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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.

SUBMITTER: Klein JD

PROVIDER: S-EPMC4277277 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Klein Jonathon D JD Sherrill Jeremy B JB Morello Gabriella M GM San Miguel Phillip J PJ Ding Zhenming Z Liangpunsakul Suthat S Liang Tiebing T Muir William M WM Lumeng Lawrence L Lossie Amy C AC

PloS one 20141226 12

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 ...[more]

PMID: 25542004

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Project description:Introduction: Though heavy alcohol drinking has been well characterized as causing a variety of injuries, recent epidemiological evidence in humans suggests moderate consumption may provide beneficial effects. For example, there exists a J- or U-shaped relationship between the level of alcohol intake and cardiovascular disease risk. We investigated the underlying mechanisms of these positive consequences by identifying which genes are responsive to moderate alcohol intake in the liver, the primary site for alcohol metabolism. Methods: Twelve female, inbred, alcohol-preferring (iP10a) rats were split equally between chronic water exposure and voluntary chronic ethanol exposure. Hepatic cholesterol and triglyceride levels were analyzed both histologically and biochemically. Hepatic transcriptomes were paired-end sequenced on the Illumina HiScanSQ system. Reads were analyzed and mapped using CLCbio Genomics Workbench 4.9. We confirmed altered expression of a subset of genes using TaqMan-based qRT-PCR. To quantify DNA methylation, we first digested DNA with methylation sensitive restriction enzymes and then performed qPCR using TaqMan assays surrounding the digest sites. Calculating M-NM-^TCt between a mock digest and digest determines the percent methylation in that locus. Results: Voluntary alcohol consumption in iP10a rats modeled moderate consumption in humans. These levels did not result in intrahepatic fat accumulation. Sequencing produced ~1.2 Gb of sequence per sample, and 65% of reads mapped uniquely. Using a FDR corrected p value of 0.05 we found 250 altered transcripts. Ontology analysis of genes with a fold change M-bM-^IM-%1.3 identified many cholesterol synthesis genes and cytoskeleton subunit genes, all of which were down-regulated. Of the 28 genes reanalyzed by qRT-PCR, altered expression was confirmed in 24 genes including the majority of the cholesterol synthesis and cytoskeleton subunit genes. Lastly, we profiled methylation throughout the promoter and gene body of four genes elicited in the RNA-Seq experiment. We found that alcohol caused demethylation at all loci; however this loss happened in a site-specific, tightly regulated manner. Conclusion: Voluntary consumption in the iP10a animals models moderate consumption in humans, does not produce intrahepatic fat accumulation, and causes down-regulation of a majority of cholesterol synthesis genes. Moderate alcohol also results in a tightly-regulated demethylation effect. Our results explain, at least in part, the J- or U-shaped relationship between level of alcohol intake and cardiovascular disease risk. We sequenced 12 female iP10a rat hepatic transcriptomes providing 6 biological replicates for water control and 6 for ethanol treatment.

Project description:Introduction: Though heavy alcohol drinking has been well characterized as causing a variety of injuries, recent epidemiological evidence in humans suggests moderate consumption may provide beneficial effects. For example, there exists a J- or U-shaped relationship between the level of alcohol intake and cardiovascular disease risk. We investigated the underlying mechanisms of these positive consequences by identifying which genes are responsive to moderate alcohol intake in the liver, the primary site for alcohol metabolism. Methods: Twelve female, inbred, alcohol-preferring (iP10a) rats were split equally between chronic water exposure and voluntary chronic ethanol exposure. Hepatic cholesterol and triglyceride levels were analyzed both histologically and biochemically. Hepatic transcriptomes were paired-end sequenced on the Illumina HiScanSQ system. Reads were analyzed and mapped using CLCbio Genomics Workbench 4.9. We confirmed altered expression of a subset of genes using TaqMan-based qRT-PCR. To quantify DNA methylation, we first digested DNA with methylation sensitive restriction enzymes and then performed qPCR using TaqMan assays surrounding the digest sites. Calculating ΔCt between a mock digest and digest determines the percent methylation in that locus. Results: Voluntary alcohol consumption in iP10a rats modeled moderate consumption in humans. These levels did not result in intrahepatic fat accumulation. Sequencing produced ~1.2 Gb of sequence per sample, and 65% of reads mapped uniquely. Using a FDR corrected p value of 0.05 we found 250 altered transcripts. Ontology analysis of genes with a fold change ≥1.3 identified many cholesterol synthesis genes and cytoskeleton subunit genes, all of which were down-regulated. Of the 28 genes reanalyzed by qRT-PCR, altered expression was confirmed in 24 genes including the majority of the cholesterol synthesis and cytoskeleton subunit genes. Lastly, we profiled methylation throughout the promoter and gene body of four genes elicited in the RNA-Seq experiment. We found that alcohol caused demethylation at all loci; however this loss happened in a site-specific, tightly regulated manner. Conclusion: Voluntary consumption in the iP10a animals models moderate consumption in humans, does not produce intrahepatic fat accumulation, and causes down-regulation of a majority of cholesterol synthesis genes. Moderate alcohol also results in a tightly-regulated demethylation effect. Our results explain, at least in part, the J- or U-shaped relationship between level of alcohol intake and cardiovascular disease risk.

Project description:BackgroundNeural activity within the prefrontal cortex (PFC) is altered by alcohol and alcohol-associated stimuli and is mediated by genetic susceptibility to alcoholism. However, very little is known about how genetic risk of excessive drinking might mediate neural firing in the PFC during alcohol consumption.MethodsTo determine how genetic risk influences alcohol seeking, intake, and neural activity, a Pavlovian alcohol consumption task was used-the 2-Way Cued Access Protocol (2CAP). Alcohol-preferring "P" rats and relatives of their (heterogeneous) founding Wistar population were used for these studies. After acquisition of 2CAP, extinction of responding for alcohol was evaluated by substituting water for alcohol. Following these experiments, in vivo electrophysiological recordings were obtained during 2CAP from the PFC in a separate cohort of Wistar and P rats implanted with moveable tetrode microdrives.ResultsP and Wistar rats increased daily alcohol seeking and intake with P rats consuming roughly twice as much alcohol as Wistar. Both rat populations decreased seeking behavior during extinction. However, P rats displayed persistent increases in seeking after controlling for intake versus Wistar. Higher firing rates (FRs) were observed in P rats prior to 2CAP and throughout alcohol and water consumption compared with Wistars that were matched for alcohol-drinking history. Differences in FR were driven, in part, by a larger percentage of neurons in P rats versus Wistars that increased FR compared with those that decreased, or did not change.ConclusionsThese data provide additional evidence of increased alcohol consumption and persistent alcohol seeking in P versus Wistar rats. Differences in PFC neural firing observed in P rats prior to drinking could be heritable and/or related to an enhanced response to alcohol-associated contextual cues. FR differences observed during alcohol drinking might be related to an augmented sensitivity of PFC neurons to orally consumed alcohol.

Project description:The goal of this study was to identify candidate genes that may influence alcohol consumption by comparing gene expression in 5 brain regions of alcohol-naïve iP and P.NP rats. Background: Selectively bred P (alcohol preferring) and NP (alcohol non-preferring) rats differ greatly in alcohol preference, in part due to a highly significant QTL on chromosome 4. Reciprocal congenic strains in which the iP chromosome 4 QTL interval was transferred to the iNP background (NP.P) and the iNP chromosome 4 QTL was transferred to the iP background (P.NP) exhibited alcohol consumption scores that correlated with the introgressed interval. The goal of this study was to identify candidate genes that may influence alcohol consumption by comparing gene expression in 5 brain regions of alcohol-naïve iP and P.NP rats. Methods: RNA from the amygdala, nucleus accumbens, hippocampus, caudate putamen, and frontal cortex from each of 8 iP and 8 P.NP rats was labeled and analyzed on Affymetrix Rat Genome 230 2.0 microarrays. Expression levels were normalized using robust multi-chip average (RMA), and differential gene expression was measured in individual brain regions and in the average of the five brain regions. Differential gene expression was validated using quantitative real-time PCR. Meta-analysis was applied to compare microarray data from this experiment with data from the reciprocal congenic strains NP.P vs. iNP (Carr et al, 2007). Results: We detected between 72 (nucleus accumbens) and 89 (hippocampus) cis-regulated probe sets within the QTL that significantly differed between the strains in the five brain regions. There was significant overlap among the regions; 157 cis-regulated probe sets were detected in at least one brain region, of which 104 showed differential expression in more than one region. Fewer trans-regulated probe sets were detected, ranging from 7 in the amygdala to 54 in the caudate putamen, and most of these differed in only one region; only 10 of the 85 trans-regulated probe sets differed in more than one region. To increase the power to detect differentially expressed genes, data from the five discrete brain regions of each animal were averaged; in this analysis we detected 141 cis-regulated probe sets and 207 trans-regulated probe sets. Meta-analysis comparing the present results from iP vs. P.NP rats with an earlier experiment that used the reciprocal congenic NP.P vs. iP demonstrated that 74 cis-regulated probe sets were differentially expressed in the same direction and with a consistent magnitude of difference in both experiments. No consistent trans-regulated probe sets were identified. Conclusions: Cis-regulated candidate genes for alcohol consumption that lie within the chromosome 4 QTL were identified and confirmed by meta-analysis with the reciprocal congenic NP.P vs iNP study. These genes are strong candidates for producing the difference in alcohol preference and consumption between the iP and iNP rats. There was little evidence for consistent trans-acting effects. Keywords: comparison of gene expression profiles for strain1 (P rat) vs. strain2 (P.NP rat)

Dataset Information

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

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

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