Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis
<p>DSS-induced colonic injury and inflammation in WT and IEC AMPK KO mice. (<b>A</b>) Experimental timeline for 4% DSS-induced colitis in female WT and IEC AMPK KO mice. Mice were treated with 4% DSS in drinking water for four consecutive days. Changes in (<b>B</b>) body weight and (<b>C</b>) colon length in WT and IEC AMPK KO mice on day 4 of 4% DSS-induced colitis (<span class="html-italic">n</span> = 5–10 mice/group). (<b>D</b>) In vivo paracellular intestinal epithelial permeability of WT and IEC AMPK KO mice on day 4 during 4% DSS-induced colitis protocol. Dextran flux was determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage (<span class="html-italic">n</span> = 10–14 mice/group). (<b>E</b>) Expression of mRNA for Il-6, TNF-α and IL-1β in the colon from WT and IEC AMPK KO mice on day 4 (<span class="html-italic">n</span> = 16–23 mice/group). The results are pooled from four independent experiments. Data are expressed as means ± SD. Statistical analysis was performed by one-way ANOVA with the post hoc Bonferroni’s multiple comparisons test; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001 indicate a significant change relative to the untreated condition.</p> "> Figure 2
<p>Absence of IEC AMPK delays intestinal epithelial recovery after DSS-induced injury. (<b>A</b>) Experimental timeline for 2.5% DSS-induced colitis in female WT and IEC AMPK KO mice. Mice were treated with 2.5% DSS in drinking water for five consecutive days, followed by six days of regular water. (<b>B</b>) Survival rate during the experimental period (<span class="html-italic">n</span> = 36–41 mice/group). The results are pooled from three independent experiments. (<b>C</b>) Evolution of body weight during the progression of colitis induced by 2.5% DSS in WT and IEC AMPK KO mice (<span class="html-italic">n</span> = 17–20 mice/group). The results are pooled from two independent experiments. (<b>D</b>) Colon length on day 10 following DSS challenge (<span class="html-italic">n</span> = 4–8 mice/group). (<b>E</b>) In vivo paracellular intestinal epithelial permeability of WT and IEC AMPK KO mice on day 9 during 2.5% DSS-induced colitis protocol. Dextran flux was determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. Data are presented as mean dextran flux fold change relative to respective control mice (<b><span class="html-italic">n</span></b> = 6–7 mice/group). (<b>F</b>) Expression of mRNA for tight junction proteins occludin and ZO-1 in the distal colon of WT and IEC AMPK KO mice on day 9 of the recovery phase (<span class="html-italic">n</span> = 5–8 mice/group). (<b>G</b>) Representative images of H&E-stained sections of Swiss-rolled colonic specimens (with proximal gut in the center and distal colon in the exterior) from IEC AMPK KO and WT mice on day 9. Pictures are representative of 10–13 mice/group. Ulceration is highlighted in blue. (<b>H</b>) Higher magnification images of colon sections. Scale bars = 50 µm. (<b>I</b>) Histological damage score (<span class="html-italic">n</span> = 10–13 mice/group) and quantification of percentage of colon ulceration (<span class="html-italic">n</span> = 5–7 mice/group). (<b>J</b>) Expression of mRNA and (<b>K</b>) plasma levels for pro-inflammatory cytokines Il-6, TNF-α and IL-1β in IEC AMPK KO and WT mice on day 9 (<span class="html-italic">n</span> = 5–7 mice). Data are expressed as means ± SD. Statistical analysis was performed by using a Student’s <span class="html-italic">t</span> test, non-parametric Mann–Whitney test, one-way ANOVA or two-way ANOVA with Bonferroni post hoc test; ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001 indicate a significant difference relative to the untreated condition; # <span class="html-italic">p</span> < 0.05, ## <span class="html-italic">p</span> < 0.01 and ### <span class="html-italic">p</span> < 0.001 a significant difference between genotype; n.s., not significant.</p> "> Figure 2 Cont.
<p>Absence of IEC AMPK delays intestinal epithelial recovery after DSS-induced injury. (<b>A</b>) Experimental timeline for 2.5% DSS-induced colitis in female WT and IEC AMPK KO mice. Mice were treated with 2.5% DSS in drinking water for five consecutive days, followed by six days of regular water. (<b>B</b>) Survival rate during the experimental period (<span class="html-italic">n</span> = 36–41 mice/group). The results are pooled from three independent experiments. (<b>C</b>) Evolution of body weight during the progression of colitis induced by 2.5% DSS in WT and IEC AMPK KO mice (<span class="html-italic">n</span> = 17–20 mice/group). The results are pooled from two independent experiments. (<b>D</b>) Colon length on day 10 following DSS challenge (<span class="html-italic">n</span> = 4–8 mice/group). (<b>E</b>) In vivo paracellular intestinal epithelial permeability of WT and IEC AMPK KO mice on day 9 during 2.5% DSS-induced colitis protocol. Dextran flux was determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. Data are presented as mean dextran flux fold change relative to respective control mice (<b><span class="html-italic">n</span></b> = 6–7 mice/group). (<b>F</b>) Expression of mRNA for tight junction proteins occludin and ZO-1 in the distal colon of WT and IEC AMPK KO mice on day 9 of the recovery phase (<span class="html-italic">n</span> = 5–8 mice/group). (<b>G</b>) Representative images of H&E-stained sections of Swiss-rolled colonic specimens (with proximal gut in the center and distal colon in the exterior) from IEC AMPK KO and WT mice on day 9. Pictures are representative of 10–13 mice/group. Ulceration is highlighted in blue. (<b>H</b>) Higher magnification images of colon sections. Scale bars = 50 µm. (<b>I</b>) Histological damage score (<span class="html-italic">n</span> = 10–13 mice/group) and quantification of percentage of colon ulceration (<span class="html-italic">n</span> = 5–7 mice/group). (<b>J</b>) Expression of mRNA and (<b>K</b>) plasma levels for pro-inflammatory cytokines Il-6, TNF-α and IL-1β in IEC AMPK KO and WT mice on day 9 (<span class="html-italic">n</span> = 5–7 mice). Data are expressed as means ± SD. Statistical analysis was performed by using a Student’s <span class="html-italic">t</span> test, non-parametric Mann–Whitney test, one-way ANOVA or two-way ANOVA with Bonferroni post hoc test; ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001 indicate a significant difference relative to the untreated condition; # <span class="html-italic">p</span> < 0.05, ## <span class="html-italic">p</span> < 0.01 and ### <span class="html-italic">p</span> < 0.001 a significant difference between genotype; n.s., not significant.</p> "> Figure 3
<p>Altered cell proliferation but not adherence in AMPK-deficient Caco2 cells. (<b>A</b>) Representative H&E-stained colon from IEC AMPK KO and WT mice at 5 days after γ irradiation with 10 Gy. Pictures are representative of 6–10 mice/group. White arrow indicated dead crypts and * regenerated crypts. Scale bars = 50 µm. (<b>B</b>) Quantification of crypt length, and dead and regenerated crypts 5 days after irradiation (<span class="html-italic">n</span> = 6–10 mice/group). (<b>C</b>) Epithelial cell proliferation was assessed by immunostaining of Ki-67 during the recovery phase (day 8 to day 11) of DSS-induced colitis in IEC AMPK KO and WT mice. The graph shows the percentage of Ki-67-positive cells over total crypt length (<span class="html-italic">n</span> = 3–4 mice/group). The results were pooled from two triplicates. (<b>D</b>) Representative images of Ki67-stained colon sections following 2.5% DSS treatment on days 9 and 11. Pictures are representative of 3–4 mice/group. Scale bars = 50 µm. (<b>E</b>) WT and AMPK-deficient Caco2 cells proliferation was evaluated by xCELLigence real-time cell analysis (RTCA). Data are expressed as a cell index value corresponding to the proportion of the plate surface occupied by adherent cells. The results are representative of three replicates. (<b>F</b>) Proliferation was assessed with the use of the slope (h<sup>−1</sup>) of data collected with the xCELLigence software (<span class="html-italic">n</span> = 3 per genotype). (<b>G</b>) Adhesion of WT and AMPK-deficient Caco2 cells. The results are pooled from two independent experiments. Data are expressed as means ± SD. Statistical analysis was performed by using a Student’s <span class="html-italic">t</span> test, one-way ANOVA or two-way ANOVA with Bonferroni post hoc test; *** <span class="html-italic">p</span> < 0.001 indicates a significant difference relative to the untreated condition; ## <span class="html-italic">p</span> < 0.01 and ### <span class="html-italic">p</span> < 0.001 indicate a significant difference between genotype.</p> "> Figure 3 Cont.
<p>Altered cell proliferation but not adherence in AMPK-deficient Caco2 cells. (<b>A</b>) Representative H&E-stained colon from IEC AMPK KO and WT mice at 5 days after γ irradiation with 10 Gy. Pictures are representative of 6–10 mice/group. White arrow indicated dead crypts and * regenerated crypts. Scale bars = 50 µm. (<b>B</b>) Quantification of crypt length, and dead and regenerated crypts 5 days after irradiation (<span class="html-italic">n</span> = 6–10 mice/group). (<b>C</b>) Epithelial cell proliferation was assessed by immunostaining of Ki-67 during the recovery phase (day 8 to day 11) of DSS-induced colitis in IEC AMPK KO and WT mice. The graph shows the percentage of Ki-67-positive cells over total crypt length (<span class="html-italic">n</span> = 3–4 mice/group). The results were pooled from two triplicates. (<b>D</b>) Representative images of Ki67-stained colon sections following 2.5% DSS treatment on days 9 and 11. Pictures are representative of 3–4 mice/group. Scale bars = 50 µm. (<b>E</b>) WT and AMPK-deficient Caco2 cells proliferation was evaluated by xCELLigence real-time cell analysis (RTCA). Data are expressed as a cell index value corresponding to the proportion of the plate surface occupied by adherent cells. The results are representative of three replicates. (<b>F</b>) Proliferation was assessed with the use of the slope (h<sup>−1</sup>) of data collected with the xCELLigence software (<span class="html-italic">n</span> = 3 per genotype). (<b>G</b>) Adhesion of WT and AMPK-deficient Caco2 cells. The results are pooled from two independent experiments. Data are expressed as means ± SD. Statistical analysis was performed by using a Student’s <span class="html-italic">t</span> test, one-way ANOVA or two-way ANOVA with Bonferroni post hoc test; *** <span class="html-italic">p</span> < 0.001 indicates a significant difference relative to the untreated condition; ## <span class="html-italic">p</span> < 0.01 and ### <span class="html-italic">p</span> < 0.001 indicate a significant difference between genotype.</p> "> Figure 4
<p>Altered cell spreading in AMPK-deficient Caco2 cells. (<b>A</b>) Representative acquisitions of monolayer of WT and AMPK-deficient Caco2 cells after wound scratch. Wound delimitations are indicated with dotted lines. Pictures are representative of two independent experiments performed in triplicate. (<b>B</b>) Quantification of relative wound width between WT and AMPK-deficient Caco2 cells was assessed by measuring the distance between the boundaries of the migrating cells over time. The results are representative of two independent experiments performed in triplicate. (<b>C</b>) Representative image of wound closure two days after injury on polarized confluent WT and AMPK-deficient Caco-2 cells. Pictures are representative of 20 wounds (4 wounds/transwell, 5 transwells per condition) from three independent experiments (scale bars = 200 μM). (<b>D</b>) Variations of TEER during wound closure upon damage on polarized confluent WT and AMPK-deficient Caco-2 cells. The results are pooled from 5 transwells per condition in three independent experiments. Data are expressed as means ± SD. Statistical analysis was performed by using two-way ANOVA with Bonferroni post hoc test; # <span class="html-italic">p</span> < 0.05 and ### <span class="html-italic">p</span> < 0.001 indicate a significant difference between genotype; n.s. not significant.</p> "> Figure 5
<p>Altered goblet cell restitution after DSS-induced colitis in IEC AMPK KO cells. (<b>A</b>) High magnification of colon sections stained with Alcian Blue from WT and IEC AMPK KO mice untreated or after DSS-induced colitis on day 10. Pictures are representative of 4–6 mice/group. Scale bars = 50 µm. (<b>B</b>) Quantification of the number of goblet cells within the crypts of colons from WT and IEC AMPK KO mice untreated or after DSS challenge on day 10 (<span class="html-italic">n</span> = 4–6 mice/group). Goblet cell number was normalized by crypt depth (goblet cells/μm crypt depth) (<b>C</b>) Expression of mucin-encoding gene Muc2 (<span class="html-italic">n</span> = 3–8 mice/group). Data are expressed as means ± SD. Statistical analysis was performed by using a Student’s <span class="html-italic">t</span> test or one-way ANOVA with Bonferroni post hoc test; # <span class="html-italic">p</span> < 0.05 and ## <span class="html-italic">p</span> < 0.01 indicate a significant difference between genotype.</p> "> Figure 6
<p>Effect of metformin treatment during the recovery phase on the recovery from DSS-induced colitis in WT and IEC AMPK KO mice. (<b>A</b>) Experimental timeline for mice treated with 2.5% DSS in drinking water for five consecutive days, followed by four days of metformin (2 mg/mL) in drinking water or regular water. (<b>B</b>) Changes in body weight (<span class="html-italic">n</span> = 6–9 mice/group). (<b>C</b>) In vivo intestinal epithelial permeability on day 9. Dextran flux was determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. Data are presented as mean dextran flux fold change relative to respective control mice (<span class="html-italic">n</span> = 6–9 mice/group). (<b>D</b>) Lipocalin-2 (Lcn-2) levels in feces from WT and IEC AMPK KO mice during the course of DSS-induced colitis and treatment with metformin (2 mg/mL) in drinking water (<span class="html-italic">n</span> = 6–9 mice/group). (<b>E</b>) Experimental timeline for mice treated with 2.5% DSS in drinking water for five consecutive days, followed by five days of metformin (10 mg/mL) in drinking water or regular water. (<b>F</b>) Levels of total AMPKα and AMPKα-Thr172 phosphorylation in full-thickness colon biopsies from WT and IEC AMPK KO mice on metformin (10 mg/mL) in drinking water or regular water (<span class="html-italic">n</span> = 3 mice/group). (<b>G</b>) Quantification of AMPKα-Thr172 phosphorylation in Western blot from three replicates. (<b>H</b>) Changes in body weight (<span class="html-italic">n</span> = 6–9 mice/group). (<b>I</b>) In vivo intestinal epithelial permeability on day 10. Dextran flux was determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. Data are presented as mean dextran flux fold change relative to respective control mice (<span class="html-italic">n</span> = 6–9 mice/group). (<b>J</b>) Representative images of H&E-stained colon sections on day 10 from IEC AMPK KO and WT DSS-treated mice administrated H<sub>2</sub>O or 10 mg/mL metformin during the recovery phase. Pictures are representative of 3–4 mice/group. Scale bars = 50 µm. (<b>K</b>) Histological damage score and quantification of percentage of ulceration related to total colon length in IEC AMPK KO and WT mice upon DSS-induced epithelial damage and metformin (10 mg/mL) treatment (<span class="html-italic">n</span> = 3–4 mice/group). Data are expressed as means ± SD. Statistical analysis was performed by one-way ANOVA or two-way ANOVA with Bonferroni post hoc test; * <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01 indicate a significant difference relative to the untreated condition; # <span class="html-italic">p</span> < 0.05, ## <span class="html-italic">p</span> < 0.01 and ### <span class="html-italic">p</span> < 0.001 indicate a significant difference between genotype and §§ <span class="html-italic">p</span> < 0.01 and §§§ <span class="html-italic">p</span> < 0.001 indicate a significant difference between metformin-treated mice.</p> "> Figure 6 Cont.
<p>Effect of metformin treatment during the recovery phase on the recovery from DSS-induced colitis in WT and IEC AMPK KO mice. (<b>A</b>) Experimental timeline for mice treated with 2.5% DSS in drinking water for five consecutive days, followed by four days of metformin (2 mg/mL) in drinking water or regular water. (<b>B</b>) Changes in body weight (<span class="html-italic">n</span> = 6–9 mice/group). (<b>C</b>) In vivo intestinal epithelial permeability on day 9. Dextran flux was determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. Data are presented as mean dextran flux fold change relative to respective control mice (<span class="html-italic">n</span> = 6–9 mice/group). (<b>D</b>) Lipocalin-2 (Lcn-2) levels in feces from WT and IEC AMPK KO mice during the course of DSS-induced colitis and treatment with metformin (2 mg/mL) in drinking water (<span class="html-italic">n</span> = 6–9 mice/group). (<b>E</b>) Experimental timeline for mice treated with 2.5% DSS in drinking water for five consecutive days, followed by five days of metformin (10 mg/mL) in drinking water or regular water. (<b>F</b>) Levels of total AMPKα and AMPKα-Thr172 phosphorylation in full-thickness colon biopsies from WT and IEC AMPK KO mice on metformin (10 mg/mL) in drinking water or regular water (<span class="html-italic">n</span> = 3 mice/group). (<b>G</b>) Quantification of AMPKα-Thr172 phosphorylation in Western blot from three replicates. (<b>H</b>) Changes in body weight (<span class="html-italic">n</span> = 6–9 mice/group). (<b>I</b>) In vivo intestinal epithelial permeability on day 10. Dextran flux was determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. Data are presented as mean dextran flux fold change relative to respective control mice (<span class="html-italic">n</span> = 6–9 mice/group). (<b>J</b>) Representative images of H&E-stained colon sections on day 10 from IEC AMPK KO and WT DSS-treated mice administrated H<sub>2</sub>O or 10 mg/mL metformin during the recovery phase. Pictures are representative of 3–4 mice/group. Scale bars = 50 µm. (<b>K</b>) Histological damage score and quantification of percentage of ulceration related to total colon length in IEC AMPK KO and WT mice upon DSS-induced epithelial damage and metformin (10 mg/mL) treatment (<span class="html-italic">n</span> = 3–4 mice/group). Data are expressed as means ± SD. Statistical analysis was performed by one-way ANOVA or two-way ANOVA with Bonferroni post hoc test; * <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01 indicate a significant difference relative to the untreated condition; # <span class="html-italic">p</span> < 0.05, ## <span class="html-italic">p</span> < 0.01 and ### <span class="html-italic">p</span> < 0.001 indicate a significant difference between genotype and §§ <span class="html-italic">p</span> < 0.01 and §§§ <span class="html-italic">p</span> < 0.001 indicate a significant difference between metformin-treated mice.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Antibodies
2.2. Mice
2.3. DSS-Induced Acute Colitis
2.4. Histopathological Analysis
2.5. In Vivo Intestinal Permeability Assay
2.6. Quantification of Lipocalin-2
2.7. Cell Culture and Measure of TEER
2.8. Cell Adhesion Assay and xCELLigence Real-Time Cell Analysis
2.9. Scratch Wound Assay
2.10. Western Blotting
2.11. Quantitative Real-Time PCR Analysis
2.12. Statistical Analysis
3. Results
3.1. IEC-Specific AMPK α1/α2 Deficiency Influenced Intestinal Inflammation upon DSS-Induced Epithelial Injury
3.2. Loss of IEC AMPK α1/α2 Caused Impaired Recovery from DSS-Induced Epithelial Injury
3.3. AMPK Signaling Supports Epithelial Proliferation Following DSS-Mediated Injury
3.4. Absence of AMPK Is Not Detrimental for Cell Spreading Response in Caco2 Cells but Necessary for Mature Barrier Establishment
3.5. Impaired Restitution of Mucus-Producing Goblet Cells in the Colon of IEC AMPK KO Mice Following DSS-Induced Colitis
3.6. Metformin Ameliorated DSS-Induced Colitis in an IEC AMPK-Independent Manner
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Olivier, S.; Diounou, H.; Pochard, C.; Frechin, L.; Durieu, E.; Foretz, M.; Neunlist, M.; Rolli-Derkinderen, M.; Viollet, B. Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis. Cells 2022, 11, 590. https://doi.org/10.3390/cells11040590
Olivier S, Diounou H, Pochard C, Frechin L, Durieu E, Foretz M, Neunlist M, Rolli-Derkinderen M, Viollet B. Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis. Cells. 2022; 11(4):590. https://doi.org/10.3390/cells11040590
Chicago/Turabian StyleOlivier, Séverine, Hanna Diounou, Camille Pochard, Lisa Frechin, Emilie Durieu, Marc Foretz, Michel Neunlist, Malvyne Rolli-Derkinderen, and Benoit Viollet. 2022. "Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis" Cells 11, no. 4: 590. https://doi.org/10.3390/cells11040590