Lowering the dietary omega-6: omega-3 does not hinder nonalcoholic fatty-liver disease development in a murine model

Abstract

It is hypothesized that a high dietary n-6:n-3 (eg, 10-20:1) is partly responsible for the rise in obesity and related health ailments. However, no tightly controlled studies using high-fat diets differing in the n-6:n-3 have tested this hypothesis. The aim of the study was to determine the role that the dietary n-6:n-3 plays in non-alcoholic fatty-liver disease (NAFLD) and colitis development. We hypothesized that reducing the dietary n-6:n-3 would hinder the development of NAFLD and colitis. Male C57BL/6 J mice were fed high-fat diets, differing in the n-6:n-3 (1:1, 5:1, 10:1, 20:1), for 20 weeks. Gas chromatography-mass spectrometry was used to analyze the hepatic phospholipid arachidonic acid (AA):eicosapentaenoic acid and AA:docosahexaenoic acid. Hepatic metabolism, inflammatory signaling, macrophage polarization, gene expression of inflammatory mediators, oxidative and endoplasmic reticulum stress, and oxidative capacity were assessed as well as colonic inflammatory signaling, and gene expression of inflammatory mediators and tight-junction proteins. Although reducing the dietary n-6:n-3 lowered the hepatic phospholipid AA:eicosapentaenoic acid and AA:docosahexaenoic acid in a dose-dependent manner and mildly influenced inflammatory signaling, it did not significantly attenuate NAFLD development. Furthermore, the onset of NAFLD was not paired to colitis development or changes in tight-junction protein gene expression. In conclusion, reducing the dietary n-6:n-3 did not attenuate NAFLD progression; nor is it likely that colitis, or gut permeability, plays a role in NAFLD initiation in this model.

Keywords: Colitis; High-fat diet; Murine model; Nonalcoholic fatty-liver disease; Omega-6:omega-3; α-Linolenic acid.

Figure 1

Impact of dietary n-6:n-3 on murine NAFLD development. (A) AA:EPA, and (B) AA:DHA in hepatic phospholipids, (C) liver weight, (D) hepatic lipid accumulation, (E) hepatic mRNA expression of metabolism-related genes, as well as (F) representative hepatic H&E (10x & 40x) and oil red O images (20x) displaying steatosis and inflammatory infiltrate (n=10/group). Scale bars represent 200 µm. Treatment groups included a control diet (AIN-76A Mod) and four HFDs differing in the n-6:n-3 (1:1, 5:1, 10:1, and 20:1) consumed for a 20-week period. Diets not sharing a common letter differ significantly from one another (P≤.05). Data are presented as means (±SEM).

Figure 2

Representative hepatic western blots for markers of (A) inflammatory signaling, as well as gene expression of (B) macrophage and (C) inflammatory markers (n=10/group). Treatment groups included a control diet (AIN-76A Mod) and four HFDs differing in the n-6:n-3 (1:1, 5:1, 10:1, and 20:1) consumed for a 20-week period. All western blots were run under the same experimental conditions. Diets not sharing a common letter differ significantly from one another (P≤.05). Data are presented as means (±SEM).

Figure 3

Representative hepatic western blots for markers of (A) oxidative stress, (B) ER stress, and (C) oxidative capacity (n=10/group). All western blots were run under the same experimental conditions. Treatment groups included a control diet (AIN-76A Mod) and four HFDs differing in the n-6:n-3 (1:1, 5:1, 10:1, and 20:1) consumed for a 20-week period. Diets not sharing a common letter differ significantly from one another (P≤.05). Data are presented as means (±SEM).

Figure 4

Representative colonic western blots for markers of (A) inflammatory signaling, gene expression of (B) inflammatory and (C) tight-junction markers, as well as (D) representative colonic H&E images (20x) (n=10/group). All western blots were run under the same experimental conditions. Scale bars represent 200 µm. Treatment groups included a control diet (AIN-76A Mod) and four HFDs differing in the n-6:n-3 (1:1, 5:1, 10:1, and 20:1) consumed for a 20-week period. Diets not sharing a common letter differ significantly from one another (P≤.05). Data are presented as means (±SEM).