Impact of peripheral circadian misalignment and alcohol on the resiliency of intestinal barrier and microbiota

Abstract

Circadian organization is involved in many gastrointestinal tract (GIT) functions such as the maintenance of intestinal barrier integrity. There is compelling evidence that perturbation of the circadian clock decreases intestinal epithelial cells' resiliency to alcohol-induced injury. One of the most common causes of circadian misalignment is wrong-time eating (largest meal at dinner) in modern societies. Yet, few studies have examined the importance of peripheral circadian rhythms of the GIT to alcohol consumption. Eating patterns during physiologic rest time, defined as wrong-time eating (WTE), misalign the peripheral circadian clock of the GIT and the body's central clock. This study aims to fill this knowledge gap by testing the hypothesis that: (1) WTE worsens alcohol-induced disruption of intestinal barrier integrity and (2) decreased intestinal barrier resiliency to alcohol effects by WTE-disrupted circadian is, at least in part, due to microbiota dysbiosis. Alcohol (20% v/v) and a restricted timed-food paradigm were administered to PERIOD2 luciferase (PER2:LUC) reporter BL/6 mice for 10 weeks. Intestinal barrier integrity, intestinal (stool) microbiota, and microbial metabolites (cecal-derived) were examined. Peripheral circadian misalignment exacerbated alcohol-induced disruption of intestinal barrier integrity (tight junctional proteins) leading to increased intestinal permeability (p < 0.05). In addition, alcohol consumption changed the intestinal microbiota community, decreasing beneficial short-chain fatty acid-producing taxa. Further, we recapitulated the in vivo phenotype in a colonic organoid model and demonstrated that microbial metabolites from circadian-disrupted, alcohol-fed mice mediate decreased resiliency of intestinal epithelial barrier function. Peripheral circadian misalignment through food timing decreases the resiliency of the intestinal barrier to alcohol-induced injury and this effect is mediated through dysbiotic microbiota metabolites.

Keywords: Circadian disruption; intestinal permeability; microbiota, colonic organoid.

Figure 1.

Circadian rhythms of colon tissue. (a) Bioluminescence of PER2:LUC in RTE and WTE colon tissue with control (H₂O). (b) Bioluminescence of PER2:LUC in RTE and WTE colon tissue with alcohol (EtOH). (c) The period of WTE mice has increasing variability than the periods of RTE in colon tissue. There is a significant difference in the period between RTE and WTE colon tissue. Experiments were performed in triplicate (n = 3). Purple bars represent H₂O groups and blue bars represent EtOH groups. Two-way ANOVA (results in box) was conducted, and effects are indicated on each graph when significant: *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2.

Effect of altered food timing and alcohol consumption on urinary sugar excretion to assess intestinal barrier integrity. (a) Urinary sucralose exhibited a significant effect of food timing and sex but no interaction. (b) urinary lactulose exhibited a significant effect on food timing but no interaction. (c) Sucralose:lactulose ratio was significantly impacted by alcohol treatment, food timing, and sex. (d) urinary sucrose was significantly impacted by food timing and sex, but there was no interaction. (e) Urinary mannitol exhibited a significant effect on food timing and sex but no interaction. (f) Lactulose:mannitol (LM) ratio was significantly impacted by sex, but there was no difference of food timing or alcohol treatment nor was there an interaction. Between n = 7–13 mice/treatment group. Three-way ANOVA (results in box) was conducted, and effects are indicated on each graph when significant: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 3.

Effect of altered food timing and alcohol consumption on AJC proteins. (a-c) section of colonic tissue labeled with DAPI (blue) and ZO-1 (green). ZO-1 in colon tissue is decreased with altered food timing and alcohol treatment. Between n = 19–21 mice/treatment group. (d-f) section of colonic tissue labeled with DAPI (blue) and occludin (green). Occludin in colon tissue decreases with altered food timing and alcohol treatment. (d) Immunofluorescent staining of occludin in colon tissue (left) and cell fluorescent measurement (right), in which alcohol treatment significantly affected occludin expression. (e) Western blot analysis of occludin in its cytoplasmic fraction. Food timing and alcohol treatment effects are significant. (f) Western blot analysis of occludin in its membrane fraction. Food timing, treatment, and their interaction effects are significant. Between n = 18–21 mice/treatment group. Three-way ANOVA (results in box) was conducted, and effects are indicated on each graph when significant: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (g) Section of colonic tissue labeled with DAPI (blue) and E-cadherin (green). E-cadherin in colon tissue is decreased with alcohol treatment. Alcohol treatment effects were significant. Between n = 18–21 mice/treatment group. Two-way ANOVA (results in box) was conducted, and effects are indicated on each graph when significant: *p < 0.05 and **p < 0.01.

Figure 4.

Short-chain fatty acid (SCFA) content in cecal content supernatant of mice. n = 4 pooled samples from each respective group, RTE H₂O (blue bars) and WTE EtOH (red bars). Two-way ANOVA was conducted, and the results of multiple comparison tests are indicated on the graph when significant: *p < 0.05.

Figure 5.

Relative abundance of short-chain fatty acid (SCFA)-producing taxa in the microbiota of RTE H₂O and WTE EtOH mice. Left image: overall percent abundance of SCFA-producing taxa is decreased in the WTE EtOH group (red bar) compared to the RTE H₂O (blue bar). Right image: overall percent abundance of SCFA-producing taxa is decreased in the WTE EtOH group (left) and much of the changes are occurring in Lachnospiraceae, an important butyrate producer (right).

Figure 6.

Organoid permeability increases with alcohol and cecal supernatant treatment. (a) FITC-dextran (green) was analyzed at 20x and analyzed for net fluorescence. The presence of green dye inside the lumen of the organoids is indicative of increased paracellular permeability. (b) Net fluorescence levels are significant when comparing the RTE H₂O control (blue bar) to the treatment groups: RTE H₂O cecal supernatant (green bar, p = 0.0102), 0.2% EtOH (red bar, p = 0.0006), and WTE EtOH cecal supernatant (purple bar, p<0.0001). P-values are indicated on each graph when significant: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 7.

Effect of food timing and alcohol treatment on AJC protein expression in organoids. (a) Immunofluorescent staining of tight junction protein ZO-1 and adherens junction protein E-cadherin (green). (b) No differences in ZO-1 or E-cadherin total fluorescence, however, there is a decrease in the WTE EtOH group. N = 5 mice/treatment group. (c) Western blot analysis of ZO-1 relative density. No difference due to low n but a similar trend of decreased ZO-1 expression in the WTE EtOH group. Representative data with n = 2 mice/treatment group. (d) Western blot analysis of occludin relative density. No difference due to low n but a similar trend of decreased occludin expression in the WTE EtOH group. The blue bar represents the RTE H₂O group, while the red bar represents the WTE EtOH group. Representative data with n = 2 mice/treatment group.