Animals and treatments
Two animal experiments were performed. In experiment 1, 27 male 9-week-old C57BL/6J mice (Janvier Labs, Saint-Berthevin, France) were purchased. Three mice were housed in one individually ventilated cage. In experiment 2, four male 12-week-old C57BL/6J mice (Janvier Labs) were housed in one individually ventilated cage. Mice were kept in a pathogen-free environment with a 12 h daylight cycle and free access to food and water. The acclimatisation period lasted 1 week on a standard diet (AIN-93M; ssniff, Soest, Germany). The experiments were approved by and performed in accordance with the guidelines of the local ethics committee of Université catholique de Louvain. Housing conditions were as specified by the Belgian Law of 29 May 2013 regarding the protection of laboratory animals (Agreement no LA 1230314).
In experiment 1, mice were randomised based on body composition assessed by NMR (LF50 minispec; Bruker, Rheinstetten, Germany) to minimise differences (initial mean body weight ± SEM was 24.46 ± 0.23 g). No blinding procedure was followed. The groups (n = 9) were: (1) group-fed a control diet (D12450K; Research Diets, New Brunswick, NJ, USA) containing 10% kJ fat; (2) group-fed a WD (D12451; Research Diets) containing 45% kJ fat and 17% kJ sucrose; and (3) group-fed a WD plus vildagliptin (Cayman, Ann Arbor, MI, USA [supplied by Sanbio, Uden, the Netherlands]) in the drinking water (0.6 mg/ml according to previous studies , corresponding to approximately 50 mg kg−1 day−1). To discriminate the effect of vildagliptin from the effect of the WD on DPP-4 activity, we pretreated with vildagliptin for 2 weeks. In the third week, the WD was introduced. A scheme of the experimental design is shown in electronic supplementary material (ESM) Fig. 1.
After 8 weeks and 6 h of fasting, glycaemia was measured using a glucometer (Roche Diagnostics, Basel, Switzerland) on blood from the tail. Mice were anaesthetised with isoflurane gas (Abbot, Lake Bluff, IL, USA). Portal blood was collected. Mice were necropsied after cervical dislocation. Liver, adipose tissue and caecal content and tissue were weighed. Blood, liver, caecal content and tissue, ileum and colon were collected, frozen in liquid nitrogen and stored at −80°C until analysed.
Details of experiment 2 are given below in the section Precision-cut liver slices.
Portal active GLP-1 (7-36 amide and 7-37), insulin and total GIP were determined in plasma collected in an EDTA tube with a DPP-4 inhibitor (DDP4-010; Millipore, Darmstadt, Germany) using a multiplex immunoassay kit (Bio-Plex; Bio-Rad, Nazareth, Belgium).
Analysis of gut microbiota composition
Genomic DNA was extracted from the caecal content using a QIAamp DNA Stool Mini Kit (Qiagen, Hildren, Germany), including a bead-beating step. The composition of the gut microbiota was analysed by Illumina sequencing of the 16S rRNA gene (Illumina, San Diego, CA, USA). Illumina sequencing was performed as previously described [28, 29]. The V5–V6 region of the 16S rRNA gene was amplified by PCR using modified primers. The amplicons were purified, quantified and sequenced using the Illumina MiSeq system to produce 2 × 300 bp sequencing products, at the University of Minnesota Genomics Center. Subsequent bioinformatics and biostatistics analyses were performed as previously described . The full protocol and statistical analyses are described in ESM Methods.
In addition, in the caecal content, some components of the gut microbiota were analysed by quantitative real-time PCR (qPCR) as previously described . Primers are presented in ESM Table 1.
Gene expression analyses
Total RNA was isolated from tissues and qPCR was performed using the StepOne System (Applied Biosystems, Waltham, MA, USA) to analyse expression of the following genes: Cd3g, Cd11c (also known as Itgax), Cd68, Cd163, Claudin2 (also known as Cldn2), DefA, F4/80 (also known as Adgre1), Il1b, Il6, Il10, Ki67 (also known as Mki67), Lyz1, Mcp1 (also known as Ccl2), Muc2, Ocln, Pla2g2a, Reg3g, Tcf4, Tnfα (also known as Tnf) and Zo1 (also known as Tjp1). Data were analysed using the 2-ΔΔCT method and expression was normalised to Rpl19 (see ESM Methods and ESM Table 2 for further details).
Short-chain fatty acids
Caecal content was diluted 1:6 in LAL reagent water (Lonza, Walkersville, MD, USA) and homogenised with a tissue lyser (Qiagen) for 4 min without beads to avoid bacteria disruption. Samples were centrifuged (8000 g, 2 min). Cell-free supernatants were used for the quantification of acetate, butyrate and propionate as previously described  and for the analyses of TLR agonists as described below.
TLR-2 and TLR-4 agonists
TLR-2 and TLR-4 agonists were measured using HEK-Blue reporter cell lines according to the manufacturer’s instructions (InvivoGen, San Diego, CA, USA). Cells were exposed to the supernatants of caecal content and the protocol previously described was followed .
Histological analysis of the intestine
Crypt depth and villus length were measured after H&E staining. Sections were digitised (Leica SCN400; Leica, Wetzlar, Germany) and images were captured using Leica Image Viewer software, version 4.0.4. At least ten measurements per mouse were made.
Precision-cut liver slices
After 1 week of acclimatisation, mice in experiment 2 were anesthetised with ketamine (100 mg/kg body weight) (Nimatek; Eurovet, Bladel, Netherlands) and xylazine (100 mg/kg body weight) (Rompun; Bayer, Leverkusen, Germany). Precision-cut liver slices (PCLS) were prepared as previously described [32, 33]. PCLS were incubated without (control medium) or with vildagliptin (0.6 mg/ml) in oxygenated and supplemented Waymouth’s medium for 4 h at 37°C under agitation. PCLS were frozen in dry ice until analysed.
Bacterial growth conditions
Growth curves of Oscillibacter valericigenes DSM18026 and L. reuteri 100-23 in the presence of vildagliptin (0.6 mg/ml) or absence of vildagliptin (control broth) were determined (ESM Methods).
To measure DPP-4 activity 5 ml of overnight cultures were harvested and processed as described below.
DPP-4 activity was measured by the cleavage of para-nitroanilide (PNA) from Gly-Pro-PNA (Sigma, St. Louis, MO, USA). Samples (20–50 mg) were suspended in TRIS-base buffer (50 mmol/l, 1% N-octylglucoside, pH 8.3), homogenised for 2 min with a tissue lyser and centrifuged (3000 g, 20 min). The supernatant (20 μl) was incubated with Gly-Pro-PNA. The enzymatic activity was measured in a kinetic analysis of 30 min at 37°C (380 nm) (SpectraMax M2; Molecular Devices, San Jose, CA, USA) and quantified with a standard curve of free PNA. In tissues and bacterial samples, the values were normalised by the amount of protein (Bradford method).
The number of mice per group was based on previous experiments to measure the primary outcomes (the WD increased the body weight) with the minimum number of animals . Analyses were performed by one-way ANOVA followed by post hoc Tukey’s tests (GraphPad Prism, version 5; GraphPad, La Jolla, CA, USA). For the growth curves, two-way ANOVA followed by Bonferroni’s post hoc test was performed. A χ2 test was used for categorical data (active GLP-1 and TLR-4 agonist) (Statgraphics Plus, version 5.1; Statgraphics, The Plains, VA, USA). Data of microbiota composition were analysed using Welch’s t test and the false discovery rate was applied for p value correction (q value) according to the Benjamini–Hochberg procedure. Ecological descriptors and data from the PCLS experiment were analysed using Welch’s t test. Multiple correlation analyses were performed in R version 3.1 (www.R-project.org), with adjustment of p values according to the Benjamini–Hochberg procedure Results were considered statistically significant at p < 0.05. Any exclusion decision was supported by Grubbs’ test. Plots were generated using GraphPad showing the mean ± SEM. Each dot represents one biological replicate.