At the start of the diet intervention, the mice were divided into two groups and were fed a powdered high- or a low-fat purified diet. After 2, 4, and 8 weeks of diet intervention, 6 mice per diet group, per time point were sacrificed after they were anaesthetized with a mixture of isofluorane (1.5%), nitrous oxide (70%) and oxygen (30%). All mice were sacrificed in the postprandial state and diurnal variability was avoided by harvesting small intestines at the same time of the day for both diet groups. The small intestines were divided into three equal parts along the longitudinal axis (proximal, middle and distal part of the small intestine). Small intestinal epithelial cells were scraped, snap-frozen in liquid nitrogen, and stored at -80°C until RNA isolation. Body weight was recorded weekly. The mice that were sacrificed after 8 weeks of diet intervention were all subjected to an oral glucose tolerance test (OGTT) at week 7. Therefore, after 6-hours fasting, all mice received 0.5 ml of a 20% glucose solution via an oral gavage and blood glucose was measured after 15, 30, 45, 60, 90 and 150 minutes using Accu-Chek blood glucose meters (Roche Diagnostics, Almere, The Netherlands). To determine food intake, non-absorbable chromic oxide was supplemented to the diets for one week (week 5 of diet intervention). At the end of this week feces was quantitatively collected during 48 hours and fecal chromic oxide levels were determined as previously described [15 (link)]. These fecal chromic oxide levels were then used to calculate the energy intake per mouse per day on a high-fat and low-fat diet. An outline of this study design is presented in Additional file
For immunohistochemical analysis, an identical low-fat and high-fat diet intervention study was performed for 2 weeks (n = 12 per diet). Small intestines were again excised, divided into three equal parts, cut open longitudinally, and washed with PBS. Thereafter, the small intestinal parts were fixed in 10% buffered formalin and embedded in paraffin.