Understanding how fibre reduces food intake and adiposity

Project Summary

The purpose of this project was to examine how two different dietary fibres, pectin and oligofructose inhibit high fat food intake at the level of the gut and how these responses compare to a mixed 4-fibre diet, more typical of normal human consumption. For this, we examined how the gut microbiome and the gut epithelium responded.

The study involved using C57Bl/6J mice purchased from Charles River Laboratories UK at 4 weeks of age and from the same breeding unit. They were held in paired housing for 6 weeks being fed a normal chow diet, ad libitum with free access to water, within the Medical Research Facility at the University of Aberdeen, before starting the experiment.

Groups of 10 mice, remaining in paired housing, were put on one of 8 treatments, following a 2-week acclimatization period, during which mice were fed a low fat refined + 10% cellulose diet (LF + 10% Cellulose). One group of mice were maintained on this diet for the 8-week intervention period. The remaining mice were transferred on to one of the following diet treatments: High fat (HF) refined 10% cellulose diet (HF + 10% Cellulose) or High Fat refined diets where the cellulose was replaced with either high (10%) or low (2%) levels of pectin (HF + Pectin), or Fructo-oligosaccharide (FOS) (HF + FOS), or Mixed Fibre (HF + Mixed Fibre). The 10% Mixed fibre diet contained 2.5% pectin, 2.5% FOS, 2.5% inulin and 2.5% beta glucan. For the 2% fibre treatments, Cellulose was added at 8%.

Fibres

Apple pectin: Cat no. 93854-1KG; Merck Life Science UK Limited, The Old Brickyard, New Road, Dorset SP8 4XT, UK.

Fructooligosaccharide, (FOS), Orafti®P95; BENEO GmbH, Maximilianstr. 10, 68165 Mannheim, Germany.

FOS + inulin: Synergy 1; BENEO GmbH, Maximilianstr. 10, 68165 Mannheim, Germany.

Oat beta-glucan: Cat no. NIGECER000241; Nutraceuticals Group, The Old Smithy, 7 High Street, Merstham, Surrey, RH1 3BA, UK.

Diet recipes

Study Data

Body weights were recorded weekly and fat and lean mass measured by Echo MRI scanning at the start. Fat mass of the epididymal and retroperitoneal depots were weighed at the end of the experiment. Bloods were collected by cardiac puncture, into K2EDTA-coated tubes and immediately chilled on ice then plasma prepared and stored frozen. Gut tissues were harvested and weighed. Gut epithelial cells were prepared by scraping the PBS-flushed luminal walls and saved in tubes containing RNAlater® (Merck cat no. R0901, Merck Life Science UK Limited, Gillingham, Dorset, UK). Caecal, colon and faecal material was collected and stored at -70C. Liver samples were frozen on dry ice and stored at -70C.

Analyses

SCFA levels

SCFA levels in faecal samples were measured by capillary gas chromatography using the technique developed by Richardson et al., (1989) with helium used as the carrier gas. Samples were diluted in distilled water and 2-ethylbutyric acid (5 mmol/L) was added as internal standard. The extraction of samples was then carried out in diethyl ether and derivatised with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Analysis was carried out on Agilent GC HP-1 capillary columns.

Microbiota

Microbial DNA was extracted using FastDNA® SPIN Kit for Faeces (MP Biomedicals 116570200, MP Biomedicals SARL, Illkirch, France) and was processed according to the manufacturer’s instructions. The bacterial DNA extracted were used as templates to amplify the V1-V2 variable regions of the 16S rRNA gene employing forward primer MiSeq-27F (5′-AATGATACGGCGACCACCGAGATCTACACTATGGTAATTCCAGMGTTYGATYMTGGCTCAG-3′) and MiSeq-338R (5′-CAAGCAGAAGACGGCATACGAGAT-barcode-AGTCAGTCAGAAGCTGCCTCCCGTA GGAGT-3′) which also contain adaptors used for downstream Illumina sequencing. Also included in each of the samples amplified is a unique 12-base pair barcode on the reverse primer for sample identification.

The extracted DNA samples were amplified using the New England Biolabs Q5® High-Fidelity DNA Polymerase (Hertfordshire, UK). For each of the extracted DNA samples, 4 individuals (quadruplets) of 25µl PCR reaction mixtures were prepared comprising of a mixture of 5X Q5 Buffer (5µl), 10 mM dNTPs (0.5µl), 10 µM F Primer (1.25µl), 10 µM R Primer (1.25µl), Template DNA (1µl), Q5 High-Fidelity DNA Polymerase (0.25µl), and Nuclease-Free Water (15.75µl). PCR conditions were set at 2 minutes at 98°C, then 20 cycles of 30 seconds at 98 °C, 30 seconds at 50 °C, 90 seconds at 72 °C; then a final 5-minute extension at 72 °C followed by a holding temperature of 4 °C. Following verification of satisfactory amplified products by agarose gel electrophoresis, the 25µl quadruplicate for each sample was pooled into 1.5 ml sterile microcentrifuge tubes and ethanol precipitated. Quantification of the amplicons was carried out using the Qubit dsDNA HS Assay Kit (Invitrogen, CA, USA, Q32854). An equimolar master mix required for Illumina MiSeq sequencing was prepared using equal molar quantities from each DNA sample. Sequencing of the amplified PCR products of the V1-V2 region of the 16S rRNA gene was carried out on the Illumina MiSeq machine. Sequencing was carried out by the Centre of Genome Enabled Biology and Medicine (CGEBM) of the University of Aberdeen. Illumina MiSeq sequenced data obtained from the CGEBM were analysed using the Mothur software package (Schloss et al., 2009) and based on Mothur Miseq standard operating procedure (Kozich et al., 2013).

Plasma hormones

The plasma hormones GLP-1 (total), IL-6, insulin, leptin, PYY and TNFα were analyses using a Milliplex Mouse Metabolic Hormone Expanded Panel kit number MMHE-44K, 96-Well Plate Assay, following the manufacturer’s instructions and using a BioRad Bioplex 200 instrument (Bio-Rad Laboratories Ltd. The Junction, Station Road, Watford, Hertfordshire, WD17 1ET, UK).