Nine pairs of adult MZ twins who were native residents in Tongzhou District of Beijing were enrolled in our study. The twin pairs grew up in the same family, without diagnosed diabetes, and not taking antibiotics or other medications that may influence the gut microbiome in the last two weeks before coming to hospital. The twin pairs were excluded as long as one of them is pregnant, or with tumor history, or with mental disease, or with recent history of diarrhea or intestinal infection.
The participants were divided into two twin-pair groups (a and b). Clinical and laboratory measurements were conducted. Visceral adipose tissue (VAT) was assessed. Fecal samples were collected to analyze the microbiome composition by 16S rDNA gene amplicon sequencing. Liquid chromatography mass spectrometry was performed to detect the metabolites.
Clinical and laboratory measurements
The nurses first administered questionnaires, inquiring into each participant’s medical history, smoking and drinking habits, and intake of medications. Then, venous blood samples were obtained after 8 to 10 h of fasting. Blood samples were analyzed for serum levels of HbA1c, glucose, insulin, triglycerides, total low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, glutamic-pyruvic transaminase enzyme (ALT), glutamic-oxaloacetic aminotransferase(AST), gamma-glutamyltransferase (GGT), and serum creatinine. Physical examinations were also performed, including body weight, body height, waist and hip circumference. Visceral adipose fat (VAT) was evaluated for each participant (Inbody 770, Biospace Co. Ltd.).
Blood samples were also obtained and sent to Beijing Genomics Institute to extract DNAs and identify the egg type. Short tandem repeats were applied to identify and confirm the egg type.
Microbiome composition analysis
Total genome DNA from fecal samples was extracted using Soil DNA Kit according to manufacturer’s protocols. DNA concentration was monitored by Qubit® dsDNA HS Assay Kit.
20–30ng DNAs were used to generate amplicons. V3 and V4 hypervariable regions of prokaryotic 16S rDNA were selected for generating amplicons and following taxonomy analysis. The concentration of DNA library was validated by Qubit3.0 Fluorometer. Quantify the library to 10 nM, DNA libraries were multiplexed and loaded on an Illumina MiSeq or NovaSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was performed using paired-end. Image analysis and base calling were conducted by the Control Software embedded in the instrument.
The effective sequences were used in final analysis. Sequences were grouped into operational taxonomic units (OTUs) using the clustering program VSEARCH (1.9.6) against the UNITE ITS database (https://unite.ut.ee/) pre-clustered at 97% sequence identity. The Ribosomal Database Program (RDP) classifier was used to assign taxonomic category to all OTUs at confidence threshold of 0.8. The RDP classifier used the UNITE ITS database which has taxonomic categories predicted to the species level.
Fecal metabolites analysis
Various metabolites, including methanol, acetonitrile, 2-chlorophenylalanine, formic acid, ammonium formate and ddH2O, were detected based on liquid chromatography mass spectrometry (LC/MS) in fecal samples.
Fecal samples were thawed at 4 °C. 100 µL of each sample was transferred into 1.5 mL centrifuge tubes, and 400 µL of methanol (pre-cooled at − 20 °C) were added to each tube and vortex for 60 s. Then, the mixtures were centrifuged for 10 min at 12,000 rpm 4 °C and all supernatant in each tube was transferred into another 1.5 mL centrifuge tube, and samples were blow-dried by vacuum concentration. The processed supernatant was dissolved with 150 µL 2-chlorobenzalanine (4 ppm) methanol aqueous solution (4 °C), and filtered through a 0.22 μm membrane to obtain the prepared sample extracts for LC-MS.
Chromatographic separation was accomplished in an Thermo Ultimate 3000 system equipped with an ACQUITY UPLC® HSS T3 (150 × 2.1 mm, 1.8 μm, Waters) column maintained at 40 °C. The temperature of autosampler was 8 °C. Gradient elution of analytes was carried out with 0.1% formic acid in water (C) and 0.1% formic acid in acetonitrile (D) or 5 mM ammonium formate in water (A) and acetonitrile (B) at a flow rate of 0.25 mL/min. Injection of 2µL of each sample was done after equilibration. An increasing linear gradient of solvent B (v/v) was used as follows: 0–1 min, 2% B/D; 1–9 min, 2–50% B/D; 9–12 min, 50–98% B/D; 12–13.5 min, 98% B/D; 13.5–14 min, 98–2% B/D; 14–20 min, 2% D-positive model (14–17 min, 2% B-negative model).
The ESI-MSn experiments were performed on the Thermo Q Exactive Focus mass spectrometer with the spray voltage of 3.8 kV and − 2.5 kV in positive and negative modes, respectively. Sheath gas and auxiliary gas were set at 45 and 15 arbitrary units, respectively. The capillary temperature was 325 °C, respectively. The Orbitrap analyzer scanned over a mass range of m/z 81–1000 for full scan at a mass resolution of 70,000. Data dependent acquisition (DDA) MS/MS experiments were performed with HCD scan. The normalized collision energy was 30 eV. Dynamic exclusion was implemented to remove some unnecessary information in MS/MS spectra.
Database management and statistical analysis were carried out using SAS 9.4 software (Cary, NC). The central tendency (spread) was represented by the arithmetic mean (SD). To compare means and proportions, paired t-test and the χ2-statistic were applied, respectively. Significance was a 2-tailed α-level of 0.05 or less.