Diurnal feeding behaviour drives 24h rhythms within intestinal epithelial cells and the gut microbiome
Diurnal feeding behaviour drives 24h rhythms within intestinal epithelial cells and the gut microbiome
In order to determine the influence of diurnal feeding behaviour on the IEC clock and the circadian transcriptome we established an interval feeding regime.  Mice were provided with 8 small meals a day at 3 hourly intervals for 16 days (Figure 1A  ).  This resulted in an equal spread of food consumption across each mealtime  (Figure 1B).  In contrast, ad libitum fed mice consumed the majority of their food at night, with approximately 25% of their food eaten during the light period, as expected (Figure 1B).  After 16 days RNA was extracted from Colonic intestinal epithelial cells (IECs). Colonic IECs were extracted using established methods (PMID: 32888430) .  Dissected colons were opened out longitudinally and sectioned into four pieces, washed in ice cold PBS and incubated in HBSS containing 2% FBS on ice.  After 20min, the tissue was transferred to HBSS containing 2mM EDTA and 1mM dithiothreitol and placed in a shaking incubator (180rpm, 37degrees C, 15 min).  Subsequently, samples were vigorously agitated for 30s to dissociate the epithelium from the basement membrane before being passed through a 70μm filter.  The effluent was centrifuged (432 x g, 4 degrees C, 5 min) and the pellet, containing IECs, re-suspended in 350μL RLT plus buffer (Qiagen) and stored at -20 degrees C.  RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. For purity checks, isolated colonic cells were labelled with PE labelled EpCAM (G8.8, eBioscience) and BV510 labelled CD45 (30-F11, Biolegend) 1:200 following a 20 min Fc block (anti mouse CD16/CD32 eBioscience, 1:100).  Cells were washed with and then re-suspended in FACS buffer (PBS+5% FBS) before analysis on an BD LSR II. All experimental procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986, subject to local ethical review and approval from the University of Manchester Animals Welfare and Ethical Review Body (AWERB). Mice were housed in the Biological Services Facility (BSF) at the University of Manchester with regulated temperature and humidity.  Mice were housed under a 12:12 light:dark cycle in light controllable cabinets, whereby zeitgeber time 0 (ZT0) refers to lights on, and ZT12 refers to lights off. Unless stated otherwise, 8-12 week old, male, C57BL/6J mice (Charles River Laboratories) were used and were co-housed.    Interval feeding Food availability was restricted to a short feeding window every 3 h.  Upon initiating the interval feeding regime, during the 4 meals in the light phase (ZT2, ZT5, ZT8 and ZT11) food hoppers were made available for a 30 min window.  For the 4 meals during the dark phase (ZT14, ZT17, ZT20 and ZT23), food hoppers were made available for a 10 min window. On the fifth day onwards (in response to observations that mice were consuming more food during the mealtime immediately after lights on, compared to later mealtimes during the light phase) the ZT2 meal was reduced to 20 min, and the dark phase meals were increased to 12.5 min to maintain equal light and dark food intake. Food was weighed after each feeding window and body weight was tracked.  Food was delivered manually, or using a programmable automated system (TSE systems).  Large intact food pellets were provided during meals, to avoid smaller fragments falling onto the cage floor, and cages were regularly checked to ensure there was no residual food on the floor. RNA was extracted using chloroform, then precipitated using isopropanol. After washing in 75% ethanol, the RNA pellet was re-suspended in RNase free water.   RNA was extracted from IECs and quality was determined using a 2200 TapeStation (Agilent Technologies). Library preparation and sequencing was performed by the University of Manchester Genomic Technologies Core Facility. Libraries were generated using the TruSeq Stranded mRNA assay (Illumina) according to the manufacturer's protocol.