Altered hepatic retinyl ester concentration and acyl composition in response to alcohol consumption

Abstract

Retinoids (vitamin A and its metabolites) are essential micronutrients that regulate many cellular processes. Greater than 70% of the body's retinoid reserves are stored in the liver as retinyl ester (RE). Chronic alcohol consumption induces depletion of hepatic retinoid stores, and the extent of this has been correlated with advancing stages of alcoholic liver disease. The goal of this study was to analyze the mechanisms responsible for depletion of hepatic RE stores by alcohol consumption. A change in the fatty-acyl composition of RE in alcohol-fed mice was observed within two weeks after the start of alcohol consumption. Specifically, alcohol-feeding was associated with a significant decline in hepatic retinyl palmitate levels; however, total RE levels were maintained by a compensatory increase in levels of usually minor RE species, particularly retinyl oleate. Our data suggests that alcohol feeding initially stimulates a futile cycle of RE hydrolysis and synthesis, and that the change in RE acyl composition is associated with a change in the acyl composition of hepatic phosphatidylcholine. The alcohol-induced change in RE acyl composition was specific to the liver, and was not seen in lung or white adipose tissue. This shift in hepatic RE fatty acyl composition is a sensitive indicator of alcohol consumption and may be an early biomarker for events associated with the development of alcoholic liver disease.

Fig. 1

Alcohol feeding is associated with an alteration in the fatty acyl composition of hepatic retinyl ester. A diagram showing the alcohol feeding protocol used in this study is provided (A). Tissue was collected from mice in the control group after 3 or 7 weeks of consuming the alcohol-free liquid diets. Tissue was collected from alcohol-fed mice after the adaptation period and after consuming 6.4% alcohol for 1, 2 and 4 weeks. Representative HPLC chromatograms are shown for control (B) and alcohol-fed mice (C) for liver tissue collected immediately after the alcohol adaptation period. Numbered peaks correspond to 1) retinol, 2) retinyl acetate (internal standard), 3) retinyl linoleate, 4) retinyl oleate, 5) retinyl palmitate and 6) retinyl stearate. Quantification of retinyl esters revealed a significant decline in total hepatic retinyl ester concentration after 2 and 4 weeks of consuming 6.4% alcohol (D). Hepatic levels of retinyl palmitate were significantly decreased following the alcohol adaptation period (E). Hepatic levels of retinyl oleate were significantly increased in association with alcohol consumption (F). We also observed alcohol-induced changes in the hepatic levels of the less abundant retinyl ester species, retinyl linoleate (G) and retinyl stearate (H). Data analyzed by one-way ANOVA; * p < 0.05 versus control.

Fig. 2

The change in hepatic fatty acyl composition of retinyl ester is reversible following the withdrawal of alcohol from the diet. Hepatic retinyl esters were examined in mice fed with the control liquid diet (control), the alcohol-containing liquid diet for the adaptation period (alcohol), or the alcohol-containing liquid diet for the adaptation period and then an alcohol-free diet for a further 4 weeks (recovery). The total hepatic retinyl ester (A), retinyl palmitate (B), retinyl oleate (C), and retinyl linoleate (D) levels are shown. Data analyzed by one-way ANOVA; * p < 0.05 versus control.

Fig. 3

Alcohol feeding is associated with an alteration in the fatty acyl composition of plasma retinyl ester. Circulating levels of retinyl palmitate are not significantly different in control and alcohol-fed mice (A). However, alcohol feeding is associated with a significant increase in the circulating level of retinyl oleate (B). The retinyl palmitate:retinyl oleate ratio is significantly altered in alcohol-fed mice (C). Data analyzed by Student’s t-test; * p < 0.05 versus control.

Fig. 4

Hepatic LRAT protein expression is increased by chronic alcohol consumption. Representative Western blot data for LRAT and β-ACTIN are shown for liver tissue collected during chronic alcohol feeding (A). Quantification of LRAT (B) protein expression relative to β-actin is also shown, indicating that chronic alcohol consumption increases LRAT protein expression level. Data analyzed by Student’s t-test; * p < 0.05 versus control.

Fig. 5

Altered fatty acyl composition of retinyl ester is observed using multiple protocols for chronic alcohol feeding. The relative retinyl ester content for hepatic retinyl palmitate and retinyl oleate is shown in all panels. Chronic consumption of the high-fat formulation of the alcohol-free Lieber–DeCarli liquid diet was not associated with a decrease in hepatic retinyl palmitate or increased retinyl oleate levels (A). However, chronic consumption of the high-fat formulation of the alcohol-containing Lieber–DeCarli liquid diet was associated with a significant decline in hepatic retinyl palmitate levels, with a corresponding increase in retinyl oleate (B). A significant decline in hepatic retinyl palmitate levels and an increase in retinyl oleate was also observed in mice consuming the alcohol-containing low-fat formulation of the Lieber–DeCarli diet (C), as well as in those mice provided with alcohol in their drinking water (D). Data analyzed by Student’s t-test; * p < 0.05 versus control.

Fig. 6

Hepatic phosphatidylcholine levels and GPAT expression in control and alcohol-fed mice. The total hepatic PC level in control and alcohol-fed mice is shown (A). Composite PC species were further categorized according to their relative abundance and graphed as major (B; > 1.0 nmol/g), minor (C; 1 to 0.05 nmol/g), and low abundance PCs (D; < 0.05 nmol/g). The mRNA expression level of Gpat1 and Gpat4, relative to the reference gene 18S, was also measured in control and alcohol-fed mice (E). Data analyzed by Student’s t-test; * p < 0.05 versus control.