Male C57BL/6J mice (age 4 weeks) were purchased from Harlan (Horst, The Netherlands) and were housed in the light and temperature-controlled animal facility (12/12 (light/dark), 20°C) of Wageningen University. They had free access to water and received standard laboratory chow (RMH-B, Arie Blok BV, Woerden, The Netherlands) for two weeks, followed by the low-fat diet for three weeks to adapt to the purified diets. All experiments were approved by the Ethical Committee on animal testing of Wageningen University. To investigate the effect of dietary fat on development of obesity and insulin resistance and on small intestinal gene expression in C57BL/6J mice, we used low-fat (reference) and high-fat diets that are based on 'Research Diets' formulas D12450B/D12451, with adaptations regarding type of fat (palm oil in stead of lard) and carbohydrates to mimic the fatty acid and carbohydrate composition of the average human diet in Western societies (Research diet services, Wijk bij Duurstede, The Netherlands). Thus, the high-fat diet mimics the ratio of saturated to monounsaturated to polyunsaturated fatty acids (40:40:20) in a human diet. The complete composition of the diets is given in Additional file 1 . It should be noted that in these diets the energy density of all nutrients, except fat and starch, is equal.
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 file2 .
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.
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.
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