Deletion of intestinal epithelial AMP-activated protein kinase alters distal colon permeability but not glucose homeostasis

Abstract

Objective: The intestinal epithelial barrier (IEB) restricts the passage of microbes and potentially harmful substances from the lumen through the paracellular space, and rupture of its integrity is associated with a variety of gastrointestinal disorders and extra-digestive diseases. Increased IEB permeability has been linked to disruption of metabolic homeostasis leading to obesity and type 2 diabetes. Interestingly, recent studies have uncovered compelling evidence that the AMP-activated protein kinase (AMPK) signaling pathway plays an important role in maintaining epithelial cell barrier function. However, our understanding of the function of intestinal AMPK in regulating IEB and glucose homeostasis remains sparse.

Methods: We generated mice lacking the two α1 and α2 AMPK catalytic subunits specifically in intestinal epithelial cells (IEC AMPK KO) and determined the physiological consequences of intestinal-specific deletion of AMPK in response to high-fat diet (HFD)-induced obesity. We combined histological, functional, and integrative analyses to ascertain the effects of gut AMPK loss on intestinal permeability in vivo and ex vivo and on the development of obesity and metabolic dysfunction. We also determined the impact of intestinal AMPK deletion in an inducible mouse model (i-IEC AMPK KO) by measuring IEB function, glucose homeostasis, and the composition of gut microbiota via fecal 16S rRNA sequencing.

Results: While there were no differences in in vivo intestinal permeability in WT and IEC AMPK KO mice, ex vivo transcellular and paracellular permeability measured in Ussing chambers was significantly increased in the distal colon of IEC AMPK KO mice. This was associated with a reduction in pSer425 GIV phosphorylation, a marker of leaky gut barrier. However, the expression of tight junction proteins in intestinal epithelial cells and pro-inflammatory cytokines in the lamina propria were not different between genotypes. Although the HFD-fed AMPK KO mice displayed suppression of the stress polarity signaling pathway and a concomitant increase in colon permeability, loss of intestinal AMPK did not exacerbate body weight gain or adiposity. Deletion of AMPK was also not sufficient to alter glucose homeostasis or the acute glucose-lowering action of metformin in control diet (CD)- or HFD-fed mice. CD-fed i-IEC AMPK KO mice also presented higher permeability in the distal colon under homeostatic conditions but, surprisingly, this was not detected upon HFD feeding. Alteration in epithelial barrier function in the i-IEC AMPK KO mice was associated with a shift in the gut microbiota composition with higher levels of Clostridiales and Desulfovibrionales.

Conclusions: Altogether, our results revealed a significant role of intestinal AMPK in maintaining IEB integrity in the distal colon but not in regulating glucose homeostasis. Our data also highlight the complex interaction between gut microbiota and host AMPK.

Keywords: AMPK; Intestinal epithelial barrier (IEB); Metformin; Microbiota; Obesity; Permeability.

Graphical abstract

Figure 1

AMPKα1 and AMPKα2 deletion in mouse GI tract. (A) Western blotting analysis of AMPKα1, α2, β1, β2, γ1, and γ2 expression in intestinal epithelial cells (IECs) isolated from the duodenum, jejunum, ileum, and colon of WT and IEC AMPK KO mice. β-actin was used as a loading control. IEC-specific AMPK deletion was obtained by expressing Cre-recombinase driven by the villin promoter. Measurement of (B) body weight, (C) intestine length, (D) colon length, (E) total transit time, and (F) feces humidity in WT and IEC AMPK KO mice. n = 6–8 mice. All of the data are expressed as means ± SEM. Statistical analysis was performed using Student's t test. Black bars, WT mice (WT); white bars, IEC AMPK KO mice (KO).

Figure 2

Absence of IEC AMPK induced hyperpermeability in the distal colon. (A) In vivo paracellular intestinal epithelial permeability of WT and IEC AMPK KO mice on regular control diet (CD) determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. n = 3–7 mice per genotype from three independent experiments. (B) In vivo transcellular intestinal epithelial permeability of WT and IEC AMPK KO mice on CD determined by measuring the activity of HRP in the plasma 4 h after oral gavage. n = 4–6 mice from three independent experiments. (C) Ex vivo paracellular permeability in the jejunum, ileum, proximal colon, and distal colon from WT and IEC AMPK KO mice on CD evaluated in Ussing chambers by measuring FITC-dextran flux through intestinal segments for 3 h. n = 7 mice. Statistical analysis was performed using Student's t test. (D) Ex vivo transcellular permeability in the jejunum, ileum, proximal colon, and distal colon from WT and IEC AMPK KO mice on CD evaluated in Ussing chambers by measuring the activity of HRP in the basolateral medium for 3 h. n = 7 mice. (E) Expression of mRNA for tight junction proteins ZO-1, ZO-2, occludin, claudin 1, claudin 2, and claudin 3 in entire distal colon biopsies from WT and IEC AMPK KO mice. (F) Expression of mRNA for IL-6, TNF-α, and IL-1β in entire distal colon biopsies from WT and IEC AMPK KO mice. n = 4–7 mice. All of the data are expressed as means ± SEM. Statistical analysis was performed using Student's t test. ∗p < 0.05 and ∗∗∗p < 0.001 indicate a significant increase relative to the respective WT intestinal segment. Black bars, WT mice (WT); white bars, IEC AMPK KO mice (KO). (G) Schematic summarizing the stress polarity signaling (SPS) pathway showing the impact of AMPK activation/deletion on Ser425 GIV phosphorylation and barrier integrity. (H) Schematic diagram displays the workflow to assess the activation of the SPS pathway in situ in the murine colons as determined by assessing the abundance of epithelial pS245GIV. (I) Representative images are presented from the proximal colon (upper section) and distal colon (lower section) of WT (WT) and IEC AMPK KO (KO) mice without metformin treatment (H₂O) and after administration of 2 mg/ml of metformin in drinking water for 5 days (Met). (J) Violin plots displaying the % area and intensity of pS245 GIV staining as determined by IHC Profiler (each dot represents the average of measurements in 4 ROIs, which were assessed in 3–5 images taken from each sample; n = 3 in H₂O group; n = 5 in Met group). Error bars represent SEM. Statistical significance was determined by three-way ANOVA; ∗∗p < 0.01 and ∗∗∗∗p < 0.0001.

Figure 3

Deletion of IEC AMPK did not exacerbate permeability and inflammation in the colon after a HFD challenge. (A) Experimental timeline for control diet (CD) or high-fat diet (HFD) challenge for 10 weeks in male WT and IEC AMPK KO mice. (B) In vivo paracellular intestinal epithelial permeability of WT and IEC AMPK KO mice on HFD determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. n = 3–7 mice from three independent experiments. (C) Ex vivo paracellular permeability in the distal colon of WT and IEC AMPK KO mice on a CD or HFD. Permeability was evaluated in Ussing chambers by measuring FITC-dextran flux through intestinal segments for 3 h. n = 7–12 mice. (D) Expression of mRNA for ZO-1 in the intestinal epithelial layer (IEL) fraction isolated from the distal colon of WT and IEC AMPK KO mice fed a CD or HFD. n = 7 mice. (E) Expression of mRNA for IL-6, TNF-α, and IL-1β in the lamina propria layer (LPL) fraction isolated from the distal colon of WT and IEC AMPK KO mice fed a CD or HFD. n = 7 mice. (F) Circulating IL-6, TNF-α, and IFN-γ levels in WT and IEC AMPK KO mice fed a CD or HFD. n = 5–7 mice. All of the data are expressed as mean fold change relative to WT. Statistical analysis was performed by two-way ANOVA with Bonferroni's post hoc test. ∗p < 0.05 and ∗∗p < 0.01 indicate a significant difference in respective WT intestinal segment and diet challenge. $p < 0.05 and $$$p < 0.001 indicate diet effect within genotype. CD, control diet; HFD, high-fat diet; black bars, WT mice; white bars, IEC AMPK KO mice.

Figure 4

Deletion of IEC AMPK did not worsen HFD-induced obesity. (A) Body weight monitoring in WT and IEC AMPK KO mice on control diet (CD) or high-fat diet (HFD) for 10 weeks. n = 5–7 mice. (B) Body weight, body composition examination (expressed as a percentage of fat mass and lean mass relative to total body mass), body fat mass, and body lean mass measured by nuclear magnetic resonance in WT and IEC AMPK KO mice at the end of CD and HFD challenge. n = 5–7 mice. (C) Daily food intake of WT and IEC AMPK KO mice on CD or HFD. (D) Daily drink intake of WT and IEC AMPK KO mice on CD or HFD. (E) Daily spontaneous locomotor activity of WT and IEC AMPK KO mice. (F) Energy expenditure of WT and IEC AMPK KO mice on CD or HFD during the light and dark phases. (G) O₂ consumption of WT and IEC AMPK KO mice on CD or HFD during the light and dark phases. (H) CO₂ production of WT and IEC AMPK KO mice on CD or HFD during the light and dark phases. (I) Respiratory exchange ratio (VCO₂/VO₂) of WT and IEC AMPK KO mice fed a CD or HFD during the light and dark phases. All of the calorimetry data are representative of 5 days of measurement; n = 6 mice. All of the data are expressed as means ± SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni's post hoc test. ^oop < 0.01 and ^ooop < 0.001 indicate a diet effect in WT mice. §p < 0.05 and §p < 0.001 indicate a diet effect in IEC AMPK KO mice. $p < 0.05 and $$$p < 0.001 indicate a diet effect within genotype. ##p < 0.01 and ###p < 0.001 indicate a light/dark phase effect within genotype. CD, control diet; HFD, high-fat diet; black bars, WT mice; white bars, IEC AMPK KO mice.

Figure 5

Deletion of IEC AMPK did not worsen obesity-induced metabolic dysfunctions and did not affect the acute response to metformin. (A) Blood glucose profile during glucose tolerance test in WT and IEC AMPK KO mice on control diet (CD) or high-fat diet (HFD). AUC, area under the curve was calculated. n = 5–7 mice. (B) Blood glucose profile during insulin tolerance tests in WT and IEC AMPK KO mice on CD or HFD. n = 5–7 mice. (C) Blood glucose profile during metformin tolerance tests in WT and IEC AMPK KO mice on CD. Mice were given an oral gavage dose of 250 mg/kg of metformin and after 30 min challenged with an oral administration of glucose. n = 7–8 mice per genotype. (D) Blood glucose profile during metformin tolerance tests in WT and IEC AMPK KO mice on HFD. Mice were given an oral gavage dose of 250 mg/kg of metformin and after 30 min challenged with an oral administration of glucose. n = 5–7 mice. All of the data are expressed as means ± SEM. Statistical analysis was performed by two-way repeated measures ANOVA or two-way ANOVA with Bonferroni's post hoc test. Black bars, WT mice; white bars, IEC AMPK KO mice. ##p < 0.01 and ###p < 0.001 indicate a diet or metformin effect in WT mice. §p < 0.05, §§p < 0.01, and §§§p < 0.001 indicate a diet or metformin effect in IEC AMPK KO mice. $p < 0.05, $$p < 0.01, and $$$p < 0.001 indicate a diet or metformin effect within genotype. CD, control diet; HFD, high-fat diet; black bars, WT mice; white bars, IEC AMPK KO mice.

Figure 6

Inducible deletion of gut AMPK did not exacerbate the development of HFD-induced obesity and metabolic dysfunctions. (A) Experimental timeline for tamoxifen treatment and control diet (CD) or high-fat diet (HFD) challenge for 16 weeks in male WT and i-IEC AMPK KO mice. (B) Western blotting analysis of AMPKα, phospho-AMPKα-Thr172, ACC, and phospho-ACC-Ser79 expression in intestinal epithelial cells isolated from the ileum of WT and i-IEC AMPK KO mice at the completion of CD or HFD challenge. β-actin was used as a loading control. (C) Body weight monitoring in WT and i-IEC AMPK KO mice on CD or HFD for 16 weeks. n = 7–13 mice. (D) Body weight and body composition examination (expressed as a percentage of fat mass and lean mass relative to total body mass) in WT and i-IEC AMPK KO mice at the end of CD or HFD challenge. n = 7–13 mice. (E) Blood glucose profile during glucose tolerance tests in WT and i-IEC AMPK KO mice on CD or HFD. AUC, area under the curve was calculated. n = 6–11 mice. All of the data are expressed as means ± SEM. Statistical analysis was performed by two-way repeated measures ANOVA or two-way ANOVA with Bonferroni's post hoc test. ##p < 0.01 and ###p < 0.001 indicate a diet effect in WT mice. §§p < 0.01 and §§§p < 0.001 indicate a diet effect in i-IEC AMPK KO mice. $p < 0.05 and $$$p < 0.001 indicate a diet effect within genotype. CD, control diet; HFD, high-fat diet; black bars, WT mice; dashed bars, i-IEC AMPK KO mice.

Figure 7

Effect of inducible deletion of IEC AMPK on gut microbiota in mice on CD and HFD. (A) Principal coordinate analysis (PCoA) based on the unweighted UniFrac distance between samples from WT and i-IEC AMPK KO mice on control diet (CD) and high-fat diet (HFD). (B) The Shannon index representing α diversity of microbial communities of WT and i-IEC AMPK KO mice on CD and HFD. Box plots for each α diversity metric and pairwise comparisons between the groups using t tests with pooled SD and Bonferroni's correction are shown. (C) Relative abundance of bacterial orders in WT and i-IEC AMPK KO mice on CD and HFD. (D) Heat map of bacterial genera abundance in each subgroup. The color scale represents the log₁₀ + 1 transformed abundance. “_f” or “_g” at the end of taxon name indicate unidentified families or genera, respectively. Linear discriminant analysis (LDA) effect size (LEfSe) score was calculated to evaluate bacterial families or genera overrepresented between WT and i-IEC AMPK KO mice on CD (E) or high-fat (F) diet. n = 7–13 mice. CTL_WT: CD-fed WT mice; CTL_KO: CD-fed i-IEC AMPK KO mice; HFD_WT: HFD-fed WT mice; HFD_KO: HFD-fed i-IEC AMPK KO mice. Black bars, WT mice; gray bars, i-IEC AMPK KO mice.

Figure 8

Inducible deletion of intestinal AMPK did not worsen obesity-induced permeability and inflammation in the colon. (A) In vivo paracellular intestinal epithelial permeability of WT and i-IEC AMPK KO mice on control diet (CD) or high-fat diet (HFD) determined by measuring the amount of 4 kDa TRITC-dextran in the plasma 4 h after gavage. n = 7–13 mice. (B) Ex vivo paracellular permeability in the distal colon from WT and i-IEC AMPK KO mice on CD or HFD evaluated in Ussing chambers by measuring TRITC-dextran flux through intestinal segments for 3 h. n = 3–6 mice. (C) Expression of mRNA for IL-6, TNF-α, and IL-1β in the distal colon of WT and i-IEC AMPK KO mice on CD or HFD. n = 6–13 mice. (D) Lipocalin-2 (Lcn-2) levels in feces from WT and i-IEC AMPK KO mice on CD or HFD. n = 4–10 mice. (E) Circulating IL-6, TNF-α, and IFN-γ levels in WT and i-IEC AMPK KO mice on CD or HFD. n = 3–6 mice (F) Expression of mRNA for ZO-1 and occludin in the distal colon of WT and i-IEC AMPK KO mice on CD or HFD. n = 6–13 mice. All of the data are expressed as means ± SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate a significant increase in dextran flux relative to the respective WT intestinal segment and diet challenge. CD, control diet; HFD, high-fat diet; black bars, WT mice; dashed bars, i-IEC AMPK KO mice.

Multimedia component 1

Supplementary Figure 1: AMPKα1 and α2 expression in the duodenum crypt-villus axis and along the small intestine in global AMPKα1 and AMPKα2 KO mice. (A) Western blotting analysis of AMPKα1 and AMPKα2 expression in mouse intestinal epithelial samples from the duodenum fractionated from the villus (F1) to crypt (F4). Expression of proliferating cell nuclear antigen (PCNA), a marker of cell proliferation, was used to monitor the cellular fractionation where the proliferating cells were found in the crypt (F4). β-actin was used as a loading control. (B) Quantification of AMPKα1 and AMPKα2 expression along the duodenum crypt–villus axis (from villus [F1] to crypt [F4]) expressed as percent of F1. n = 4. Data are expressed as means ± SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni's post hoc test. ∗p < 0.05 and ∗∗p < 0.01 indicate a significant change relative to the respective F1 fraction. Black bars, AMPKα1; dashed bars, AMPKα2. (C) Western blotting analysis of AMPKα1 and AMPKα2 expression in the duodenum, jejunum, and ileum from WT and global AMPKα1 and AMPKα2 KO mice. β-actin was used as a loading control. (D) Western blotting analysis of AMPKα1 and AMPKα2 expression in the liver and skeletal muscle from WT and IEC AMPK KO mice. β-actin was used as a loading control.

Multimedia component 2

Supplementary Figure 2: Contractile activity of the GI tract in WT and IEC AMPK KO mice. (A) Frequency of spontaneous phasic contractions was evaluated for 2 min on jejunum and distal colon longitudinal muscular segments from WT and IEC AMPK KO mice in basal condition and after sequential addition of l-Name (50 μM) and atropine (1 μM). (B) Average tension of jejunum and distal colon longitudinal muscular segments from WT and IEC AMPK KO mice was measured in basal condition and after sequential addition of l-Name (50 μM) and atropine (1 μM). n = 8, 2 samples per mice, and 4 mice/group. All of the data are represented as means ± SEM. Statistical analysis was performed using two-way ANOVA with Bonferroni's post hoc test. ∗p < 0.05 indicates significant change relative to respective WT intestinal segment. Black bars, WT mice; white bars, IEC AMPK KO mice.

Multimedia component 3

Supplementary Figure 3: Analysis of small and large intestine integrity in WT and IEC AMPK KO mice. (A) Representative hematoxylin and eosin staining in the small and large intestines from WT and IEC AMPK KO mice. Scale bar, 100 μm. (B) Ultrastructure of the colon from WT and IEC AMPK KO mice observed by transmission electron microscopy. High magnification of intercellular spaces with distinguishable tight junctions are shown (white arrows). Scale bar, 0.5 μm. (C) Expression of mRNA for tight junction proteins ZO-1, ZO-2, occludin, claudin 1, claudin 2, and claudin 3 in entire jejunum, ileum, and proximal colon biopsies from WT and IEC AMPK KO mice. n = 4–7 mice. (D) Western blotting analysis and quantification of ZO-1 and occludin expression in entire jejunum, ileum, proximal colon, and distal colon biopsies of WT and IEC AMPK KO mice. β-actin was used as a loading control. (E) Expression of mRNA for IL-6, TNF-α, and IL-1β in entire jejunum, ileum, and proximal colon biopsies from WT and IEC AMPK KO mice. n = 4–7 mice. All of the data are expressed as means ± SEM. Statistical analysis was performed by using Student's t test. Black bars, WT mice (WT); white bars, IEC AMPK KO mice (KO).

Multimedia component 3

Supplementary Figure 3: Analysis of small and large intestine integrity in WT and IEC AMPK KO mice. (A) Representative hematoxylin and eosin staining in the small and large intestines from WT and IEC AMPK KO mice. Scale bar, 100 μm. (B) Ultrastructure of the colon from WT and IEC AMPK KO mice observed by transmission electron microscopy. High magnification of intercellular spaces with distinguishable tight junctions are shown (white arrows). Scale bar, 0.5 μm. (C) Expression of mRNA for tight junction proteins ZO-1, ZO-2, occludin, claudin 1, claudin 2, and claudin 3 in entire jejunum, ileum, and proximal colon biopsies from WT and IEC AMPK KO mice. n = 4–7 mice. (D) Western blotting analysis and quantification of ZO-1 and occludin expression in entire jejunum, ileum, proximal colon, and distal colon biopsies of WT and IEC AMPK KO mice. β-actin was used as a loading control. (E) Expression of mRNA for IL-6, TNF-α, and IL-1β in entire jejunum, ileum, and proximal colon biopsies from WT and IEC AMPK KO mice. n = 4–7 mice. All of the data are expressed as means ± SEM. Statistical analysis was performed by using Student's t test. Black bars, WT mice (WT); white bars, IEC AMPK KO mice (KO).

Multimedia component 4

Supplementary Figure 4: Effect of HFD on intestinal epithelial permeability of small and large intestines of WT and IEC AMPK KO mice. (A) In vivo transcellular intestinal epithelial permeability of WT and IEC AMPK KO mice on a control diet (CD) and high-fat diet (HFD) determined by measuring the activity of HRP in the plasma 4 h after oral gavage. n = 6–12 mice from three independent experiments. (B) Ex vivo transcellular permeability in the jejunum, ileum, proximal colon, and distal colon of WT and IEC AMPK KO mice on CD and HFD evaluated in Ussing chambers by measuring the HRP activity in the basolateral medium for 3 h. n = 7–12 mice. (C) Ex vivo paracellular permeability in the jejunum, ileum, proximal colon (Prox colon), and distal colon (Dist colon) of WT and IEC AMPK KO mice on CD or HFD. Permeability was evaluated in Ussing chambers by measuring FITC-dextran flux through intestinal segments for 3 h. n = 7–12. All of the data are expressed as means ± SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni's post hoc test. CD, control diet; HFD, high-fat diet; black bars, WT mice; white bars, IEC AMPK KO mice.

Multimedia component 5

Supplementary Figure 5: Impact of constitutive intestinal AMPK deletion on obesity-induced metabolic dysfunctions. (A) Food intake, (B) drink intake, and (C) spontaneous locomotor activity of WT and IEC AMPK KO mice on control diet (CD) or high-fat diet (HFD) during the light and dark phases. n = 6 mice. (D) WT and IEC AMPK KO mice were fasted overnight and gavaged with olive oil for an oral fat tolerance test. Plasma triglycerides were measured at the indicated time points. n = 15–17 mice. (E) Plasma lipid parameters in WT and IEC AMPK KO mice on CD or HFD. n = 3–10 mice per genotype. (F) Plasma GLP-1 levels in WT and IEC AMPK KO mice at basal state or 10 min after an oral challenge with a glucose/olive oil mix. n = 7–8 mice per genotype. All of the data are expressed as means ± SEM. Statistical analysis was performed by two-way repeated measures ANOVA or two-way ANOVA with Bonferroni's post hoc test. $p < 0.05, $$p < 0.01, and $$$p < 0.001 indicate a light/dark phase or diet/ bolus challenge effect within genotype. CD, control diet; HFD, high-fat diet; black bars, WT mice; white bars, IEC AMPK KO mice.

Multimedia component 6

Supplementary Figure 6: Impact of inducible intestinal AMPK deletion on obesity-induced metabolic dysfunctions. (A) Western blotting analysis of AMPKα1 and AMPKα2 expression in IEC from the duodenum, jejunum, ileum, and colon of WT and i-IEC AMPK KO mice. Inducible IEC-specific AMPK deletion was obtained by expressing a tamoxifen-inducible Cre-recombinase construct driven by the villin promoter and administering tamoxifen for 5 consecutive days. Mice were investigated 2 weeks after tamoxifen treatment. β-actin was used as loading control. (B) Body fat mass and body lean mass measured by nuclear magnetic resonance in WT and i-IEC AMPK KO mice at the end of CD or HFD challenge. n = 7–13 mice. (C) Number of macrophage, dendritic, neutrophil, and monocyte cells in visceral adipose tissue of i-IEC AMPK KO and WT mice fed a normal diet (ND) or high-fat diet (HFD). n = 3–7 per genotype. (D) Ex vivo paracellular permeability in the jejunum from WT and i-IEC AMPK KO mice on CD or HFD evaluated in Ussing chambers by measuring TRITC-dextran flux through intestinal segments for 3 h. n = 3–6 mice per genotype. Data are expressed as means ± SEM. (E) Percentage of macrophage, dendritic, neutrophil, and monocyte cells among CD45⁺ cells in the colon of i-IEC AMPK KO and WT mice on ND or HFD. n = 3–7 mice per genotype. Data are expressed as means ± SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni's post hoc test. $$$p < 0.001 indicates diet effect within genotype. CD, control diet; HFD, high-fat diet; black bars, WT mice; dashed bars, i-IEC AMPK KO mice.