A snapshot of the hepatic transcriptome: ad libitum alcohol intake suppresses expression of cholesterol synthesis genes in alcohol-preferring (P) rats

Abstract

Research is uncovering the genetic and biochemical effects of consuming large quantities of alcohol. One prime example is the J- or U-shaped relationship between the levels of alcohol consumption and the risk of atherosclerotic cardiovascular disease. Moderate alcohol consumption in humans (about 30 g ethanol/d) is associated with reduced risk of coronary heart disease, while abstinence and heavier alcohol intake is linked to increased risk. However, the hepatic consequences of moderate alcohol drinking are largely unknown. Previous data from alcohol-preferring (P) rats showed that chronic consumption does not produce significant hepatic steatosis in this well-established model. Therefore, free-choice alcohol drinking in P rats may mimic low risk or nonhazardous drinking in humans, and chronic exposure in P animals can illuminate the molecular underpinnings of free-choice drinking in the liver. To address this gap, we captured the global, steady-state liver transcriptome following a 23 week free-choice, moderate alcohol consumption regimen (∼ 7.43 g ethanol/kg/day) in inbred alcohol-preferring (iP10a) rats. Chronic consumption led to down-regulation of nine genes in the cholesterol biosynthesis pathway, including HMG-CoA reductase, the rate-limiting step for cholesterol synthesis. These findings corroborate our phenotypic analyses, which indicate that this paradigm produced animals whose hepatic triglyceride levels, cholesterol levels and liver histology were indistinguishable from controls. These findings explain, at least in part, the J- or U-shaped relationship between cardiovascular risk and alcohol intake, and provide outstanding candidates for future studies aimed at understanding the mechanisms that underlie the salutary cardiovascular benefits of chronic low risk and nonhazardous alcohol intake.

Figure 1. Histological analysis of livers.

Liver sections were stained using Oil Red O and Harris hematoxylin. (A) and (B) Representative images of H₂O animal livers. (C) and (D) Representative images of EtOH livers. There is no evidence of hepatic steatosis in the livers of EtOH animals following 23 weeks of ethanol exposure. The scale bar is 200 µm.

Figure 2. The cholesterol biosynthesis pathway.

Moderate alcohol consumption decreased expression of 15 genes throughout the cholesterol biosynthesis pathway. Blue circles denote genes with a fold change greater than −1.3, while green denote those less than −1.3. All enzymes (orange arrowheads) produced from these genes catalyzed (green arrows labeled Z) their respective chemical reactions (grey boxes) on their target compounds (purple hexagons). The first compound, Acetyl-CoA, and the last compound, Cholesterol, are denoted by black circles (top and bottom of figure, respectively). The figure also displays the cellular localization of the enzymes: the entire figure is found in the cytoplasm, pink ovals represent peroxisomes (e.g. IDI1), blue ovals are lysosomes (e.g. LSS), and pink stacks represent endoplasmic reticulum (e.g. FDFT1). Srebf1 and Srebf2, transcription factors that activate many of these genes, were both down-regulated (not displayed). Nine of these results were confirmed by qRT-PCR (Fig. 3A). Image modified from Thomson Reuter's MetaCore.

Figure 3. Confirmation of altered expression by qRT-PCR in functional pathway genes.

Taqman-based qRT-PCR was used to confirm the changes in gene expression. These graphs display the fold change (±SEM) of the candidate genes by qRT-PCR (red bars) and RNA-Seq (black bars) analysis for comparison. Numbers are relative to the water treated control rats (set at 1.0). (A) Nine of twelve genes in the cholesterol biosynthesis pathway were confirmed. (B) Pparα, a transcription factor that activates genes for cholesterol oxidation was not significantly altered by RNA-Seq analysis, but was significantly up-regulated by qRT-PCR. Srebf1, a transcriptioin factor that activates many genes in the synthesis pathway, was significantly decreased in both analyses. (C) Four cytoskeleton subunit genes were suppressed by RNA-Seq and qRT-PCR. (D) Cyp2e1, a gene induced by chronic high levels of alcohol intake that mediates various alcohol-induced injuries was not induced when measured by RNA-Seq analysis, but was significantly up-regulated by qRT-PCR. * - p-value <0.05, ** - p-value <0.005, *** - p-value <0.0001.

Figure 4. Altered expression by qRT-PCR.

(A) Irs2 and Pck1, involved in the regulation of lipid metabolism, were up-regulated by RNA-Seq analysis and confirmed by qRT-PCR. (B) and (C) confirm the genes with the greatest up-regulation and down-regulation, respectively. The direction of the change was confirmed for all 6 genes, though not necessarily to the same degree. ** - p-value <0.005, *** - p-value <0.0001.