Study population

We included 134 individuals with prediabetes and 134 individuals with normal glucose regulation. In accordance with the criteria from the WHO [10, 11], prediabetes was defined as fasting plasma glucose levels of 6.1–7.0 mmol/l or HbA 1c levels of 42–48 mmol/mol (6.0–6.5%). Normal glucose regulation was defined according to the more strict ADA criteria [12], i.e. fasting plasma glucose <5.6 mmol/l and HbA 1c <39 mmol/mol (5.7%). Individuals with known diabetes and individuals receiving any glucose-lowering drugs were ineligible for inclusion. Individuals with prediabetes were recruited from the Danish part of the Innovative Medicines Initiative – DIabetes REseraCh on patient straTification (IMI-DIRECT) (n = 63) and the Danish study of Functional Disorders (DanFunD) (n = 71) [13, 14]. At the initiation of the present study, 228 Danish individuals were eligible from IMI-DIRECT and 598 were eligible from DanFunD; all individuals fulfilling the abovementioned inclusion and exclusion criteria were included. Individuals with normal glucose regulation were recruited only from DanFunD and matched 1:1 to individuals with prediabetes with respect to sex and age (n = 134). Dietary habits could only be reliably compared between individuals with prediabetes and normal glucose regulation in a subset of the cohort (n = 142) where dietary information was obtained by identical questionnaires.

All participants gave written informed consent and both studies conformed to the principles of the Declaration of Helsinki and were approved by the Regional Scientific Ethical Committee, the Capital Region of Denmark (H-1-2011-166 and H-3-2012-015).

Biochemical analyses and anthropometrics

Biochemical analyses were performed on fasting blood samples. Plasma insulin and C-peptide were measured by chemiluminometric immunoassays (DiaSorin Liaison Analyzer, DiaSorin, Saluggia, Italy, at University of Eastern Finland, Kuopio, Finland). Plasma high-sensitivity C-reactive protein (hsCRP) was measured by an immunoturbidimetry assay (Roche/Hitachi analyser, Roche, Basel, Switzerland, at Vejle Hospital, Vejle, Denmark). HbA 1c , plasma glucose and triacylglycerol were measured at the Steno Diabetes Center, Gentofte, Denmark (IMI-DIRECT participants) or Rigshospitalet Glostrup, Copenhagen University Hospital, Denmark (DanFunD participants). HbA 1c was measured by high-performance liquid chromatography (TOSOH analyser, TOSOH, Tokyo, Japan) and plasma triacylglycerol by colorimetric slide analysis (Vitros analyser, Ortho Clinical Diagnostics, Raritan, NJ, USA). In IMI-DIRECT, plasma glucose was measured twice by a glucose hexokinase assay (Konelab Analyser, Thermo Fisher Scientific, Waltham, MA, USA) and the mean was calculated. In DanFunD, plasma glucose was measured by colorimetric slide analysis (Vitros analyser). Insulin resistance was estimated as HOMA-IR: (fasting plasma glucose [mmol/l] × fasting serum insulin [pmol/l])/135.

Body weight (kg) and height (cm) were measured in light indoor clothes and without shoes. Waist circumference was measured midway between the iliac crest and the lower costal margin.

Collection of faecal samples and extraction of faecal genomic DNA

Fresh stool samples were collected by the participants at home and were immediately frozen in their home freezer at about −20°C. Frozen samples were transported to the laboratory using insulating polystyrene foam containers or dry ice. The maximum transportation time in the insulating polystyrene foam containers was 4 h and at delivery study nurses ensured that all samples were frozen. After delivery, the samples were stored at −80°C until DNA extraction. Total faecal genomic DNA was extracted from 300 mg of faecal material using the NucleoSpin Soil kit (Macherey-Nagel, Düren, Germany) [15]. Briefly, the faecal material was suspended in SL2 buffer containing SX enhancer and cell disruption was carried out by bead beating at 30 Hz for 5 min using a TissueLyser instrument (Qiagen, Hilden, Germany).

Profiling of faecal microbiota composition by sequencing of the 16S rRNA gene

Faecal microbiota composition was profiled by sequencing the V4 region of the 16S rRNA gene on an Illumina MiSeq instrument (llumina RTA v1.17.28; MCS v2.5) with 515F and 806R primers designed for dual indexing [16] and the V2 Illumina kit (2 × 250 bp paired-end reads). Details on amplification of 16S rRNA genes are provided in the electronic supplementary material (ESM) Methods.

Illumina reads were merged using PEAR [17] and quality filtered by removing all reads that had at least one base with a q-score <20. Quality filtered reads were analysed with the software package QIIME, version 1.8.0 (http://qiime.org) [18] as described in ESM Methods. Chimeric sequences, low abundant operational taxonomic units (OTUs; relative abundance <0.002%) and OTUs that could not be aligned with PyNAST [19] were excluded from all analyses. We obtained a mean ± SD of 48,169.6 ± 11,131.7 sequences/sample (range 26,968–87,208). In total, 12,909,447 sequences and 1609 OTUs were included in the analyses. To correct for differences in sequencing depth between samples, 26,968 sequences were randomly subsampled from each sample and included in the analyses for the estimation of α- and β-diversity. OTUs that showed differential abundance among individuals with prediabetes and normal glucose regulation were blasted against the NCBI 16S ribosomal RNA sequences (Bacteria and Archaea) database to obtain a more specific annotation.

Transfer of gut microbiota to conventional mice

From the study population of 268 individuals, we selected four individuals with the poorest (fasting plasma glucose >6.1 mmol/l and HbA 1c >42 mmol/mol [6.0%]) and four individuals with the best glucose regulation (lowest levels of fasting plasma glucose). Case–controls were matched with respect to sex. Frozen stools (250 mg) were obtained from each donor. Case stools and control stools were pooled separately and re-suspended in 4.5 ml PBS prior to each transfer. Mice (48 male wild-type C57BL/6 J, Taconic, Lille Skensved, Denmark; 10 weeks of age) were co-housed with three mice per cage and kept at 22°C under a 12 h light cycle and fed ad libitum with free access to water. After 2 weeks of acclimatisation on a standard chow diet (Altromin 1310, Altromin, Lage, Germany), mice were transferred to a Western diet (D12079 mod.* customised; sucrose was the main carbohydrate source with mixed protein and fat sources) and sham-gavaged with PBS twice a week. After 3 weeks on the Western diet, mice were divided into two equal recipient groups of 18 mice each (six cages per group) and one sham-gavaged control group of 12 mice (four cages per group). The groups were stratified based on magnetic resonance determined fat mass, weight and response to a glucose challenge prior to intervention. For the following 4 weeks, mice were gavaged three times per week with 200 μl of either faecal microbiota (control or case) or PBS (sham) while remaining on the Western diet. Two mice (one case and one control) died during the experiment and were excluded from all analyses.

An OGTT was performed after 4 weeks of gavaging. The mice were fasted for 5 h and gavaged with 3 g glucose/kg lean mass. Blood glucose was measured in tail vein blood at 0, 15, 30, 60, 90 and 120 min using standard Contour Next Test Strips (Bayer Contour, Leverkusen, Germany). Plasma insulin was measured before the glucose bolus and during the OGTT using an electrochemiluminescence assay (Meso Scale Diagnostics, Rockville, MD, USA). Colonisation of the recipient mice by the human gut microbiota was tested by extraction of total genomic DNA from faecal samples collected before and at the end of the 4 week gavage period, as well as caecal samples at the end of the experiment, followed by sequencing of the 16S rRNA gene as described above. Faecal and caecal samples were randomised before DNA extraction.

All animal experiments were conducted in accordance with national Danish guidelines (Amendment number 1306 of 23 November 2007) as approved by the Danish Animal Inspectorate, Ministry of Justice, permission number 2014-15-2934-01027. Mice were kept under specific-pathogen-free conditions and experimental protocols were validated by in-house standard operation procedures.

Transfer of gut microbiota to germfree mice

From the IMI-DIRECT study, we selected two male donor pairs each consisting of one individual with screen-detected type 2 diabetes (according to WHO criteria) and one with normal glucose regulation (according to ADA criteria). Germfree Swiss Webster mice were bred in-house. Five to six 10-week-old male Swiss Webster mice fed regular chow diet were transplanted with faeces from each donor. Mice were kept in individually ventilated cages (ISOcage N System, Tecniplast, Buguggiate, Italy) with a maximum of five mice per cage under a 12 h light cycle and a room temperature of 21°C. Water was given ad libitum. Frozen stools (500 mg) obtained from each human donor were suspended in 5 ml of reduced PBS. The mice were divided into two groups based on body weight and colonised by oral gavage with 200 μl of faecal slurry from each donor. Two weeks after colonisation an intraperitoneal GTT was performed. Mice were fasted for 4 h and injected with d-glucose (2 g/kg body weight). Blood glucose was measured in tail vein blood at 0, 15, 30, 60, 90 and 120 min with a Contour Next EZ glucometer (Bayer, Leverkusen, Germany). Additional blood samples were collected at 0, 15 and 30 min to analyse plasma insulin levels by insulin ELISA Crystal Chem (Downers Grove, IL, USA). Colonisation of the recipient mice by the human gut microbiota was examined in caecal samples collected at the end of the experiment as described above. Caecal samples were randomised before DNA extraction.

All animal procedures were approved by the Gothenburg Animal Ethics Committee (152-2015).

Statistical analyses