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Contributed by Philippe J. Sansonetti, August 1, 2016 (sent for review February 28, 2016; reviewed by Fredrik Bäckhed, Lora V. Hooper, and Peter Turnbaugh)
Article Figures & SI
Figures
- Fig. S1.
Metabolic parameters after 1 mo of diet. (A) Body weight gain (%) in mice fed SD and HF diet for 4 wk. (B) Epidydymal fat pads (percent of total body weight) in SD-fed and HF-fed mice for 4 wk. (C) Daily food intake (kilocalories per mouse) in SD-fed and HF-fed mice for 4 wk. (D) Plasma glucose (mg/dL) after an oral glucose load (1 g/kg in 0.9% NaCl) in SD-fed and HF-fed mice for 4 wk. All values are means ± SEM. Means with asterisks are significantly different from values of SD-fed mice (**P < 0.01). NS, not significant.
- Fig. 1.
HF diet shapes the spatial segregation and the composition of intestinal microbiota. (A) Representative photographs of FISH analyses with the pan-bacterial probe Eub338 (red) and the SFB-1008 probe (green) in ileum sections of SD- and HF-fed mice. Nuclei stained with DAPI (blue). (Scale bar, 50 μm.) (B) PCoA plot showing microbiota communities cluster with diet determined by unweighted UniFrac analysis. SD cecum (blue), SD feces (green), HF cecum (black), HF feces (red). (C) Average relative abundance at phylum level in cecal and fecal contents, and in microdissected ileum content of SD- and HF-fed mice.
- Fig. S2.
HF diet shapes the spatial segregation and the composition of intestinal microbiota. Representative photographs of FISH analyses with the pan-bacterial probe Eub338 (red) in unwashed duodenum and jejunum sections of SD- and HF-fed mice. Nuclei stained with DAPI (blue). (Scale bars, 50 μm.)
- Fig. S3.
HF diet modifies the genus microbiota composition in cecal and fecal contents. Individual relative abundance at genus level in cecal and fecal contents of SD- and HF-fed mice (n = 5).
- Fig. S4.
HF diet modifies the microbiota composition in LCM ileum. Average relative abundance at genus level in microdissected ileum content of SD- and HF-fed mice (n = 5).
- Fig. S5.
HF diet alters specifically the ileal Cftr chloride secretion pathway. (A) Measurement in Ussing chamber of the short-circuit current increase (Forskolin-induced Isc, μA/cm2) after serosal challenging of duodenum and jejunum tissues with 10 µmol/L forskolin (n = 8–9 per group). NS, not significant. (B) Gene expression relative of Cftr from HS and HF/HS diets (n = 8 per group) compared with SD-fed mice in ileum. Results were normalized to those for the Gapdh gene expression. All values are means ± SEM (**P < 0.01); NS, not significant. (C) Tissue conductance measurements in duodenum and jejunum (n = 7–11 per group). Conductance was studied in Ussing chamber after 30 min (HF/TD ratio). Values are expressed as a ratio of HF-fed mice values compared with SD-fed mice. NS, not significant. (D) Representative photographs of immunofluorescence staining of claudin-7 protein in unwashed ileum sections of SD- and HF-fed mice. Nuclei stained with DAPI (blue). (Scale bars, 50 μm.)
- Fig. 2.
HF diet alters differently AMP gene expression in duodenum, jejunum, and ileum. (A) Gene expression relative to AMP genes from HF-fed mice compared with SD-fed mice in duodenum, jejunum, and ileum (n = 10–12 per group). Results were normalized to those for the Gapdh gene expression. All values are means ± SEM. Means with asterisks are significantly different from values of SD-fed mice (**P < 0.01, ***P < 0.001). NS, not significant. (B) Representative photographs of immunofluorescence staining of Reg3γ protein (green) in unwashed duodenum, jejunum, and ileum sections of SD- and HF-fed mice. Nuclei stained with DAPI (blue). (Scale bars, 50 μm.)
- Fig. 3.
HF diet alters ileal permeability and affects the Cftr chloride secretion pathway. (A) Measurement in Ussing chamber of the increase in short-circuit current (ΔIsc, μA/cm2) induced after serosal challenging of ileum tissue with 10 µmol/L forskolin. Values are expressed as a ratio of HF-fed mice values compared with SD-fed mice (Forskolin-induced Isc ratio). (B) Gene expression relative of electrolyte transporters genes from HF-fed mice compared with SD-fed mice in ileum (n = 8–11). Results were normalized to those for the Gapdh gene expression. All values are means ± SEM. (C) Representative photographs of immunofluorescence staining of CFTR (green) and NKCC1 (red) proteins in unwashed ileum sections of HF- and SD-fed mice. Nuclei stained with DAPI (blue). (Scale bars, 50 μm.) (D) Tissue conductance measurements in duodenum, jejunum and ileum (n = 7–11 per group). Conductance was studied in Ussing chamber after 30 min (HF/TD ratio). Values are expressed as a ratio of HF-fed mice values compared with SD-fed mice. Means with asterisks are significantly different from values of SD-fed mice (*P < 0.05, **P < 0.01). NS, not significant.
- Fig. 4.
HF diet alters the mucus secretion pathway in ileum. (A) Representative photographs of immunofluorescence staining of MUC2 protein in unwashed and Carnoy fixed ileum sections. The red arrows indicate accumulation of MUC2 at the surface of the goblet cells. (Scale bars, 50 µm.) (B) Gene expression relative to Meprin-β from HF-fed mice compared with SD-fed mice in ileum (n = 8–11). Results were normalized to those for the Gapdh gene expression. All values are means ± SEM. (**P < 0.01) (C) Representative photographs of immunofluorescence staining of Meprin-β in unwashed and Carnoy fixed ileum sections of SD- and HF-fed mice. (Scale bars, 50 µm.) Means with asterisks are significantly different from values of SD-fed mice.
- Fig. S6.
HF diet alters the mucus secretion pathway in ileum but not in duodenum and jejunum. (A) Gene-expression relative of mucus from HF-fed mice compared with SD-fed mice in ileum (n = 7–12). Results were normalized to those for the Gapdh gene expression. All values are means ± SEM. NS, not significant. (B) Representative photographs of immunofluorescence staining of MUC2 protein in unwashed and Carnoy fixed duodenum and jejunum sections in SD-fed mice and in HF-fed mice. (Scale bars, 50 µm.)
- Fig. 5.
Reversing from the HF diet to the SD corrects the bacterial spatial segregation and recovers the expression of the AMP Cftr and Ppar-γ genes. (A) Representative photographs of FISH analyses with the pan-bacterial probe Eub338 (red) in ileum sections of mice switched from HF diet to SD for 30 d. Nuclei stained with DAPI (blue). (Scale bar, 50 μm.) (B) Graphs showed the gene expression relative to AMP and electrolytes transporters of HF-fed mice vs. HF-fed mice for 2 mo and HF-rev mice vs. HF-fed mice for 2 mo compared with SD-fed mice in the ileum (n = 4). Results were normalized to those for the Gapdh gene expression. All values are means ± SEM. Means with stars are significantly different from values of SD-fed mice (*P < 0.05, ***P < 0.001). NS, not significant. (C) PCoA plot showing microbiota communities cluster in feces of mice determined by unweighted UniFrac analysis. SD-fed mice (blue), HF-fed mice (red), and HF-rev mice (black).
- Fig. 6.
Ppar-γ pathway regulates the expression of AMP and Cftr and activation by rosiglitazone corrects the HF-diet effects in the ileum. (A) Gene expression relative of Ppar-γ in the ileum (n = 8–11). HF-rosi and SD-rosi are mice treated with rosiglitazone for 1 wk. Results were normalized to those for the Gapdh gene expression. (B) Gene expression relative of Cftr in ileum (n = 8–10 per group). Results were normalized to those for the Gapdh gene expression (C) Gene expression relative of AMP in the ileum (n = 8–10 per group). Results were normalized to those for the Gapdh gene expression and compared with the mean target gene expression in SD mice. All values are means ± SEM. Means with asterisks are significantly different from values of SD-fed mice following one-way ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001). NS, not significant.
- Fig. 7.
Rosiglitazone restores the spatial distribution of the microbiota in the ileum. (A) Representative photographs of FISH analyses with the pan-bacterial probe Eub338 (red) in ileum sections of SD-rosi and HF-rosi mice. Nuclei stained with DAPI (blue). (Scale bars, 50 μm.) (B) Representative photographs of immunofluorescence staining of MUC2 protein in ileum sections of SD-rosi and HF-rosi mice. (Scale bars, 50 µm.) (C) Representative photographs of immunofluorescence staining of Meprin-β in ileum sections of SD-rosi and HF-rosi mice. (Scale bars, 50 µm.) (D) Representative photographs of FISH analyses with the pan-bacterial probe Eub338 (red) in ileum sections of PPAR-γVillinCre+ mice under SD feeding, and their littermate control mice PPAR-γVillinCre− mice under SD feeding. Nuclei stained with DAPI (blue). (Magnification: 100×.) (E) Gene expression level of PPAR-γVillinCre+ mice mice under SD feeding compared with PPAR-γVillinCre− mice under SD feeding in the ileum. Results were normalized to those for the Gapdh gene expression. All values are means ± SEM.
Tables
Diets Proteins (%kcal/kg) Fat (%kcal/kg) Carbohydrates (%kcal/kg) Calories (kcal/kg) SD (R03-40) 30.3 16.2 53.5 2,827 HF (231HF) 21 71 8 5,053 HS 22.5 11.5 66 3,979 HF/HS (230HF) 13 61 26 5,317 Name Forward (5′→3′) Reverse (5′→3′) gapdh CTTATCAGGCCAAGTATGATG CAACCTGGTCCTCAGTGTAGC muc2 TACGCTCTCCACCAGTTCCT CAGCTCTCGATGTGTGTGTAGGT muc3 TGGTCAACTGCGAGAATGGA TACGCTCTCCACCAGTTCCT meprin-β CAGGCAAGGAACACAACTTC TCTGTCCCGTTCTGGAAAG klf4 AGAGGAGCCCAAGCCAAAGAGG CCACAGCCGTCCCAGTCACAGT retnlß TGGCTGTGGATCGTGGGATA TAAACCATTCGGCAGCAGCG mmp-7 CTGCCACTGTCCCAGGAAG GGGAGAGTTTTCCAGTCATCG lyz GAGACCGAAGCACCGACTATG CGGTTTTGACATTGTGTTCGC reg3γ CCATCTTCACGTAGCAGC CAAGATGTCCTGAGGGC pla2g4A GGCCTTTGGCTCAATACAGGT C ACAGTGGCATCCATAGAAGGCA ang4 TGGCCAGCTTTGGAATCACTG GCTTGGCATCATAGTGCTGACG defa3 TCCTCCTCTCTGCCCTCGT GACCCTTTCTGCAGGTCCC defa5 GTCCAGGCTGATCCTATCCA GATTTCTGCAGGTCCAAAA defa20 GACCTGCTCAGGACGACTTT GCCTCAGAGCTGATGGTTGT cftr AAGGCGGCCTATATGAGGTT AGGACGATTCCGTTGATGAC nkcc1 CAAGGGTTTCTTTGGCTAT TCACCTGAGATATTTGCTCC slc26A3 TTCCCCTCAACATCACCATCC GTAAAA TCGTTCTGAGGCCCC slc26A6 CCAAAC ATA GGAGGCAATCC GGTATCCTGTGCGTGAATGGCTC nhe3 TGGCAGAGACTGGGATGATAA CGCTGACGGATTTGATAGAGA ano1 AGCAGGCTTCTGACCATCAC CACGTCCAGACGACACAAGA ppar-γ CCAGCATTTCTGCTCCACAC ATTCTTGGAGCTTCAGGCCA
Data supplements
Supporting Information
High fat alters the small intestinal ecosystem
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