Dietary nicotinic acid supplementation ameliorates chronic alcohol-induced fatty liver in rats

Abstract

Background: Alcohol abuse frequently causes niacin deficiency in association with the development of alcoholic liver disease. The objective of the present study was to determine whether dietary nicotinic acid (NA) deficiency exaggerates and whether dietary NA supplementation alleviates alcohol-induced fatty liver.

Methods: Male Sprague-Dawley rats were pair-fed with 4 isocaloric liquid diets: control, ethanol (EtOH), EtOH with dietary NA deficiency, and EtOH with dietary NA supplementation, respectively, for 8 weeks. The control and EtOH diets contained normal levels of NA (7.5 mg/l). Dietary NA deficiency (0 mg NA/l) was achieved by removing NA from the vitamin mix, while NA was added to the liquid diet at 750 mg/l for dietary NA supplementation.

Results: Chronic EtOH feeding induced significant lipid accumulation in the liver, which was not worsened by dietary NA deficiency, but was ameliorated by dietary NA supplementation. Liver total NAD, NAD(+) , and NADH levels were remarkably higher in the NA supplemented group than the NA deficient or EtOH alone groups. Dietary NA supplementation to EtOH-fed rats increased the protein levels of hepatic cytochrome P450 4A1 (CYP4A1) and acyl-coenzyme A oxidase 1 without affecting their mRNA levels. Interestingly, we found dietary NA supplementation reduced the ubiquitination level of CYP4A1. In addition, hepatic fatty acid synthase expression was reduced, while the serum β-hydroxybutyrate and adiponectin concentrations were significantly elevated by dietary NA supplementation. Moreover, dietary NA supplementation modulated EtOH-perturbed liver and serum metabolite profiles.

Conclusions: These results demonstrate that alcoholic fatty liver was not exaggerated by dietary NA deficiency, but was ameliorated by dietary NA supplementation. Increased hepatic fatty acid oxidation and decreased hepatic de novo lipogenesis contribute to the effects of dietary NA supplementation.

Keywords: Alcoholic Fatty Liver; Lipid Metabolism; Nicotinic Acid.

Figure 1

Dietary nicotinic acid supplementation ameliorates alcohol-induced hepatic lipid accumulation in rats. (A) Oil red O staining of the neutral lipids in the liver of Ctrl, EtOH, EtOH/ND, and EtOH/NS rats. Lipid droplets are stained with red color. (B) Quantitative measurements of hepatic concentrations of triglycerides, free fatty acids, and cholesterol. Data are expressed as mean ± SD from 6-8 rats. Statistical differences were analyzed by ANOVA followed by Student-Newman-Keuls test. Means without a common letter differ at p < 0.05, and the absence of a symbol indicates no significant difference. BW, body weight; Ctrl, control group; EtOH, ethanol group; EtOH/ND, ethanol with dietary nicotinic acid deficiency group; EtOH/NS, ethanol with dietary nicotinic acid supplementation group.

Figure 2

Hepatic NAD levels in rats after 8 weeks of liquid diet feeding. Liver total NAD, NAD⁺, and NADH concentrations as well as the NAD⁺/NADH ratio in the Ctrl, EtOH, EtOH/ND, and EtOH/NS rats were measured. Data are expressed as the mean ± SD from 6-8 rats. Means without a common letter differ at p < 0.05. BW, body weight; Ctrl, control group; EtOH, ethanol group; EtOH/ND, ethanol with dietary nicotinic acid deficiency group; EtOH/NS, ethanol with dietary nicotinic acid supplementation group.

Figure 3

OPLS-DA score plots of spectral data of liver and serum metabolites in rats after 8 weeks of liquid diet feeding. Metabolite profiles of liver tissue homogenates and serum were analyzed by HPLC-TOFMS. The ES+ and ES- raw data generated from HPLC-TOFMS were analyzed using the MassHunter Qualitative Analysis Program (vB.05.01, Agilent). The resulting data were then subjected to OPLS-DA to establish characteristic metabolomic profiles associated with different groups. (A) OPLS-DA score plots of liver metabolites. (B) OPLS-DA score plots of serum metabolites. Ctrl, control group; EtOH, ethanol group; EtOH/ND, ethanol with dietary nicotinic acid deficiency group; EtOH/NS, ethanol with dietary nicotinic acid supplementation group.

Figure 4

Dietary nicotinic acid supplementation does not affect ethanol metabolism enzymes in chronic ethanol-fed rats. Representative immunoblots showing hepatic ADH, CYP2E1, catalase, and ALDH2 protein levels in Ctrl, EtOH, and EtOH/NS groups are presented with quantification of the band intensities. Data are expressed as the mean ± SD from 6 rats. Means without a common letter differ at p < 0.05, and absence of a symbol indicates no significant difference. Ctrl, control group; EtOH, ethanol group; EtOH/NS, ethanol with dietary nicotinic acid supplementation group.

Figure 5

Dietary nicotinic acid supplementation improves hepatic lipid metabolism in chronic ethanol-fed rats. (A) Representative immunoblots showing hepatic proteins related to fatty acid oxidation in Ctrl, EtOH, and EtOH/NS groups are presented with quantification of the band intensities. (B) Representative immunoblots showing hepatic proteins related to fatty acid and triglyceride synthesis are presented with quantification of the band intensities. Data are expressed as the mean ± SD from 6 rats. Means without a common letter differ at p < 0.05, and absence of a symbol indicates no significant difference. Ctrl, control group; EtOH, ethanol group; EtOH/NS, ethanol with dietary nicotinic acid supplementation group.

Figure 6

Dietary nicotinic acid supplementation does not affect the PPARα pathway, but does reduce the ubiquitination of CYP4A1 in the liver of chronic ethanol-fed rats. (A) Representative immunoblots showing hepatic protein levels of transcription factors/cofactors related to the PPARα pathway in Ctrl, EtOH, and EtOH/NS groups are presented with quantification of band intensities. (B) The mRNA levels of CYP4A1 and ACOX1 in the liver of Ctrl, EtOH, and EtOH/NS rats. (C) Ubiquitination levels of CYP4A1 in the liver of Ctrl, EtOH, and EtOH/NS rats. Hepatic CYP4A1 was immunoprecipitated and then subjected to Western blot using a rabbit polyclonal anti-ubiquitin antibody. Data are expressed as the mean ± SD from 6 rats. Means without a common letter differ at p < 0.05, and absence of a symbol indicates no significant difference. Ctrl, control group; EtOH, ethanol group; EtOH/NS, ethanol with dietary nicotinic acid supplementation group.