Study design

In this study, mice were divided into seven groups (n = 47), including H (high fat diet, n = 6), HE (H with exercise, n = 7), N (normal fat diet, n = 7), NE (N with exercise, n = 6), H_FHE (H receiving FMT from HE, n = 7), H_FNE (H receiving FMT from NE, n = 7) and N_FNE (N receiving FMT from NE, n = 7). H represents the diet-induced obesity group independent from external treatment (e.g., exercise or FMT). N represents the treatment-naïve group with normal fat diet. Comparisons were performed for each group versus H to monitor the differential outcomes of metabolic and inflammatory profiles and gut microbiota under given treatments. Mice were randomly assigned to each group except for those in the exercised groups identified during 2-week assimilation training. Details of study design are shown in Fig. 1. Animal management, exercise regimen and FMT protocol are described below.

Animal model

All experimental procedures were approved by the Institutional Animal Care and Utilization Committee of Taichung Veterans General Hospital (La-1031216 and La-1041342) and performed in accordance with institutional guidelines. Male C57BL/6JNarl mice (n = 49), aged five weeks, were received from the National Laboratory Animal Center (Taipei, Taiwan). These mice were initially group housed in 7 cages (7 mice per cage) with free access to water and food. The mice in 4 cages were provided high fat diet (HFD; composition 60 kcal% fat, 20 kcal% carbohydrates, 20 kcal% protein; 5.24 kcal/g total energy content; Research Diets Inc., New Brunswick, NJ, USA) and the mice in 3 cages were provided normal fat diet (NFD; composition 11 kcal% fat, 63 kcal% carbohydrates, 26 kcal% protein; 3.25 kcal/g total energy content; Fwusow Industry, Taichung, Taiwan). The housing conditions included constant temperature of 22 ± 1 °C and regular 12-hour light/dark cycle (lights on 8 PM to 8 AM).

After a one-week acclimation period, all mice underwent assimilation training for 2 weeks to identify those competent to be enrolled in the exercised groups. Assimilation training was carried out individually for 39 minutes every Monday and Tuesday on a motorized treadmill outside the cage, starting with a 5-minute warm-up at 7 m/min that was followed by acceleration at 3 m/min every minute to a maximum speed of 18 m/min. After 2-week assimilation training, the mice were reallocated into 7 groups (H, HE, H_FHE, H_FNE, N, NE and N_FNE). Fourteen mice adaptable to treadmill running were assigned to exercised groups (7 in HE and 7 in NE) and underwent formal exercise regimen (18 m/min, 30 min/day, 5 days/week)53 following the same warm-up program as during assimilation training for 16 weeks. During exercise sessions, HE and NE mice were pulled from their respective cages and exercised on a motorized 5-lane treadmill at zero-percent incline. The chosen exercise paradigm aimed to create a high impact training model for C57BL/6J mice51,54. Forty-nine mice were initially enrolled in the experiment. Two were removed from downstream analysis, of which one in the H group was recalcitrant to HFD (increased 8 g by the end of experiment) and one in the NE group died during exercise at week 15.

Fecal sample collection and FMT

For comparative purposes, mice feces were collected twice for 16S ribosomal RNA (rRNA) gene amplicon sequencing, once at the beginning of week 12 (i.e., before FMT) and once at the end of week 24 (i.e., after FMT). About 100 mg of feces from each mouse were stored in 1 mL InhibitEX Buffer (Qiagen, Gaithersburg, MD, USA) at −80 °C for 1 week or less until DNA extraction.

After four weeks of formal exercise regimen (i.e., at week 12), feces from exercised mice were collected fresh for daily FMT, which was conducted under sterile conditions under laminar flow hood. Feces from exercised mice were pooled by cage, and 100 mg (about 5–6 fecal pellets) were re-suspended in 1 mL sterile saline. The solution was vigorously mixed for 10 seconds before centrifugation at 800 × g for 3 min. The supernatant (about 500–600 μL) was collected and administered by oral gavage within 10 min to minimize changes in microbial contents55.

Among the sedentary mice, 14 HFD-fed and 7 NFD-fed mice were randomly designated as FMT recipients (7 H_FHE, 7 H_FNE and 7 N_FNE) (Fig. 1). According to the established microbial depletion and recolonization protocol56,57,58,59, a combination of ciprofloxacin (0.2 g/L) and metronidazole (1 g/L) was added to the drinking water of FMT recipients for two days (i.e., the weekend of week 11) before FMT, to ensure that the outcomes are compatible with clinical guidelines for FMT in humans27. Ciprofloxacin plus metronidazole was chosen because it is one of the first-line antibiotic regimens recommended for the treatment of abdominal infections in adults60. Non-FMT recipients did not receive any antibiotics. Antibiotics were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). From week 12, FMT was conducted each weekday, with each recipient administered 100 μL fecal supernatant by oral gavage until week 24.

Metabolic marker profiles of mice

For each mouse, body weight was measured weekly and intraperitoneal glucose tolerance test (IPGTT) was conducted monthly. For IPGTT, mice received intraperitoneal injection of 20% glucose with normal saline (2 g glucose/kg body mass) after 16-hour fast, and blood glucose levels at 0, 30, 60, 90, and 120 minutes were measured using GM700 (BIONIME, Taichung, Taiwan).

Food consumption per group was recorded weekly, to monitor their feeding behavior throughout the FMT experimental period. Food efficacy was estimated for each group as grams of body weight gain per 100 g food consumed.

At the end of the experimental period, mice were euthanized by carbon dioxide inhalation after overnight fast. The chest was opened and blood was drawn from the left ventricle apex of heart. Serum was obtained by low-speed centrifugation (3000 × g, 10 min, 4 °C), then stored at −80 °C and delivered to the National Laboratory Animal Center within one week for measurement of fasting blood glucose, serum alanine aminotransferase (ALT) activity, and low-density lipoprotein (LDL) cholesterol determinations using a Hitachi 7080 automatic chemistry analyzer (Hitachi Co., Ltd., Tokyo, Japan). Intra-abdominal fat and epididymal fat were manually separated and collectively weighed as fat weight.

qPCRs of inflammatory cytokines in mice

Chronic inflammation in lipid is associated with HFD-induced obesity61. To quantitatively compare inflammation status of mice, the gene expression of related inflammatory cytokines, including tumor necrosis factor (Tnf), interleukin 1 alpha (Il1a) and peroxisome proliferator-activated receptor gamma (Pparg)62,63, were measured in liver by quantitative polymerase chain reactions (qPCRs).

Total RNA was extracted from liver tissues and kept in TriPure isolation reagent (Roche Molecular Biochemicals, Mannheim, Germany). Of the total RNA, 1 μg was treated with 10 units of RQ1 RNase-Free DNase (Promega Corp., Madison, WI, USA) and purified with phenol-chloroform. The purified RNA was reverse transcribed into cDNA using Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA, USA). FastStart Universal Probe Master and Universal Probe Library probes (Roche Molecular Biochemicals) were used in qPCRs. For each inflammatory cytokine gene (i.e., Tnf, Il1a and Pparg), qPCRs were performed in triplicate for each of the 47 cDNA pools along with a no template control in parallel. The primers and probes are listed in Supplementary Table 8 (Supplementary file). The qPCR cycling conditions were: 50 °C for 2 min, 95 °C for 10 min and 40 cycles at 95 °C for 15 sec, with a final extension at 60 °C for 1 min using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative gene expression was quantified by the comparative C T (ΔΔC T ) method, employing 18S rRNA gene (Rn18s) as internal control for normalization.

Statistical analysis of mice metabolic parameters and gene expression data

Differences in body weights among groups were evaluated by two-way ANOVA. For the remaining metabolic parameters and gene expression data (including fat weight, fasting blood glucose, IPGTT, Tnf expression, Il1a expression, Pparg expression, ALT and LDL), to avoid potential bias from sporadic outliers on statistical tests (e.g., one Tnf expression was higher than average by 99 folds), the 5% truncated mean (data within 5–95% quantile) was applied. The truncated mean is a robust estimator of the arithmetic mean because it is more resistant to outliers while providing a reasonable estimate of the central tendency of data64. The 50% truncated mean is equivalent to the median, and therefore the truncated mean is often regarded as a compromise between the arithmetic mean and median. With H the diet-induced obesity group, Student’s t-test was used to assess the respective difference in the means of other groups versus H, to understand the outcomes of external treatments designed to deal with obesity.

Histological examination

HFD is associated with fatty liver in mouse and humans29,30. Therefore, mice liver and fat pad tissues were subjected to histological examination to confirm the effectiveness of HFD in terms of adipose accumulation. After euthanasia, mice livers and fat pad tissues were collected in 10% formaldehyde for histological examination by Bestjet Biotechnology Co., Ltd. (Taipei, Taiwan). Haematoxylin and Eosin (H&E) staining was applied to both tissue types and oil red O staining was applied to fat pad tissues. After staining, tissues were examined under light microscope at 200× magnification.

Fecal DNA extraction

Fecal DNA was extracted using QIAamp Fast DNA Stool Mini Kit (Qiagen). DNA quality and quantity were determined by agarose gel electrophoresis and NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE, USA). DNA was stored at −80 °C until 16S rRNA gene amplicon sequencing (at least 500 ng per sample).

16S rRNA gene amplicon sequencing and analysis

The hypervariable regions V3–V4 of bacterial 16S rRNA genes were amplified by PCR using bar-coded universal primers 341F (F, forward primer; 5′-CCTACgggNggCWgCAg-3′) and 805R (R, reverse primer; 5′-gACTACHVgggTATCTAATCC-3′). Library construction and sequencing of amplicon DNA samples were done by Genomics BioScience (Taipei, Taiwan). A pair-end library (insert size of 465 bp for each sample) was constructed using MiSeq Reagent Kit v3 according to the manufacturer’s instructions (Illumina, Wilmington, DE, USA) and high-throughput sequencing was performed on Illumina MiSeq2000 platform (Illumina).

Bioinformatics analysis of 16S rRNA gene amplicons was performed by Germark Biotechnology (Taichung, Taiwan). Briefly, USEARCH (v7.0.1090) was used to merge pair-end reads on a per-sample basis, setting 8 bp as the minimum overlap of read pair. Mothur (v1.34.3) was used for quality-filtering to retain reads with: (1) a length of 400–550 bp, (2) a minimum average quality score of 27, (3) no ambiguous base and (4) homopolymers not exceeding 8 bp. Chimera detection was performed by UCHIME65 as implemented in USEARCH using reference mode and 3% minimum divergence. Quality-filtered and non-chimeric reads were further analyzed according to UPARSE pipeline66 to generate OTUs per sample (97% identity level). OTU representative sequences were searched against the Greengenes 13_5 database using USEARCH global alignment to identify the corresponding taxonomy of the best hit. Any OTU without a hit or with only a weak hit (i.e., the average of % sequence identity and % alignment coverage <93) was excluded from following analyses.

Statistical and bioinformatics analyses of microbiota

All statistical analyses were performed using R (https://www.r-project.org/), unless otherwise specified. PCoA was conducted with R package phyloseq based on OTU-level Bray–Curtis distance measure. Between-group inertia percentage was evaluated using R package ade4 with Monte-Carlo test (with 1000 permutations) to determine the p value of PCoA results. Two-way PERMANOVA was performed by R package vegan using OTU-level Bray–Curtis distance matrix with 999 permutations. Shannon diversity index was estimated at OTU level by R package phyloseq and tested for associations with sampling time (before and after FMT), diet, exercise and FMT status by linear modeling, in which the data of H_FHE and H_FNE before FMT were categorized into H group and that of N_FNE before FMT into N group. To identify genera differentially enriched in the between-group comparisons, LEfSe was applied with α = 0.05 (Kruskal–Wallis and Wilcoxon tests) and effect size threshold of two on LDA using the stand-alone implementation (https://bitbucket.org/nsegata/lefse). Enriched genera identified by LEfSe were further filtered by a group-mean difference in relative abundance larger than 10−5 to exclude genera absent in one group and minutely abundant in another. To deliver a more intuitive perception of contrasting abundance, LEfSe results were visualized by plotting the log-2 ratio of average relative abundance between groups rather than the LDA score. The abundance profile of functional genes (i.e., COGs) of each microbial community was predicted using PICRUSt (v1.0.0). To identify functional genes with differential abundances, enrichment analysis was performed by a two-group comparison for medians using two-tailed Wilcoxon test with a Benjamini-Hochberg false discovery rate correction to adjust q-values for multiple testing. The R code for all statistical analyses is available in Supplementary dataset, and was created by the R package knitr67.