https://orcid.org/0000-0001-8643-6195
Figures & data
Figure 1. HFD-fed fat-1 mice are protected against colonic mucus layer alteration.
Mice (fat-1 transgenic and WT littermates) were fed CTL or HF diet for 11 weeks. a: Representative photographs of Carnoy-fixed colonic tissue sections stained with Alcian blue/periodic acid-Schiff from WT (top) and fat-1 (bottom) mice fed a CTL or HF diet (n=8 per group) that were used for mucus layer thickness measurements (scale bars, 100 µm). b: Colonic mRNA expression of Muc2, goblet cell differentiation factor Klf4 and Tff3 and KLF4 protein expression, involved in mucosal protection and thickening of the mucus (n=8 per group). Data are shown as mean ± SEM, and differences were analyzed by Tukey’s multiple comparison test. Bars assigned different superscript letters (a, b, c) are statistically different at p<0.05. MUC-2: mucin 2. KLF4: Krüppel-like factor 4. TFF3: Trefoil factor of 3.
Figure 2. Omega-3 tissue enrichment protects fat-1 mice against HFD-induced structure changes of the colonic mucus layer.
Mice (fat-1 transgenic and WT littermates) were fed CTL or HF diet for 11 weeks. a: Representative electron microscopy images of colons from WT and fat-1 mice (three mice per group) fed a control (CTL) or a high-fat diet (HFD). The enlarged images of the second row are from a part of the first row (scale bar, 1 mm). b: Electron microscopy images (Magnification =X50.0k) of WT and FAT1 mice (three mice per group) fed a control (CTL) or a high-fat diet (HFD).
Figure 3. Markers of the colonic endoplasmic reticulum stress are alleviated in HFD-fed fat-1 mice.
Mice (fat-1 transgenic and WT littermates) were fed CTL or HF diet for 11 weeks. a: Representative photographs (20X objective) of immunohistochemistry for BiP in the distal colon of WT (top) and fat-1 mice (bottom) fed CTL or HF diet. Colonic mRNA expression of GRP78/Bip. b: Colonic mRNA expression of Chop, Edem1 and colonic protein expressions of CHOP, eIF2α and Phospho-eIF2α (Ser51) (n = 8 per group). Data are shown as mean ± SEM, and differences were analyzed by Tukey’s multiple comparison test. Bars assigned different superscript letters (a, b, c) are statistically different at p < 0.05. BiP: Binding immunoglobulin protein. GRP78: glucose-regulated protein 78. CHOP: C/EBP Homologous Protein. Edem1: ER degradation enhancer, mannosidase alpha-like 1. eIF2α: eukaryotic initiation factor 2.
Figure 4. Impact of high fat feeding and omega-3 fatty acid tissue enrichment on colonic autophagy.
Mice (fat-1 transgenic and WT littermates) were fed CTL or HF diet for 11 weeks. a: Expression of LC3 in the colon tissue from WT and fat-1 mice fed CTL or HF diet by western blot analysis. b: Relative mRNA expression of Ulk1, P62, LC3 and Vamp8 in the colon tissue of mice as detected by qPCR. c: Representative images of LC3 and VAMP8 immunofluorescence in colon sections of WT and fat-1 mice fed the CTL or HF diet (Scale bar 20 µm). Colocalization of Vamp8 and LC3 within tissue sections was assessed from ROIs within the epithelium and calculated for the Pearson correlation coefficient with a value of 1 indicating perfect colocalization and 0 indicating no colocalization (six mice per group, >10 ROIs per mouse). Data are shown as mean ± SEM, and differences were analyzed by Tukey’s multiple comparison test. Bars assigned different superscript letters (a, b, c) are statistically different at p < 0.05. ULK1: unc51-like kinase-1. P62: Sequestosome-1. LC3: Microtubule-associated protein 1A/1B-light chain 3. VAMP8: Vesicle-associated membrane protein 8.
Figure 5. Transplantation of fat-1 microbiome decreases weight gain, enhances metabolic parameters and alleviates intestinal alteration in mice fed a HFD.
WT mice were transplanted with the microbiome of WT or fat-1 mice and fed CTL or HF diet for 12 weeks. a: Weight gain curves (n = 8 per group). b: OGTT and (inset) mean area under the curve (AUC) measured between 0 and 120 min after glucose loading (n = 6 per group). c: ITT measured between 0 and 90 min after glucose loading (n = 6 per group). d: Food intake has been carried out at 6 and 8 weeks of the feeding period (n = 8 per group). e: Intestinal permeability assay: Plasma FITC-dextran 4 (DX, 4,000 molecular weight) oral challenge was measured in mice (n = 8 per group) fed CTL or HF diet for 12 weeks. f: Direct plasma quantitation of 3-β-hydroxymyristic (OH) acid concentration by gas chromatography-mass spectrometry in mice fed CTL or HF diet for 12 weeks (n = 8 per group). Data are shown as mean ± SEM, and differences were analyzed by Tukey’s multiple comparison test. Data with different superscript letters (a, b, c) are significantly different at p < 0.05. WTW CTL: WT mice transplanted with microbiota of WT mice fed a CTL diet, WTW HFD: WT mice transplanted with microbiota of WT mice fed a HFD, WTF CTL: WT mice transplanted with microbiota of fat-1 mice fed a CTL diet, and WTF HFD: WT mice transplanted with microbiota of fat-1 mice fed a HFD. OGTT: Oral Glucose Tolerance Test. ITT: Insulin Tolerance Test.
Figure 6. Transplantation of the fat-1 microbiome prevents the increase of the thickness of the colonic mucus layer in mice fed the HF diet.
WT mice were transplanted with the microbiome of WT or fat-1 mice and fed CTL or HF diet for 12 weeks. a: Representative photographs of Carnoy-fixed colonic tissue sections stained with Alcian blue/periodic acid-Schiff of mice transplanted with the microbiome of WT or fat-1 mice and fed a CTL or HF diet (n = 8 per group) used for mucus layer thickness measurements (scale bars, 100 µm). b: Colonic mRNA expression of Muc2 and Klf4 (n = 8 per group). Data are shown as mean ± SEM, and differences were analyzed by Tukey’s multiple comparison test. Bars assigned different superscript letters (a, b, c) are statistically different at p < 0.05. WTW CTL: WT mice transplanted with microbiota of WT mice fed a CTL diet, WTW HFD: WT mice transplanted with microbiota of WT mice fed a HFD, WTF CTL: WT mice transplanted with microbiota of fat-1 mice fed a CTL diet, and WTF HFD: WT mice transplanted with microbiota of fat-1 mice fed a HFD. MUC2: mucin 2. KLF4: Krüppel-like factor 4.
Figure 7. Transplanting fat-1 microbiome protects HFD-fed WT mice against colonic endoplasmic reticulum stress.
WT mice were transplanted with the microbiome of WT or fat-1 mice and fed CTL or HF diet for 12 weeks. a: Colonic mRNA expressions of Chop, Atf4, and Edem1 I (n = 8 per group). b: Colonic mRNA expression of Lc3, Ulk1, P62 and Vamp8 (n = 8 per group). Data are shown as mean ± SEM, and differences were analyzed by Tukey’s multiple comparison test. Bars assigned different superscript letters (a, b, c) are statistically different at p < 0.05. WT mice transplanted with microbiota of WT mice fed a CTL diet, WTW HFD: WT mice transplanted with microbiota of WT mice fed a HFD, WTF CTL: WT mice transplanted with microbiota of fat-1 mice fed a CTL diet, and WTF HFD: WT mice transplanted with microbiota of fat-1 mice fed a HFD. CHOP: C/EBP Homologous Protein. ATF4: Activating transcription factor 4. Edem1: ER degradation enhancer, mannosidase alpha-like 1. LC3: Microtubule-associated protein 1A/1B-light chain 3. ULK1: Unc-51 like autophagy activating kinase. P62: Sequestosome-1. VAMP8: vesicle-associated membrane protein; 8.
Figure 8. Impact of HFD on microbiota analysis in WT mice transplanted with WT or fat-1 microbiome.
WT mice were transplanted with the microbiome of WT or fat-1 mice and fed CTL or HF diet for 12 weeks. a: The NMDS representation of the microbiota of mice transferred with control donor or fat-1 donors allows discrimination of the four groups at week 12 based on the Bray-Curtis distance. The same color code as was used. The Stress in the NMDS representation was 16.9%. b: Major bacterial genera encountered in WT mice transplanted with the microbiome of WT or fat-1 mice and fed CTL or HF diet
Figure 9. Impact of HFD on metabolites in the cecum of WT mice transplanted with WT or fat-1 microbiome.
WT mice were transplanted with the microbiome of WT or fat-1 mice and fed CTL or HF diet for 12 weeks. Metabolites were analyzed in the cecal content using NMR-based metabolomics. a: Relative concentrations of microbiota-derived metabolites. b: Relative concentrations of metabolites linked to choline metabolism and bile acids. For bile acids, the chemical shift of the peak used for quantification was indicated in ppm. c: Relative concentration of amino acids. Data are shown as mean ± SEM, and differences were analyzed by Tukey’s multiple comparison test. Bars assigned different superscript letters (a, b) are statistically different at p < 0.05.
Table 1. Identification of metabolites in cecal content NMR spectra.
Supplemental material
Supplemental Material
Download Zip (526.2 KB)Data availability statement
The Illumina MiSeq cecal microbiota analysis sequences were submitted to the Short-Read Archive with accession number PRJNA946706.