Article Figures & Data
Figures
- FIG 1
L. paracasei (Lp) and E. coli (Ec) colonization modulate intestinal lipid metabolism (n = 7 to 8 mice per group). Conventional mice were administered a microbiota-depleting antibiotic treatment before being gavaged with water (control), L. paracasei, or E. coli and maintained on a chow diet for 8 weeks. (A and B) Terminal microbiota composition assessed by conventional culture methods in the (A) feces and (B) ileum. (C and D) Terminal blood serum 1-h fasting levels of (C) TG and (D) ApoB-48 concentrations. (E) Representative confocal images (of 5 per condition) of jejunum sections stained with BODIPY showing intracellular lipid droplets. (F and G) Quantitative imaging analysis of intratissular intestinal LD using Imaris software, showing (F) mean LD number per condition and (G) LD distribution according to the size of the droplets. In panels A to D, F, and G, results are expressed as means ± standard errors of the means (SEM). Statistical significance is expressed relative to the control data unless otherwise specified. *, P < 0.05; **, P < 0.01 (one-way ANOVA).
- FIG 2
Intestinal and hepatic reprogramming of gene expression following L. paracasei (Lp) and E. coli (Ec) colonization (n = 7 to 8 mice per group). Conventional mice were administered a microbiota-depleting antibiotic treatment before being gavaged with water (control), L. paracasei or E. coli and maintained on a chow diet for 8 weeks. (A and B) terminal intestinal expression levels assessed by RT-qPCR of (A) PPAR-controlled genes and (B) lipogenic genes. (C) Liver weight. (D) Liver TG content expressed in micromoles per gram of liver weight. (E to G) Terminal hepatic expression levels assessed by RT-qPCR of (E) regulators of lipid metabolism, (F) PPAR targets, and (G) SREBP-2 targets involved in cholesterol homeostasis. (H) Liver total cholesterol content expressed in micrograms per gram of liver weight. In panels A and B and panels E to G, results are normalized to the Actin gene and expressed as means of fold change relative to the control ± SEM. In panels C, D, and H, results are expressed as means ± SEM. Statistical significance is expressed relative to the control unless otherwise specified. *, P < 0.05; **, P < 0.01 (one-way ANOVA).
- FIG 3
L. paracasei (Lp) and E. coli (Ec) impair TG secretion and lipid metabolism of cultured enterocytes. m-ICcl2 cells were polarized on transwell inserts for 14 to 21 days before infection of the upper compartment with bacteria (multiplicity of infection [MOI] 100). Supernatants and cell lysates were recovered after 16 h of infection. Control: noninfected cells. (A) Intracellular and supernatant TG levels. Results are normalized to control data and expressed as means ± SEM. (B and C) mRNA levels of genes involved in host lipid metabolism, including (B) PPAR-controlled genes and (C) SREBP-1c targets, assessed by RT-qPCR using Actin as reporter gene. Results are expressed as means of fold change ± SEM relative to the control. (D) Western blot analysis of SREBP-1 (3 experiments were performed in duplicate). Representative sample blots of total cell lysates show the cytoplasmic precursor (p125) and the nuclear form (p68) of SREBP-1 protein. (A to C) Statistical significance is expressed relative to the control unless otherwise specified. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA). A total of ≥3 experiments were performed in triplicate.
- FIG 4
L. paracasei (Lp) and E. coli (Ec) inhibit the Akt/mTOR pathway in cultured enterocytes. m-ICcl2 cells were polarized on transwell inserts for 14 to 21 days before infection of the upper compartment with bacteria (MOI of 100); cell lysates were recovered following 16 h of infection. Control: non-exposed cells. (A and B) Western blot analysis of (A) S6 signaling and (B) Akt signaling. Cell lysates were subjected to Western blot analysis for (A) phosphorylated and total endogenous S6 and S6K1 and for (B) phosphorylated and total endogenous Akt. Representative sample blots and quantifications of S6 and Akt phosphorylation levels are shown. Values are presented as ratios between levels of phosphorylated protein S6 (A) or Akt and total endogenous protein (B), normalized to control cells. (C) mRNA levels of Srebf1 and Acaca assessed by RT-qPCR using the Actin gene as the reporter gene following 16 h of coincubation with bacteria and with the mTOR activator 3-BDO. Results are expressed as means of fold change ± SEM. Statistical significance is expressed relative to the control unless otherwise specified. *, P < 0.05; **, P < 0.01 (one-way ANOVA). A total of 3 experiments were performed in duplicate.
- FIG 5
L. paracasei (Lp) and E. coli (Ec) inhibit chylomicron secretion by cultured enterocytes through increased lipid storage and catabolism, respectively. m-ICcl2 cells were polarized on transwell inserts for 14 to 21 days before infection of the upper compartment with bacteria (MOI of 100). Following 16 h of stimulation, the upper compartment cellular medium was replaced by fresh medium containing BODIPY C12 fluorescent lipid micelles for 10 min, then replaced by regular cell culture medium (time zero). Supernatants were sampled and cells were fixed for staining at the indicated time points. Control: non-exposed cells. (A) Secretion kinetics of BODIPY C12. The lower compartment cellular medium of the culture chamber was sampled at the indicated time points and the fluorescence measured. Values are expressed as means ± SEM of ratios between relative fluorescence units (RFU) of stimulated cells and RFU of control cells 4 h after the addition of lipid micelles. (B) ApoB-48 concentration in the lower compartment cell medium prior to and 6 h after the addition of lipid micelles. Results are expressed as means ± SEM. (C) Representative confocal microscopy images 4 h after the addition of lipid micelles showing incorporation of BODIPY C12 in intracellular LD (in green). Scale bar, 20 µm. (D and E) Quantitative imaging analysis of LD following the addition of lipid micelles using Imaris software. (D) LD numbers and mean sizes and proportions of LD larger than 4.95 µm3 at the indicated time points. (E) Distribution of lipid droplets according to size 4 h after the addition of lipid micelles. (A and B) A total of ≥3 experiments were performed in triplicate. (C to E) A total of 3 experiments were performed in duplicate, representing a total of 18 images per condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (panels A and B, one-way ANOVA; panels C and D, two-way ANOVA).
- FIG 6
Soluble bacterial factors partially reproduce lipid metabolism modifications induced by live bacteria on cultured enterocytes. m-ICcl2 cells were polarized on transwell inserts for 14 to 21 days before infection of the upper compartment with bacteria (MOI of 100) or exposure to bacterial culture supernatants (CS), conditioned media (CM), heat-killed (HK) bacteria, or acidic pH. Following 16 h of incubation, the fluorescent lipid secretion assay was performed as described for Fig. 5 or cell lysates were collected for gene expression analysis. (A and B) Secretion kinetics of BODIPY C12. (C) mRNA levels of genes involved in host lipid metabolism under corresponding L. paracasei (Lp) conditions. (D) Secretion kinetics of BODIPY C12. (E and F) mRNA levels of genes involved in host lipid metabolism under corresponding E. coli (Ec) conditions. In panels A, B, and D, the lower compartment cellular medium of the culture chamber was sampled at the indicated time points and the levels of fluorescence were measured. Values are expressed as means ± SEM of ratios between RFU of exposed cells and RFU of control cells 4 h after the addition of lipid micelles. In panels C, E, and F, mRNA levels assessed by RT-qPCR using the Actin gene as the reporter gene are shown. Results are expressed as means of fold change ± SEM relative to the control. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA). A total of ≥3 experiments were performed in triplicate.
- FIG 7
L. paracasei (Lp) and E. coli (Ec) colonization affect lipid secretion by enterocytes and the host’s response to a HFD (n = 7 to 8 mice per group). Conventional mice were administered a microbiota-depleting antibiotic treatment before being gavaged with water (control), L. paracasei, or E. coli and maintained on a CD or submitted to a HFD for 8 weeks. (A) Daily food intake per mouse starting from week 3 following L. paracasei or E. coli colonization. (B) Body weight gain. (C and D) Terminal blood serum 1-h fasting levels of (C) leptin and (D) total cholesterol. (E) Terminal liver TG content expressed in micromoles per gram of liver weight. (F) Terminal blood serum 1-h fasting levels of ApoB-48. (G to I) Confocal imaging of LD stained with BODIPY in jejunum sections. (G) Representative confocal images (of 5 per condition) showing intracellular lipid droplets in mice submitted to a HFD. (H and I) Quantitative imaging analysis of intratissular intestinal LD using Imaris software showing (H) LD number and (I) large LD (>4.95 µm3) number. (J to M) Intestinal expression levels assessed by RT-qPCR of (J) PPAR targets involved in fatty acid oxidation (K) and in TG and chylomicron biosynthesis and of (L) Fabp2 and (M) SREBP2 targets involved in cholesterol homeostasis. Results are normalized to the Actin gene and expressed as means of fold change relative to the control ± SEM. In panels A to F, H, and I, results are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA).
- FIG 8
Model for L. paracasei and E. coli impact on lipid absorption and secretion by enterocytes. Once taken up by the brush border of the enterocytes, FA released by dietary lipid micelles are either reesterified in TG or degraded through beta-oxidation. Newly synthetized TG are incorporated into cytosolic LD or into chylomicrons, which are then secreted in the lymphatic system. Under normal chow diet conditions, L. paracasei participates in digestion of polysaccharides, provides monosaccharides to epithelial cells, and decreases FA absorption, and the newly synthetized TG are preferentially stored in cytosolic LD. Conversely, E. coli competes for monosaccharides and disaccharides with epithelial cells and induces an increase in FA absorption and beta-oxidation, leading to reduced TG synthesis, LD size, and chylomicron secretion.
Supplemental Material
FIG S1
Simplified schematic view of lipid absorption and metabolism in enterocytes. The main steps of intracellular lipid metabolism are represented together with the main transcriptional regulators in brackets (1). Dietary triglycerides (TG) are emulsified by bile acids and digested by the pancreatic lipase in fatty acids (FA), monoacylglycerides (MAG), and diacylglycerides (DAG), which are taken up by the brush border and absorbed by passive diffusion or by the fatty acid translocase FAT/CD36 and the scavenger receptor B1 SR-B1. Dietary free cholesterol is absorbed by the cholesterol transporter NPC1L1 (2). Cholesterol absorption and metabolism are mainly regulated by SREBP-2, whose activity depends on the intracellular cholesterol content (3). FA, MAG, and DAG are reesterified in TG (4). Absorbed FA can be degraded through FA beta-oxidation and FA oxidation, and TG and chylomicrons synthesis is regulated by the three types of PPARs (PPARα, PPARβ/δ, and PPARγ) upon activation by their ligands, mainly polyunsaturated FA (5). FA de novo biosynthesis depends on LXR, ChREBP, and SREBP-1c, which are regulated by insulin signaling and glucose (6). TG are taken up by the microsomal-triglyceride transfer protein (MTTP) and incorporated with cholesterol, esterified cholesterol, and apolipoproteins in prechylomicrons, which are eventually secreted in the mesenteric lymph as chylomicrons or stored in cytosolic lipid droplets. Download FIG S1, TIF file, 0.6 MB.
Copyright © 2018 Tazi et al.
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TABLE S1
Small intestine gene expression levels assessed by RT-qPCR in mice colonized with L. paracasei or E. coli in chow diet. Download TABLE S1, DOCX file, 0.02 MB.
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TABLE S2
Hepatic gene expression levels assessed by RT-qPCR in mice colonized with L. paracasei or E. coli in chow diet. Download TABLE S2, DOCX file, 0.1 MB.
Copyright © 2018 Tazi et al.
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FIG S2
L. paracasei and E. coli modulate BODIPY C12 incorporation in intracellular LD. m-ICcl2 cells were polarized on transwell inserts for 14 to 21 days before infection of the upper compartment with bacteria (MOI of 100) was performed. (A) Supernatants were recovered after 16 h of stimulation, and cytotoxicity was assessed by measurement of LDH release. Control: non-exposed cells. A total of ≥3 experiments were performed in triplicate (one-way ANOVA). (D to G) Following 16 h of stimulation, the upper-compartment cellular medium of the culture chamber was replaced by fresh medium containing BODIPY C12 fluorescent lipid micelles and was then replaced by regular cell culture medium following 10 min of incubation. Supernatants were sampled, and cells were fixed for staining at the indicated time points. Control: non-exposed cells. (B and C) Representative confocal microscopy images (B) 1 h and (C) 2 h after the addition of lipid micelles, showing incorporation of BODIPY C12 in intracellular LD in green. Scale bar, 20 µm. (D to G) Quantitative image analysis of LD by the use of Imaris software following the addition of lipid micelles. Data represent the distribution of LD according to droplet size 1 h (D and E) and 2 h (F and G) after the addition of lipid micelles. Panels E and G are enlarged views of panels D and F, respectively, showing the proportions of LD that were ≥4.95 µm3 in size. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-way ANOVA). A total of 3 experiments were performed in duplicate. Download FIG S2, TIF file, 1.3 MB.
Copyright © 2018 Tazi et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S3
m-ICcl2 gene expression levels assessed by RT-qPCR following 16 h of infection with L. paracasei or E. coli. Download TABLE S3, DOCX file, 0.02 MB.
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TABLE S4
Impact of bacterial factors and acidic pH on m-ICcl2 enterocytes. Download TABLE S4, DOCX file, 0.1 MB.
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FIG S3
Expression levels of cytokine genes in enterocytes stimulated with L. paracasei-based and E. coli-based media. m-ICcl2 cells were polarized on transwell inserts for 14 to 21 days before infection of the upper compartment was performed with bacteria (MOI of 100) or exposure to bacterial culture supernatants (CS), conditioned media (CM), heat-killed (HK) bacteria, or acidic pH. Following 16 h of incubation, total RNAs were extracted for gene expression analysis of cytokines under (A) L. paracasei-based and (B) E. coli-based conditions using RT-qPCR with the actin gene used as a reporter gene. Results are expressed as means of fold change ± SEM relative to the control data. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA). A total of ≥3 experiments were performed in triplicate. Download FIG S3, TIF file, 1.5 MB.
Copyright © 2018 Tazi et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S4
L. paracasei and E. coli gut colonization in SPF mice submitted to a HFD following a microbiota-depleting treatment (n = 7 to 8 mice per group). Conventional mice were administered a microbiota-depleting antibiotic treatment before being gavaged with water (control), L. paracasei, or E. coli and were switched to a HFD for 8 weeks. Terminal microbiota composition was assessed by conventional culture methods in the feces (A) and ileum (B). Download FIG S4, TIF file, 0.6 MB.
Copyright © 2018 Tazi et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S5
Small intestine gene expression levels assessed by RT-qPCR in mice colonized with L. paracasei or E. coli under HFD conditions. Download TABLE S5, DOCX file, 0.1 MB.
Copyright © 2018 Tazi et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S6
Primers used for RT-qPCR (5′–3′). Download TABLE S6, DOCX file, 0.1 MB.
Copyright © 2018 Tazi et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
