Dietary α-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis via ameliorating lipid homeostasis at adipose tissue-liver axis in mice

Abstract

Low levels of n-3 polyunsaturated fatty acids (PUFAs) in serum and liver tissue biopsies are the common characteristics in patients with alcoholic liver disease. The α-linolenic acid (ALA) is a plant-derived n-3 PUFA and is rich in flaxseed oil. However, the impact of ALA on alcoholic fatty liver is largely unknown. In this study, we assessed the potential protective effects of ALA-rich flaxseed oil (FO) on ethanol-induced hepatic steatosis and observed that dietary FO supplementation effectively attenuated the ethanol-induced hepatic lipid accumulation in mice. Ethanol exposure stimulated adipose lipolysis but reduced fatty acid/lipid uptake, which were normalized by FO. Our investigations into the corresponding mechanisms demonstrated that the ameliorating effect of FO might be associated with the lower endoplasmic reticulum stress and normalized lipid metabolism in adipose tissue. In the liver, alcohol exposure stimulated hepatic fatty acid uptake and triglyceride synthesis, which were attenuated by FO. Additionally, dietary FO upregulated plasma adiponectin concentration, hepatic adiponectin receptor 2 expression, and the activation of hepatic adenosine monophosphate-activated protein kinase. Collectively, dietary FO protects against alcoholic hepatic steatosis by improving lipid homeostasis at the adipose tissue-liver axis, suggesting that dietary ALA-rich flaxseed oil might be a promising approach for prevention of alcoholic fatty liver.

Figure 1. Dietary flaxseed oil alleviates hepatic lipid accumulation in alcohol-fed mice.

Representative Oil Red O staining (200× magnification) of the liver (A) and the concentrations of hepatic triglycerides (B). Values represent the means ± SD (n = 8–10). Labeled means without a common letter are significantly different (P < 0.05). AF/CO, alcohol-fed with corn oil; AF/FO, alcohol-fed with flaxseed oil; ETH, ethanol; PF/CO, pair-fed with corn oil; PF/FO, pair-fed with flaxseed oil.

Figure 2. Dietary flaxseed oil alleviates adipose lipolysis (A) and the plasma free fatty acid concentration (B) in alcohol-fed mice.

Adipose lipolysis was estimated by measuring the free fatty acid released from epididymal WAT explants ex vivo. Values represent the means ± SD (n = 8–10). Labeled means without a common letter are significantly different (P < 0.05). AF/CO, alcohol-fed with corn oil; AF/FO, alcohol-fed with flaxseed oil; ETH, ethanol; FFA, free fatty acids; PF/CO, pair-fed with corn oil; PF/FO, pair-fed with flaxseed oil.

Figure 3. Dietary flaxseed oil improves the dysfunctional lipid metabolism of adipose tissue in alcohol-fed mice.

Immunoblot analysis of p-HSL, HSL, and ATGL proteins related to adipose lipolysis in epididymal adipose tissue (A) and their results of the densitometric analysis (B). qPCR analysis of genes related to fatty acid transport (C) and VLDL uptake (D) in epididymal adipose tissue. Values represent the means ± SD (n = 3–4); Labeled means without a common letter are significantly different (P < 0.05). AF/CO, alcohol-fed with corn oil; AF/FO, alcohol-fed with flaxseed oil; ETH, ethanol; PF/CO, pair-fed with corn oil; PF/FO, pair-fed with flaxseed oil.

Figure 4. Dietary flaxseed oil ameliorates endoplasmic reticulum stress in the epididymal adipose tissue of alcohol-fed mice.

Representative Western blot images of p-IRE1α, IRE1α, p-EIF2α, and EIF2α proteins (A) and the densitometry analysis results (B). Values represent the means ± SD (n = 3–4). Labeled means without a common letter are significantly different (P < 0.05). AF/CO, alcohol-fed with corn oil; AF/FO, alcohol-fed with flaxseed oil; ETH, ethanol; PF/CO, pair-fed with corn oil; PF/FO, pair-fed with flaxseed oil.

Figure 5. Dietary flaxseed oil improves the dysfunctional lipid metabolism in the liver of alcohol-fed mice.

qPCR analysis of liver genes involved in fatty acid uptake (A) and triglyceride synthesis (B). Immunoblot analysis of genes involved in fatty acid uptake and VLDL export in the liver (C) and their results of the densitometric analysis (D). Values represent the means ± SD (n = 3–4). Labeled means without a common letter are significantly different (P < 0.05). AF/CO, alcohol-fed with corn oil; AF/FO, alcohol-fed with flaxseed oil; ETH, ethanol; PF/CO, pair-fed with corn oil; PF/FO, pair-fed with flaxseed oil.

Figure 6. Dietary flaxseed oil improves the ethanol inhibitory effect on adiponectin signaling in alcohol-fed mice.

Immunoblot analysis of ADIPOR2, ACC, AMPK, p-AMPK (Thr¹⁷²), and p-ACC (Ser⁷⁹) proteins in the liver (A) and PPARG in epididymal adipose tissue (B). Right panels show the densitometric analysis results. Values represent the means ± SD (n = 3–4). Labeled means without a common letter are significantly different (P < 0.05). AF/CO, alcohol-fed with corn oil; AF/FO, alcohol-fed with flaxseed oil; ETH, ethanol; PF/CO, pair-fed with corn oil; PF/FO, pair-fed with flaxseed oil.

Figure 7. Schematic diagram of the potential mechanisms that underlie the protective effect of α-linolenic acid-rich flaxseed oil against alcoholic hepatic steatosis in mice.

ADIPOR2, adiponectin receptor 2; AMPK, adenosine monophosphate-activated protein kinase; ATGL, adipose triglyceride lipase; CM, chylomicrons; DGAT, diacylglycerol acyltransferase; ER, endoplasmic reticulum; FFA, free fatty acid; GPAT, glycerol-3-phosphate acyltransferase; HSL, hormone-sensitive lipase; IRE1α, inositol-requiring enzyme 1α; LPL, lipoprotein lipase; MTTP, microsomal triglyceride transfer protein; PPARG, peroxisome proliferator-activated receptor-γ; TG, triglyceride; VLDL, very low-density lipoprotein; VLDL-r, very low-density lipoprotein receptor.