR-101a-3p inhibitor, mimetics or scrambled miRNA sequence through an enema. Mice were given an enema of 100 µL 50% ethanol (v/v in ddH₂O), and then, after 2 hours, 100 µL of a vehicle or viral vector solution containing a titre of 0.5×10⁸ transducing units (TU) administered intrarectally through a 1.2 mm diameter catheter. The mice were inverted for 30 s after administration of the enema to prevent leakage. After measuring gut permeability, mice were euthanised to collect tissues for further analyses. For probiotic therapy experiments, the B6 mice were also treated with ethanolamine (1 gm/kg body weight) and probiotics L. rhamnosus HL-200 (1×10¹¹ CFUs/mice/day) or the combination for 7 days and compared with PBS-only administered controls. Also, MSD with or without probiotics HL-200 were fed to B6 mice (n=9/group) for 1 week and compared with control diet-fed controls. All the animal experiments and procedures were approved by the IACUC of Wake Forest School of Medicine and the University of South Florida.

Measurements of body weight, food and water intakes

Body weight was measured daily or weekly using a vivarium-dedicated electronic balance. Food and water intakes were also measured daily by subtracting the amount of food/ water from the leftover amount per day.

Blood glucose, serum insulin and HOMA-IR measurements

Blood glucose was measured using EvenCare ProView Blood Glucose metre (Medline Industries, Illinois, USA). Insulin was measured using ELISA kits (Alpco, New Hampshire, USA). HOMA-IR was calculated using the formula (fasting insulin (μU/L) × fasting glucose (nmol/L)/22.55).

MTT and ITT assays

MTT was done in 10 hours fasted mice by administering oral gavage of 200 µL meal solution/mouse (2 g/kg body weight containing the solution of 570 mg protein (Ensure Original nutrition powder (Abbott) and 30 mg of dextrose in 1.8 mL of cell culture grade PBS) and blood glucose levels were measured using an EvenCare blood glucometer at 0 (before), 15, 30, 60 and 120 min after meal ingestion. For ITT, 4–6 hours fasted mice were given intraperitoneal injections of 0.8 U/kg body weight of insulin (Humulin R, Eli Lilly, Indiana, USA) and blood glucose was measured at 0, 15, 30, 60 and 120 min after insulin administration.

Gut permeability assay in mice

Four hours prefasted mice were orally treated with 1 g/kg body weight FITC-dextran-4kDa, 40 kDa, LPS conjugate (4 kDa, 40 kDa and LPS from Escherichia coli O55:B5, Sigma, Missouri, USA), and blood was collected after 4 hours to take the readings. Serum was isolated from blood to measure the appearance of FITC fluorescence at 485 nm excitation and 530 nm emission, using 96-well plate reader (POLARstar Omega, BMG Labtech, North Carolina, USA), which was calculated using FITC-dextran standard curve as described by us.42

Gut permeability, endotoxaemia and microbial translocation markers in serum

Systemic gut permeability markers such as LBP (Hycult Biotech, Pennsylvania, USA) and sCD14 (R&D Systems, Minnesota, USA) were measured in serum using ELISA kits. The endotoxin quantity in the systemic circulation using the Pierce Chromogenic Endotoxin Quantitation Kit (Pierce Biotechnology, Illinois, USA), following the manufacturer’s instructions. Further, proinflammatory activity of leaked LPS in serum was measured by using the secreted embryonic alkaline phosphate (SEAP) reporter activity in HEK-Blue mTLR4 cells (InvivoGen, California, USA), as described elsewhere.44 HEK-Blue mTLR4 cells were cultured in DMEM supplemented media with 10% FBS and selective antibiotics (InvivoGen). HEK-Blue mTLR4 cells were seeded (50 000 cells/well) in 96-well plates in 100 µL of HEK-Blue detection medium. The HEK-Blue mTLR4 cells were treated with 5% mouse serum and incubated for 24 hours at 37°C, 5% CO₂. SEAP activity, an indicator of TLR4 activity, was analysed based on spectrophotometric absorbance at 650 nm in microplate reader with appropriate standard. In addition, the bacterial DNA in serum was extracted and enriched by using the QIAamp DNA microbiome kit (Qiagen, Maryland, USA) following the manufacturer’s instructions. The microbial DNA V3–V4 region was amplified using the Bact-0341F (5’- CCTACGGGNGGCWGCAG-3’) and Bact-0805R (5’- GACTACHVGGGTATCTAATCC-3’) primers. All the reactions were carried out at least in triplicates.

Microbiota analyses

Genomic DNA was extracted from mouse faeces using the Qiagen DNA Stool Mini Kit (Qiagen), and the V4 region of bacterial 16S rDNA was amplified using primers 515 F (barcoded) and 806 R.45 The amplified DNA was purified and quantified with AMPure magnetic purification beads (Agencourt, Beckman Coulter, California, USA) and Qubit-3 fluorimeter (Thermo Fisher Scientific, Massachusetts, USA), respectively. Equal amounts (8 pM) of the amplicons were applied for sequencing using the Illumina MiSeq sequencer (Miseq reagent kit V.3, Illumina, California, USA). The sequences were demultiplexed, quality filtered, clustered and analysed with the Quantitative Insights into Microbial Ecology and R-based analytical tools as we published earlier.42 45–47

Metabolomics analysis

Global metabolomics was performed using NMR spectrometry in the faecal samples, using a method described by Gratton et al 48 with slight changes. The extracted aqueous samples were mixed with phosphate buffer containing 10% D₂O and 0.1 mM trimethylsilyl propionate (TSP). NMR experiments were carried out on a Bruker Ascend 400 MHz high-resolution NMR (Bruker Biospin, Ettlingen, Germany) using a 1D first increment of a NOESY (noesygppr1d) with water suppression and a 4 s recycle delay. All NMR spectra were phased and referenced to TSP in TopSpin V.4.06 (Bruker BioSpin, Ettlingen, Germany). The NMR spectra were analysed in Amix V.4.02, and a manual pattern was created using the metabolites peaks range determined by Chenomx V.8.4 (Chenomx, Edmonton, Alberta, Canada) to extract the metabolite peak intensities. Total intensity normalisation was applied before further data analysis.

Ethanolamine operon quantification

Genomic DNA was extracted from faeces and bacterial cells using the Qiagen DNA Stool Mini Kit (Qiagen). After normalising an equal amount of DNA, the qRT-PCR analyses were performed using PowerUp SYBR Green master mix (ThermoFisher Scientific) and ethanolamine-metabolising operon genes (online supplemental table 9). The gene expression was calculated using the ^ΔΔCT method while normalised with 16S rRNA as an internal control.

Enteroids development and treatments

To develop mouse enteroids, the entire small intestine was collected from mice and flushed with precooled Dulbecco’s phosphate-buffered saline (DPBS, Lonza, Maryland, USA). The lengthwise cut-opened ileum was washed with cooled PBS and fragmented into 2 mm pieces, then transferred to a 50 mL conical flask containing cleaned precooled PBS. Tissue fragments were incubated in 25 mL of prewarmed Trypsin (Gibco, New York, USA) on a rocking platform for 15 min. After removing the trypsin, tissue pieces were washed with 10 mL precold PBS with 0.1% bovine serum albumin (BSA from Fisher Scientific) and filtered in a 50 mL conical tube through a 70 µm cell strainer (Corning, North Carolina, USA). These pieces were then centrifuged at 290 × g for 5 min at 4°C. The intestinal crypt-containing pellets were suspended in the 10 mL cold DMEM: F-12 (Gibco) and centrifuged at 500 × g for 10 min, followed by resuspending in 150 µL IntestiCult Organoid Growth Medium (StemCell Technology, Canada) with 50 µg/ mL gentamicin (Gibco). The Matrigel Matrix (Thermo Fisher Scientific) was added to the suspension, and the mixture of 50 µL was used to form a dome at the centre of a prewarmed 24-well culture plate and incubated at 37°C and 5% CO₂ for 30 min to allow the Matrigel to set. To maintain the cultures, the IntestiCult Organoid Growth Medium was changed three times per week. On day 10, the enteroids were treated with faecal condition media (FCM), metabolites and ethanolamine, with three replicates in each group. The enteroids were harvested after 2 days of treatment(s) for miRNA, mRNA and protein analyses. Experiments were repeated 2–3 times.

FCM preparation

Fresh faeces collected from mice were snap-frozen in liquid nitrogen and crushed using a mortar and pestle. The finely powdered faeces (100 mg) were suspended in 100 mL cold DMEM (Gibco) media and kept on a shaker at a speed of 200 rpm for 1 hour to mix properly in a cold room. The suspended media was filtered twice through 0.45 µm filters (Corning), and twice again through 0.22 µm sterile syringe filters in sterile conditions. The 1:40 diluted FCM was used to treat the intestinal enteroids and cells.

TEER assay in Caco-2 cell monolayers

Human intestinal epithelial Caco-2 cells (ATCC, Virginia, USA) were seeded in 12-well transwell plates containing an apical chamber made of polyester membrane filters with 0.4 µm pore size (Corning) at a density of 3×10⁵/well. The culture medium from both the apical and basolateral compartments was changed every 2 days. The cells are allowed to fully differentiate for 21 days, and fully differentiated cells with no leakage tested were challenged with FCM and metabolites for the next 8 hours with continuous measuring of TEER values, using an EVOM² Epithelial Voltmeter (WPI, Florida, USA). The blank inert resistance value (the insert with only culture media) was subtracted from the measured resistance value of each sample, and the final resistance in ohm × cm² was calculated by multiplying the sample resistance by the area of the membrane.

FITC-dextran permeability assay in Caco-2 cell monolayers

Fully differentiated Caco-2 cells up to 21 days, the FITC-dextran 4 (4 kDa; Sigma) solution (1 mg/mL) was added to the apical (upper) side of the monolayers along with treatments of corresponding FCM and metabolites. The basolateral side media was collected to determine the FITC levels using the fluorescent reader and standard curve.42

Western blots

Total proteins from tissues, enteroids and cells were extracted using homogenised lysis buffer, as mentioned in our previous publications.42 Proteins were resolved by SDS-PAGE electrophoresis and transferred to the PVDF membrane for Western blotting. Membranes were developed with primary antibodies zona occludins-1 (Zo1), Invitrogen) and Arid3a (Santa Cruz Biotechnology, Texas, USA), followed by secondary antibodies and developed with a chemiluminescent kit (ECL, Thermo Scientific) and imaged on PXi with the GeneSys software (SynGene, Maryland, USA). Tubulin was used as the internal loading control.

RNA isolation and gene expression analyses

Total and small RNAs were isolated from tissue, enteroids and cells collected and stored in RNAlater solution (Ambion, ThermoFisher) at −80°C using RNeasy Mini Kit (Qiagen). Total RNA from bacterial cells were extracted using RNAprotect bacteria reagent and RNeasy Mini Kit (Qiagen). The complementary DNA (cDNA) was synthesised from total and small RNAs using High-Capacity cDNA reverse transcription kit (Thermo Fisher Scientific). The normalised cDNA of each sample was used to run the qRT-PCR using a 7900 real-time PCR machine (Thermo Fisher Scientific) using SYBR Green master mix (Thermo Fisher Scientific) and gene-specific primers (online supplemental tables 1 and 10). The relative gene expression rate was analysed using ^ΔΔCT method normalised by 18S as an internal control. All the reactions were performed at least in triplicates.

miRNA profiling and expression analyses

For miRNA profiling, the total small RNA was extracted from enteroids treated with FCMs of B6, db/db, and DIO, using miRNeasy Mini kit (Qiagen) and analysed by the NanoString nCounter miRNA Assays, using 50–100 ng of total RNA from each sample in four replicates, and using eight positive control probes and eight negative control probes. Data were analysed using nSolver analysis software V.4.0. Each miRNA background correction count was carried out by subtracting mean+2 SD of eight negative control probes as a cut-off. The miRNA of count 50 or more after background correction was further used for analysis. The individual miRNA expression was quantified, using qRT-PCR by converting small RNAs to cDNA using TaqMan miRNA Reverse Transcription kit (ThermoFisher) and TaqMan 2X Universal PCR Master Mix, No AmpErase UNG (ThermoFisher) on 7900 real-time PCR machine. The TaqMan primers were used for specific miRNAs, and RNU6B was used as an internal control as mentioned in online supplemental table 11; and relative expression was calculated using ^ΔΔCT method.

miRNA transfection to Caco-2 cells

Caco-2 cells were grown up to 80%–90% confluency and were transfected with miRIDIAN mimetic negative control #1, miRIDIAN mimetic mmu-miR-101a-3p and miRIDIAN hairpin inhibitor mmu-miR-101a-3p (Horizon Discovery, Pennsylvania, USA) as mentioned in online supplemental table 11, using Lipofectamine 3000 transfection kit (ThermoFisher). The cells were harvested after 48 hours for miRNA, mRNA and protein expression analyses.

Zo1-over-expression in Caco-2 cells

Caco-2 cells were grown up to 80%–90% confluency and were transfected with precision lentiORF RFP positive control viral particles and precision lentiORF TJP1 w/Stop codon viral particles as mentioned in online supplemental table 11 using DharmaFECT kb DNA transfection reagent kit (Horizon Discovery) and subjected for TEER and FITC-dextran assay. The cells were harvested after 48 hours for Zo1 mRNA expression analyses.

Promoter luciferase assay

Caco-2 cells seeded in a 96-well plate at a density of 2×10⁴ cells/ well were transfected with miR-101a-3p promoter pMK-RQ (KanR) reporter vector of 4 different nucleotide sequence (−1 to −500, −1 to −1000, −1 to −1500 and −1 to −2000 bps) from its TSS (transcription start site) and pMK-RQ (control vector) using Lipofectamine 3000 transfection kit (ThermoFisher) as mentioned in online supplemental table 11. The TSS of miR-101a-3p is located at position: chr1 65067704 (genome version hg38) as reported earlier.49 The cells were collected at various time points (0, 1, 3, 5, 7 and 10 hours) after transfection. The expression level of firefly luciferase reporter gene was measured using Pierce Firefly Luciferase Glow Assay kit.

mRNA stability assay

Caco-2 cells were transfected with mimetic miR-101a-3p and inhibitor miR-101a-3p, and cDNA was synthesised after harvesting the transfected cells at 0, 1, 3, 6, 12, 24 and 48 hours time points. The half-life of the Zo1 mRNA expression was calculated using a formula: E(t)=E₀×(1/2)^(t/t _1/2 ⁾, where ‘E(t)’ stands for expression of Zo1 at time point ‘t’ after transfection with mimetic miR-101a-3p and inhibitor miR-101a-3p, ‘E₀’ stands for the initial expression level of Zo1 before transfection, ‘t’ stands for the time elapsed after transfection, ‘t_1/2’ stands for the half-life in the Zo1 expression on transfection with mimetic miR-101a-3p and inhibitor miR-101a-3p.

Chromatin immunoprecipitation protein-DNA pulldown assay

Fully differentiated 21-day old Caco-2 cells were treated with 10 µM ethanolamine for 48 hours, and then washed with PBS buffer containing inhibitors (PBSI)- 0.5 mM PMSF (Phenylmethylsulphonyl fluoride), 1 mM sodium vanadate, 0.5 mM Dithiotreitol, 1 µg/mL Leupeptin, 25 mM β-glycerophosphate, 10 mM sodium fluoride, then were harvested and suspended in two package cell volume of buffer A with inhibitors (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1.5 mM magnesium chloride, 10 mM potassium chloride, 300 mM sucrose, 0.5% NP-40) and kept on ice for 10 min. Cells were pelleted by centrifuging at 2600 g for 30 s and resuspended in 2/3 package cell volume with buffer B containing inhibitors (20 mM HEPES, pH 7.9, 1.5 MgCl₂, 420 mM sodium chloride, 0.2 mM EDTA, 2.5% glycerol). After sonication and centrifugation at 10 400 g for 5 min, the supernatant was isovolumetrically diluted with buffer D-containing inhibitors (20 mM HEPES, pH 7.9, 100 mM potassium chloride, 0.2 mM EDTA, 8% glycerol). Total protein (400 µg) of nuclear extract was normalised among the samples, and two drops (40 µL) of streptavidin-agarose bead were suspended, and 4 µg of 5’-biotinylated-pmiR-101a-3p (−1000 to −500 bp promoter sequence of miR-101a-3p and scrambled sequence as control as mentioned in online supplemental table 11) in 500 µL of PBSI. After rocking this mixture for 2 hours and centrifuging at 550 g for 1 min, the pellet was washed with PBSI for three times and was resuspended in 40 µL of 2X Laemmli sample buffer and incubated for 95°C for 5 min to obtain beads.

Beads were placed onto the polyethylene filter in the Pierce 0.8 mL Centrifuge Columns (Thermo Scientific) and were washed three times by adding 200 µL of washing buffer (50 mM ammonium bicarbonate solution) and centrifugation at 1000 × g for 1 min for each time. The bottom end of the column was capped, and 200 µL of washing buffer containing 10 mM dithiothreitol solution was added. With the top capped, the column was agitated on a tube rotator for 1 hour at 37°C. The bottom cap was removed, and the tube was centrifuged at 1000 × g for 1 min to remove the supernatant. Beads were then incubated in 200 µL of 30 mM iodoacetamide solution for 45 min at room temperature in the dark. Beads were washed with 200 µL of washing buffer three times; further, 200 µL of digestion buffer (50 mM ammonium bicarbonate solution) was added, and the tube was incubated overnight at 37°C. The enzyme reaction was quenched by adding 10 µL of 20% formic acid, and the column was centrifuged to collect flow-through in the collection tube. Beads were washed with 100 µL of 50% acetonitrile containing 0.1% formic acid twice, and the flow-through was combined with the initial eluent. Beads were washed again with 80% acetonitrile containing 0.1% formic acid, and flow-through was collected in the same tube. The solution was dried under vacuum and then prepared in 5% acetonitrile with 1% formic acid and was injected in an liquid chromatography and tandem mass-spectrometry (LC-MS/MS) system consisting of an Orbitrap Velos Pro Mass Spectrometer (Thermo Scientific) and a Dionex Ultimate-3000 nano-UPLC system (Thermo Scientific). Peptides were separated on an Acclaim PepMap 100 (C18, 5 µm, 100 Å, 100 µm × 2 cm) trap column and an Acclaim PepMap RSLC (C18, 2 µm, 100 Å, 75 µm x 50 cm) analytical column employing a linear gradient consisting of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) where the gradient was from 5% B at 0 min to 40% B at 105 min. MS spectra were acquired by data-dependent scans consisting of MS/MS scans of the ten most intense ions from the full MS scan with a dynamic exclusion option, which was 30 s. To identify proteins, spectra were searched against the UniProt human protein FASTA database (20 258 annotated entries, Feb 2018), using the Sequest HT search engine with the Proteome Discoverer V.2.2 (Thermo Scientific). Search parameters were as follows: (1) FT-trap instrument; parent mass error tolerance, 10 ppm; fragment mass error tolerance, 0.6 Da (monoisotopic); enzyme, trypsin (full); number maximum missed cleavages and (2) variable modifications, +15.995 Da (oxidation) on methionine; static modification, +57.021 Da (carbamidomethyl) on cysteine.

Bacterial ethanolamine metabolisation screening

We screened 25 human-origin probiotics (5 Lactobacilli, 5 Streptococci, 9 Erysipelatoclostridia, 1 Clostridium, 5 Enterococci and 5 Bifidobacteria) strains for their ethanolamine-metabolising capabilities. These strains were characterised for their genetic identity by DNA sequencing and general safety profiles like devoid of a pathogenic island in the genome and antibiotic-resistant properties along with probiotic attributes such as antibacterial activities, bile acid tolerance and epithelial cell adhesion as published earlier.50 Bacterial streaks were grown for 48 hours in the MRS agar media plates containing 10 mM ethanolamine. Then plates were overlaid with 5 mL 500 mM ethanolamine MRS agar and incubated at 37°C for 1 hour, followed by adding 5 mL of 2,4-dinitrophenylhydrazine in each plate and further incubating for 3 min. Then the solution was discarded, and 5 mL of 5 M potassium hydroxide was added. Pink to purple zones were developed around the bacterial streaks, demonstrating the conversion of ethanolamine to acetaldehyde was quantified. The L. rhamnosus HL-200 (HL-200) probiotic was freshly cultured, counted and equal numbers (10⁹ cfu/mL) administered to ethanolamine and MSD-fed mice.

Human samples

The obese stool samples were obtained from Valued Epigenetics Glycaemic Improvements through Weight Loss study (ClinicalTrials.gov Identifier: NCT02869659), and normal weight control stool samples were acquired from Mediterranean Diet and the Gut Microbiome (ClinicalTrials.gov Identifier: NCT03269032). Stool samples were collected, processed and stored with the same protocol and kits from Dr. Yadav’s lab.

Statistical analyses

Different datasets were analysed by Student’s t-test and one/two-way analysis of variance. Alpha-diversity indices and bacterial abundance between the two groups were compared using an unpaired two-tailed t-test. Hierarchical clustering and heat-maps based on average linkage on Euclidean distance, depicting the patterns of abundance and log values were constructed within R V.6.0, using the ‘heatmap.2’, pheatmap and ‘ggplots’ packages. RFA and principal component analysis (PCA) were analysed in R programming V.6.0 using packages ‘randomForest’, ‘ggplot2’, ‘caret’, ‘psych’, ‘ggbiplots’, ‘nnet’ and ‘devtools’. PCA was applied using all features of the NMR spectra with a PLS-tool box (Eigenvector Research) in Matlab (MathWorks). Welch t-test was applied for statistical significance analysis for metabolites in Amix V.3.9 (Bruker Biospin), and a false discovery rate were applied to control the family wised error. The heatmaps, Volcano Plot, Pathway analysis for pathways and dendrogram were carried out in MetaboAnalyst V.3.0. Unless otherwise stated, all the values presented here are means±SEM. A p<0.05 was considered statistically significant.