The type of dietary fat modulates intestinal tight junction integrity, gut permeability, and hepatic toll-like receptor expression in a mouse model of alcoholic liver disease

Abstract

Background: Interactions between the gut, immune system, and the liver, as well as the type of fat in the diet, are critical components of alcoholic liver disease (ALD). The goal of the present study was to determine the effects of saturated fat (SF) and unsaturated fat (USF) on ethanol (EtOH)-induced gut-liver interactions in a mouse model of ALD.

Methods: C57BL/6N mice were fed Lieber-DeCarli liquid diets containing EtOH and enriched in USF (corn oil) or SF (medium chain triglycerides:beef tallow). Control mice were pair-fed on an isocaloric basis. Liver injury and steatosis, blood endotoxin levels, intestinal permeability, and tight junction (TJ) integrity, as well as hepatic Toll-like receptor (TLR) gene expression, were evaluated.

Results: After 8 weeks of EtOH feeding, liver injury and steatosis were observed in USF + EtOH group compared with control and SF + EtOH. Significantly increased intestinal permeability in conjunction with elevated blood endotoxin levels were observed in the ileal segments of the mice fed USF + EtOH. USF diet alone resulted in down-regulation of intestinal TJ protein mRNA expression compared with SF. Importantly, alcohol further suppressed TJ proteins in USF + EtOH, but did not affect intestinal TJ in SF + EtOH group. The type of fat in the diet alone did not affect hepatic TLR expression. Compared with control animals, hepatic TLR (TLR 1, 2, 3, 4, 7, 8, 9) mRNA expression was significantly (p < 0.05) increased in USF + EtOH, but not in SF + EtOH group. Notably, TLR5 was the only up-regulated TLR in both SF + EtOH and USF + EtOH groups.

Conclusions: Dietary fat is an important cofactor in alcohol-associated liver injury. We demonstrate that USF (corn oil/linoleic acid) by itself results in dysregulation of intestinal TJ integrity leading to increased gut permeability, and alcohol further exacerbates these alterations. We postulate that elevated blood endotoxin levels in response to USF and alcohol in conjunction with up-regulation of hepatic TLRs combine to cause hepatic injury in ALD.

Copyright © 2011 by the Research Society on Alcoholism.

Figures

Fig. 1

Composition of the experimental Lieber-DeCarli liquid diets. Saturated fat (SF) diet was enriched with medium chain triglyceride (MCT) oil:beef tallow fat, 82:18 ratio. Unsaturated fat (USF) diet was enriched with corn oil. Soybean oil was used in both diets to provide essential free fatty acids. The control (SF and USF) diets contained 43% of calories from carbohydrate, 17% from protein, 40% from fat. SF+EtOH and USF+EtOH diets contained 35% of calories from EtOH to replace the calories from carbohydrate.

Fig. 2

Food consumption and body weight of mice fed Lieber-DeCarli liquid diets. C57BL/6N male mice were fed Lieber-DeCarli diets for 10 weeks (1th week – SF and USF control diets, 2nd week – gradual increase in EtOH followed by 8 weeks – 5% EtOH). Control animals were pair-fed with equal amounts of isocaloric liquid diets. (A) Mice fed USF diet consumed significantly more food compared to SF diet fed animals during the 1st week (14.2+0.51 vs 11.61+0.89 g/per day, p<0.05, Student’s t-test). 5% EtOH feeding significantly reduced food consumption by both USF+EtOH and SF+EtOH groups. (B) USF diet feeding resulted in markedly increased body weight compared to SF diet feeding. Supplementation with EtOH led to gradually reduced body weight in both USF+EtOH and SF+EtOH groups.

Fig. 3

Effects of saturated and unsaturated diets on liver injury and steatosis in response to chronic alcohol feeding. (A) Liver injury was evaluated by plasma ALT activity. USF+EtOH feeding resulted in significant elevation of plasma ALT levels compared to control pair-fed animals as well as to SF+EtOH fed group (44.91+2.81 vs 21.43+1.19 and 27.27+1.92 IU/L, respectively, *p<0.05, one-way ANOVA with Tukey’s post hoc test). Values are mean+SEM, n=6 animals/per group. (B) Liver hematoxylin-eosin staining demonstrated severe micro- and macrovesicular steatosis in USF+EtOH fed mice compared to control pair-fed animals as well as SF+EtOH fed groups. Arrows indicate the fat droplets (x200 final magnification). (C) Significant triglyceride accumulation was observed in the livers of USF+EtOH fed mice compared to control pair-fed fed animals as well as to SF+EtOH fed group (100.2+8.1 vs 39.93+5.59 and 68.76+7.96 mg/g liver, respectively, *p<0.05, one-way ANOVA with Tukey’s post hoc test). (D) Liver nonesterified fatty acid (NEFA) measurement. NEFA levels were not significantly different between the experimental groups.

Fig. 4

Hepatic markers of inflammation, macrophage infiltration and oxidative stress in response to alcohol and saturated and unsaturated diets. EtOH feeding induced hepatic inflammation, macrophage infiltration and reactive oxygen species (ROS) accumulation in both SF+EtOH and USF+EtOH groups compared to control pair-fed animals with more prominent elevation in USF+EtOH group. (A) Inflammation marker: TNF-α mRNA levels. (B) Macrophage infiltration marker: MCP-1 mRNA levels. (C) Oxidative stress marker: Cyp2e1 mRNA levels. All mRNA levels were assessed by RT-PCR. The relative mRNA expression was analyzed using 2-ΔΔCt method by normalizing with 18s mRNA expression. Results are given as fold changes in EtOH fed groups compared to control pair-fed animals. Values in control groups were set as 1. Values are mean+SEM, n=6 animals/per group, *p <0.05, Student’s t-test. (D) Western blot analysis of CYP2E1 protein expression. Expression of β-actin was used as a loading control, n=2 animals/group. (E) Quantitative analysis of CYP2E1 levels demonstrated increase in CYP2E1 expression in the livers of mice fed SF+EtOH and USF+EtOH diets compared to their control pair-fed animals. The intensity of protein bands was quantified by densitometry using the NIH ImageJ software (NIH, Bethesda, MD). (F) ROS accumulation in the livers assessed by dihydroethidium staining. ROS accumulation was markedly increased in USF+EtOH group compared to control pair-fed animals and SF+EtOH fed littermates. Liver cryostat sections were incubated with 10 uM dihydroethidium for 15 min. Red fluorescence was formed upon oxidation by ROS, and intensity was detected by fluorescence microscope.

Fig. 5

Plasma endotoxin levels and intestinal permeability in response to alcohol and saturated and unsaturated fat diets. (A) Plasma endotoxin levels assessed by plasma LPS (lipopolysaccharides) measurement. LPS levels were significantly increased in USF+EtOH compared to USF pair-fed control animals (0.48+0.15 vs 0.10+0.02, *p<0.05). LPS levels in SF+EtOH group appeared to be increased compared to control SF pair-fed control animals (0.21+0.05 vs 0.13+0.02, p=0.18), but did not reach statistical significance. Values are mean+SEM, n=6 animals/per group, one-way ANOVA with Tukey’s post hoc test. (B) Intestinal permeability of the ileum segment. Significantly increased intestinal permeability was observed in mice fed USF+EtOH compared to USF pair-fed control group. There were no differences in ileum permeability in SF+EtOH compared to SF pair-fed control animals. Ileum permeability was determined ex vivo using fluorescent dextran FD-4. Data are expressed as fold changes in EtOH fed groups compared to control pair-fed animals. FD-4 permeability in control groups was set as 1. Values are mean+SEM, n=6-8 animals/per group, *p <0.05, Student’s t-test.

Fig. 6

Effects of saturated and unsaturated diets on ileum tight junction protein and protein-adaptor mRNA levels in response to chronic alcohol feeding. USF diet significantly down-regulated mRNA levels of (A) ileum tight junction proteins and (B) protein-adaptors compared to SF diet. EtOH supplemented to the USF diet resulted in further suppression of these proteins. Intestinal tight junction protein mRNA expression was assessed by RT-PCR. The relative mRNA levels were analyzed using 2-ΔΔCt method by normalizing with 18s mRNA expression. Results are presented as fold changes over SF group set as 1. Values are mean+SEM, n=6 animals/per group, *p <0.05, one-way ANOVA with Tukey’s post hoc test.

Fig. 7

Effects of saturated and unsaturated diets on hepatic TLR mRNA levels in response to chronic alcohol feeding. No significant differences in hepatic TLR mRNA expression were observed between the SF or USF diet groups. After 8 weeks of alcohol feeding, hepatic TLR 1, 2, 3, 4, 7, 8, 9 mRNA levels were significantly up-regulated in the USF+EtOH fed mice compared to USF pair-fed control animals. Only TLR5 mRNA was up-regulated in both SF+EtOH and USF+EtOH groups compared to their respective pair-fed groups. Hepatic TLR mRNA levels were assessed by RT-PCR. The relative mRNA expression was analyzed using 2-ΔΔCt method by normalizing with 18s mRNA expression. Results are presented as fold changes over SF group set as 1. Values are mean+SEM, n=6 animals/per group, *p <0.05, one-way ANOVA with Tukey’s post hoc test.