Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Abstract

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.

Fig. 1.

Relative levels of plasma and liver fatty acid metabolites in alcohol-fed mice. (A) The changes in plasma and hepatic total FFAs are shown for alcohol-fed mice (n = 11) relative to control mice (n = 8). In control mice, plasma total FFA levels are unchanged, whereas there is a significant increase in hepatic FFA levels for alcohol-fed mice. (B) Plasma and liver total FA-CoA levels are decreased in alcohol-fed mice. (C) Alcohol feeding is associated with a higher level of total FAEE in the liver; however there was no significant change in the plasma. Data are means ± SEM. Heat maps showing the relative change in plasma (left column) and hepatic lipids (right column) of different carbon-chain lengths are provided for FFA (D), FA-CoA (E), and FAEE (F). *P < 0.05 alcohol-fed versus control mice.

Fig. 2.

Increased unsaturated fatty acid levels in alcohol-fed mice. (A) Total hepatic saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) levels in control and alcohol-fed mice, demonstrating increased levels of MUFA and PUFA following alcohol feeding. (B) Relative levels of fatty acids in the liver, expressed as a percentage of total FFA. Following alcohol feeding, there was a relative decrease in SFA and a relative increase in PUFA. The relative amount of MUFA in the fatty acid pool was unchanged. Data are means ± SEM. *P < 0.05 alcohol-fed versus control mice.

Fig. 3.

Alcohol feeding is associated with altered expression of genes involved in FA metabolism. Relative mRNA expression of several genes associated with FA metabolism is shown. Data are means ± SEM. *P < 0.05 alcohol-fed versus control mice.

Fig. 4.

Relative levels of sphingolipids in the plasma and liver of alcohol-fed mice. (A) Plasma levels of sphingosine and sphinganine were significantly lower in alcohol-fed mice (n = 11) relative to control mice (n = 8), with no change in SO-1P or SA-1P observed. (B) Liver levels of sphingosine and sphinganine were higher in alcohol-fed mice, whereas SO-1P was decreased, and SA-1P was unchanged. (C) Total ceramide levels were unchanged in the plasma of alcohol-fed mice; however there was a significant increase in total ceramide in the liver. (D) A heat map showing relative changes in individual ceramide molecules with different fatty acyl chain lengths is shown for plasma (left column) and liver (right column). (E) Kdsr expression is decreased in the liver of alcohol-fed mice. All data are means ± SEM. *P < 0.05 alcohol-fed versus control mice.

Fig. 5.

Relative plasma and hepatic levels of AEA and 2-AG in alcohol-fed mice (n = 11) versus control mice (n = 8). (A) Plasma levels of AEA and 2-AG in alcohol-fed mice relative to control mice are shown; there was no effect of alcohol feeding observed. (B) Liver levels of AEA were significantly increased in the alcohol-fed mice relative to control mice; however there was no change in 2-AG observed. (C) The expressions of Daglb, Faah, and Magl were significantly deceased in liver of alcohol-fed mice. Data are means ± SEM. *P < 0.05 alcohol-fed versus control mice.

Fig. 6.

Decreased Elovl5 expression is associated with increased hepatic C18:3 FFA. (A) Elovl5 gene expression is decreased to ∼40% of control in the liver of alcohol-fed mice. (B) The Elovl5 substrate C18:3 is significantly increased in liver of alcohol-fed mice. Data are means ± SEM. *P < 0.05 alcohol-fed versus control mice.