High-fat diet-induced obesity

With the approval of Institutional Animal Care and Use Committee of Kaohsiung Chang Gung Memorial Hospital (Affidavit #2019091103), male C57BL/6 mice (12 weeks old) were fed high-fat diet (HFD; 60 kcal% fat; D12450K, Research Diet Inc. New Brunswick, NJ) or chow diet (CD; 10 kcal% fat; D1294) together with drinking water ad libitum for 6 consecutive months. All animals were housed in a specific pathogen-free vivarium. Blood glucose levels were quantified using Biochemistry-SP-430 (Arkray Inc., Tokyo, Japan). Animals were euthanatized to dissect bone tissue.

µCT analyses of body adipose and bone mass

Anesthetized mice were µCT scanned (50 kV, 1-mm AI filter, 0.9° rotation, and 2 frames) using 1176 Skyscan system (Bruker, Billerica, MA). Thirty-five-µm pixel size radiographs of whole body were reconstructed into three-dimension images. Muscle and adipose were manually contoured using SKYSCAN® CT-Analysis platform and Micro-CT Mouse Phantom (QRM-70137, Freiburg, Germany) as a reference. Volumes of visceral and subcutaneous fat of the regions of interest (binarization thresholds, 73– 90) were calculated using the software, according to the manufacturer's instructions. In some experiments, femur and tibiae specimens were µCT scanned to capture 300 slices of 9-µm pixel size radiographs, as previously described [22]. Upon calibration with a calcium hydroxyapatite phantom (QRM-70134), bone mineral density (BMD, mg/cm3), trabecular volume (BV/TV, %), trabecular number (Tb.N/mm), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp), structure model index (SMI), cortical BMD, cortical thickness (Ct.Th, mm), and cortical porosity (%) of the specimens were calculated using in-house software.

Biomechanical strength analyses

Biomechanical analyses of femurs were conducted using SHIMADZU Electromechanical Tester (EX-SX, Shimadzu, Kyoto, Japan). Upon placing bone tissue onto a two-jig holder (jig span, 0.5 cm), the middle parts of the specimens were 3-point bended under a load of 50-N, which was displaced at 10 mm/min. The tester's in-house TRAPEZIVMX software was utilized to calculate breaking force (N) and breaking energy (J), which were normalized with the cross-section areas of middle part of the specimens.

16S rRNA sequencing

Fresh feces of mice were harvested upon fasting for 12 h; with all protocols were conducted under sterile conditions. Fecal DNA was isolated using QIAamp PowerFecal DNA Kits (Qiagen, Germantown, MA). To prepare multiplexed SMRTbell Library, a total of 1 ng/μl DNA was used to PCR amplify the V1-V9 regions of full length 16S rRNA genes using specific primers (forward, 5′Phos-GCATCAGRGTTYGATYMTGGCTCAG-3′; reverse, 5′Phos-RGYTACCTTGTTACGACTT-3′). PCR products were purified using AMPure PB beads (100–265-900; PacBio®, Menlo Park, CA); and the SMRTbell library was mixed with sequencing primer v4. Sequel II Binding Kits (102–194-200; PacBio®) were used for primer annealing, and polymerase binding. Gene sequencing was conducted to produce HiFi readouts (predicted accuracy, 30) using PacBio Sequel IIe (circular consensus sequence mode).

Gene readout processing, clustering, and annotation

PacBio Workflow in SMRT Link (minimal prediction accuracy, 0.9 and minimal number passes, 3) was used to process the readouts of Circular Consensus Sequence. In brief, DADA2 (version 1.20) pipeline was utilized for quality filtration, dereplication, chimera removal, and algorithm of amplicon sequence variants (ASVs) from full-length 16S rRNA gene. Annotation of taxonomy classification was conducted using QIIME2 software (version 2021.4) through retrieving NCBI database. Sequence similarities in ASVs against 16S ribosomal RNA database were analyzed using QIIME2 together with MAFFT software. To normalize the sequence depth among specimens, ASVs were rarefied to minimal sequence depth. Bioinformatics analyses were conducted using R package software (version 3.6.0), including ggplot2, factoextra, phyloseq, vegan3d, Rtsne, mixOmics, metagenomeSeq together with Wilcoxon rank-sum test, ALDEx2, and MicroEco. KEGG pathways for functional abundances of bacterial taxa with PICRUSt2, Tax4Fun2, and FAPROTAX (version1.2.4) was used. CPCoA, PCoA, and Heatmap were plotted using TBtools, and ImageGP [23].

UHPLC-MS analyses for serum metabolome

Peripheral blood was drawn using an intracardiac needle. A total of 50 µl serum was mixed with 1000 µl mixture of methanol, acetonitrile, and water (2:2:1 in volume), centrifuged at 10,000 × g for 15 min. Supernatants (10 µl) were eluted through Acquity BEH C18 column (Waters, Milford, MA) with 0.1% formic acid and acetonitrile at 0.25 ml/min for ultrahigh performance liquid chromatography with tandem mass spectrometry (MS) (Orbitrap Elite; Thermo Fischer Scientific. Waltham, MA). MS data acquisition was conducted using positive mode (MS profile resolution, 6000) together with a default data-dependent acquisition (resolution, 15,000), a scan range of 70 – 1000 m/z with the normalized collision energy set to 25. ProteoWizard software was utilized to convert MS data into mzXML format. Data extraction, alignment, and integration was conducted using R package and XCMS software. MS2 bioinformation engine was used for metabolite annotation (cutoff, 0.5) [24].

Fecal microbiota transplantation

Six grams of fresh feces from age-matched CD-fed mice were mixed with 6 ml sterile Ringer's solution. The mixtures were centrifuged (500 × g, 15 min) and fecal microbiota supernatants were harvested for transplantation. Each HFD-fed mouse was transplanted with 0.5 ml fecal microbiota supernatant from CD-fed mice via oral gavage (3 transplantations/week) for 5 consecutive months. In a subset of experiments, 12-week-old female C57BL/6 mice were anesthetized and bilaterally ovariectomized to induce osteoporosis. One week postoperatively, each ovariectomized mouse was transplanted with 0.5 ml fecal microbiota supernatants from aged-matched sham mice via oral gavage for 3 consecutive months (3 transplantations/week).

TMAO treatment

Twelve-week-old male C57BL/6 mice were fed on 0.2% TMAO (317594; Sigma-Aldrich, St Louis, MO) in sterile drinking water and chow diet ad libitum for consecutive 3 months. Control mice received the same diet without TMAO. Animals were euthanatized and peripheral blood and bone tissue were biopsied at the end of study.

ELISA

Serum was mixed with a mixture of methanol/acetonitrile and formaldehyde (15:85) and centrifuged at 14,000 × g for 5 min at 4 °C. The supernatants were eluted through SUPELCO Ascentis C18 column and tandem mass spectrometry to quantify TMA and TMAO. In some experiments, serum TMAO (AMS.E03T0904; Amsbio, Cambridge, MA), L-carnitine (ab83392; Abcam, Cambridge, UK), and FMO3 (MBS9347271; MyBioSource, San Diego, CA) were quantified using specific ELISA kits.

Ex vivo osteogenic and osteoclastogenic differentiation

Bone-marrow mesenchymal cells were isolated from FMT recipient mice or TMAO-fed mice or control mice. A total of 2 × 105 cells/well (24-well plates) were cultured in osteogenic medium (A1007201; Thermal Fisher Scientific) with 10% fetal bovine serum (FBS) for 18 days, as previously described [22]. Mineralized matrices were stained using von Kossa Stain Kits (ab150687; Abcam, Cambridge, UK). Areas of mineralized matrix in 3 random fields of each well from 6 wells of each experiment were measured using Zeiss Image Analysis System. In a subset of experiments, bone-marrow macrophages (5 × 104 cells/well, 48-well plates) were incubated in αMEM with 10% FBS, 20 ng/ml M-CSF and 40 ng/ml RANKL (R&D Systems, Minneapolis, MN) for 10 days [22]. Osteoclasts were stained using tartrate-resistant acid phosphatase staining kits (MK300; Takara Bio Inc., Shiba, Japan). TRAP-stained osteoclasts in 9 random fields from 3 wells were counted.

Histomorphometry and immunohistochemistry

Animals were intraperitoneally injected 50 mg/kg calcein 3 days and 9 days before the end of experiment. Calcein-labeled mineral deposition in the sections of methyl acrylate-embedded bone specimens were evaluated using fluorescence microscopy. Nine random fields of 3 sections of each specimen were selected to calculate mineral apposition rate (MAR, µm/day) and bone formation rate (BFR/BS, µm3/µm2/day), as previously described [25]. Sections of paraffin-embedded bone specimens were stained using alkaline phosphatase and tartrate-resistant acid phosphatase histochemical staining kits and hematoxylin and eosin stain to calculate osteoblast number (Ob.N/mm), osteoclast number (Oc.N/mm), and adipocyte number (Ad.N). In some experiments, sections of paraffine-embedded colon specimens were stained using Periodic Acid-Schiff Stain Kits (ab150680; Abcam, Cambridge, UK) or using primary CLDN-1 (SAB3500438; Sigma-Aldrich, St Louis, MO), TJP-1 (ab276131; Abcam), and IL-17 (ab79056; Abcam) antibodies together with Super Sensitive™ IHC Detection Systems (BioGenex Laboratories; Fremont, CA). Immunostained goblet cells in 9 random fields of 3 different sections of each animal were calculated.

Osteoblast culture

Murine MC3T3-E1 osteoblasts (105 cells/well, 24-well plates) were incubated in osteogenic medium (DMEM with 10% FBS, 50 µg/ml ascorbic acid, 1 mM β-glycerophosphate), supplemented with 50 µM, 100 µM, 200 µM and 300 µM TMAO for 1 day or 18 days. In a subset of experiments, osteoblasts were incubated in osteogenic medium with 200 µM TMAO or 5 µM GSK2606414 (5107; Tocris Bioscience, Bristol, UK) or 2.5 µM nicotinamide riboside (72340; Sigma-Aldrich, St Louis, MO). Mineralized matrices were stained using von Kossa staining kits. Areas of mineralized matrix were calculated from 9 random fields of 3 wells per experiment.

Osteoblast growth and senescence assay

Growth of osteoblasts (2 × 104 cells/well, 96-well plates) were quantified using BrdU Cell Proliferation Assay Kit (#6813; Cell Signaling Technology; Danvers, MA). Senescent cells were stained using Senescence β-Galactosidase Staining Kits (SA-β-gal; #9860; Cell Signaling Technology). SA-β-gal-stained senescent cells in 9 random fields of 3 different wells per experiment were counted.

RT-PCR

Extraction and reverse transcription of total RNA from 106 osteoblasts was conducted using PureLink RNA Mini Kit and High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), respectively. PCR was conducted using ABI StepOne Plus 96-Well Real-Time PCR Detection System (Thermo Fisher Scientific), with Applied Biosystems™ TaqMan™ Universal PCR Master Mix and specific primers (Supplementary Table 1). The relative fold change in mRNA expression was calculated using the ΔΔCt method, with 18S rRNA as calibrator.

Mitochondrial respiration and ATP production

Seahorse XFe Analyzer together with Seahorse XFp Cell Mito Stress Test Kits (Agilent, Santa Clara, CA) were utilized to quantify mitochondrial respiration capacity. Upon 105 cells were incubated in cartridge with Seahorse XF DMEM Assay Medium, 2 µg/ml oligomycin, 2 µM FCCP, and 2 µM antimycin and rotenone were added to the cartridge to inhibit complex activities. Oxygen consumption rate was calculated automatically. ATP production of 106 osteoblasts were quantified using ATP Assay Kits (ab83355, Abcam), according to the manufacturer's manuals.

Western blotting

Primary antibodies PERK (#3192; Cell Signaling Technology), mTOR (#2983), RPTOR (#2280), FAM134B (#61011), ATF4 (#11815), Mfn2 (#9482), Actin (#4967), phosphorylated PERK (#PA5-102853; Thermal Fisher Scientific), LC3-II (#PA1-16930), ATF5 (#PA5-17988), Opa1 (ab42364, Abcam), and Pierce™ Fast Western Blot Kits (Thermal Fisher Scientific) were utilized for immunoblotting of cell lysates. To characterize aggregated Atf5, 100 µg lysate was ultracentrifuged (125,000 × g, 1 h) to harvest protein aggregates; and proteins from the pellets were separated using SDS-PAGE, followed by Atf5 immunoblotting [22].

Fluorescence microscopy and transmission electron microscopy

Endoplasmic reticulum stress and mitochondria in osteoblasts (102 cells/slide) were stained using ER-Tracker™ (E34251; Thermo Fisher Scientific), MitoTracker™ Green FM (M7514; Thermo Fisher Scientific), respectively. Autophagosome and mitophagosome in cells were stained using CYTO-ID Autophagy Detection Kits (ENZ-51031; Enzo Life Sciences, Farmingdale, NY) and Mitophagy Detection Kits (MD01; Dojindo Laboratories, Tokyo, Japan), respectively. In some experiments, osteoblasts were fixed by 3% glutaraldehyde, post-fixed by 1% OsO4, epoxy resin embedded, and cut into 50-nm sections, which were further coated by gold particles. ER ultrastructure in osteoblasts was investigated using Hitachi SU8229 TEM System.

Statistical analysis

The distribution and the differences in CD and HFD-fed mice or in vehicle and TMAO-fed mice were analyzed using Kolmogorov–Smirnov test and Student t-test, respectively. The differences in 3 or 4 groups were analyzed using ANOVA test and post hoc Bonferroni test. P value < 0.05 is considered significant difference.