• Energy Research

BackgroundGlutathione S‐transferase A4‐4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4‐hydroxynonenal (4‐HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild‐type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4−/− mice).MethodsAdult male WT and GSTA4−/− mice were fed chow (N = 10 to 12) or high‐fat Lieber‐DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH‐fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4‐HNE adduction and by comprehensive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) of purified carbonylated proteins.ResultsChronic EtOH intake significantly increased hepatic 4‐HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro‐inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4−/− mice increased B‐cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4−/− mice exhibited panlobular rather than periportal distribution of 4‐HNE‐adducted proteins and increased overall 4‐HNE staining after EtOH binge. Comprehensive LC‐MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4−/− group.ConclusionsThese data suggest long‐term adaptation to EtOH in WT mice does not occur in GSTA4−/− mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B‐cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.

Abstract Background Chronic alcohol consumption is a significant cause of liver disease worldwide. Several biochemical mechanisms have been linked to the initiation and progression of alcoholic liver disease (ALD) such as oxidative stress, inflammation, and metabolic dysregulation, including the disruption of NAD+/NADH. Indeed, an ethanol-mediated reduction in hepatic NAD+ levels is thought to be one factor underlying ethanol-induced steatosis, oxidative stress, steatohepatitis, insulin resistance, and inhibition of gluconeogenesis. Therefore, we applied a NAD+ boosting supplement to investigate alterations in the pathogenesis of early-stage ALD. Methods To examine the impact of NAD+ therapy on the early stages of ALD, we utilized nicotinamide mononucleotide (NMN) at 500 mg/kg intraperitoneal injection every other day, for the duration of a Lieber-DeCarli 6-week chronic ethanol model in mice. Numerous strategies were employed to characterize the effect of NMN therapy, including the integration of RNA-seq, immunoblotting, and metabolomics analysis. Results Our findings reveal that NMN therapy increased hepatic NAD+ levels, prevented an ethanol-induced increase in plasma ALT and AST, and changed the expression of 25% of the genes that were modulated by ethanol metabolism. These genes were associated with a number of pathways including the MAPK pathway. Interestingly, our analysis revealed that NMN treatment normalized Erk1/2 signaling and prevented an induction of Atf3 overexpression. Conclusions These findings reveal previously unreported mechanisms by which NMN supplementation alters hepatic gene expression and protein pathways to impact ethanol hepatotoxicity in an early-stage murine model of ALD. Overall, our data suggest further research is needed to fully characterize treatment paradigms and biochemical implications of NAD+-based interventions.

We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.In the HXB/BXH recombinant inbred (RI) rats, correlation analysis of brain gene expression levels with alcohol consumption in a two-bottle choice paradigm, and filtering based on behavioral and gene expression quantitative trait locus (QTL) analyses, generated a list of candidate genes. A literature-based, functional analysis of the interactions of the products of these candidate genes defined pathways linked to presynaptic GABA release, activation of dopamine neurons, and postsynaptic GABA receptor trafficking, in brain regions including the hypothalamus, ventral tegmentum and amygdala. The analysis also implicated energy metabolism and caloric intake control as potential influences on alcohol consumption by the recombinant inbred rats. In the human populations, polymorphisms in genes associated with GABA synthesis and GABA receptors, as well as genes related to dopaminergic transmission, were associated with alcohol consumption.Our results emphasize the importance of the signaling pathways identified using the non-human animal models, rather than single gene products, in identifying factors responsible for complex traits such as alcohol consumption. The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans. The importance of a well-defined phenotype is also illustrated. Our results also suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.

Although ethanol has been considered to be an anxiolytic agent, consumption of ethanol has also been shown to increase plasma adrenocorticotropin and glucocorticoids. The corticotrophin-releasing factor (CRF) receptor 1alpha (CRF-R1) is a G protein-coupled receptor that activates adenylyl cyclase (AC), leading to adrenocorticotropin (and subsequently glucocorticoid) release into the circulation. There are nine members of the membrane-bound AC family, and the type 7 AC (AC7) is most sensitive to ethanol, which enhances the responsiveness of AC7 to G protein-coupled receptor activation. We determined the time course of ethanol's effect on plasma adrenocorticotropin and corticosterone levels in male and female AC7 transgenic (Adcy7(huTG)) mice (in which AC7 is overexpressed in neural tissue) and AC7 heterozygous knockdown [Adcy7(+/-)] mice (in which AC7 is underexpressed in neural tissue), and their respective littermate controls [wild type (WT)]. CRF-R1 mRNA and mRNA and protein for different forms of ACs were measured by using gene expression arrays, quantitative reverse transcription-polymerase chain reaction, and immunoblotting in pituitaries of all animals. Our results demonstrated increased levels of AC7 in pituitary of Adcy7(huTG) mice and decreased levels in pituitary of Adcy7(+/-) mice compared with WT animals. Male and female Adcy7(huTG) mice displayed higher plasma adrenocorticotropin and corticosterone levels than WT and/or Adcy7(+/-) mice after ethanol injection. Female mice displayed higher adrenocorticotropin and corticosterone levels after ethanol injection than males, regardless of genotype. The data provide evidence for an integral role of AC7 in the increase of plasma adrenocorticotropin and corticosterone levels during alcohol intoxication.

MicroRNAs (miRNAs) are small non-coding RNAs that bind messenger RNAs and promote their degradation or repress their translation. There is increasing evidence of miRNAs playing an important role in alcohol related disorders. However, the role of miRNAs as mediators of the genetic effect on alcohol phenotypes is not fully understood. We conducted a high-throughput sequencing study to measure miRNA expression levels in alcohol naïve animals in the LXS panel of recombinant inbred (RI) mouse strains. We then combined the sequencing data with genotype data, microarry gene expression data, and data on alcohol-related behavioral phenotypes such as 'Drinking in the dark', 'Sleep time', and 'Low dose activation' from the same RI panel. SNP-miRNA-gene triplets with strong association within the triplet that were also associated with one of the 4 alcohol phenotypes were selected and a Bayesian network analysis was used to aggregate results into a directed network model.We found several triplets with strong association within the triplet that were also associated with one of the alcohol phenotypes. The Bayesian network analysis found two networks where a miRNA mediates the genetic effect on the alcohol phenotype. The miRNAs were found to influence the expression of protein-coding genes, which in turn influences the quantitative phenotypes. The pathways in which these genes are enriched have been previously associated with alcohol-related traits.This work enhances association studies by identifying miRNAs that may be mediating the association between genetic markers (SNPs) and the alcohol phenotypes. It suggests a mechanism of how genetic variants are affecting traits of interest through the modification of miRNA expression.

Alcohol "sensitivity" has been proposed as a predictive factor for development of alcohol dependence (Schuckit et al., 2005). Most measures of alcohol sensitivity in humans and animals include a component that can be ascribed to acute functional tolerance (AFT). AFT is a form of tolerance that develops within a single period of alcohol exposure and has a genetic component. We used microarray technology as well as quantitative trait locus analysis of phenotypic and gene expression data across 30 BXD recombinant inbred strains of mice, 20 inbred strains of mice, and two replicate lines of mice selectively bred for differences in AFT, to identify differentially expressed candidate genes that contribute to predisposition to AFT. Eight candidate genes were identified by our statistical and filtering methods. The location of brain expression of these genes was mapped using the Allen Brain Atlas (http://www.brain-map.org), and the transcript location and molecular pathway analysis indicated that brain structures and biochemical pathways implicated in long-term potentiation and memory might also participate in the generation of acute functional alcohol tolerance.

We previously reported that acute functional tolerance (AFT) to the hypnotic effects of alcohol was significantly correlated with drinking in the dark (DID) in the LXS recombinant inbred panel, but only in mice that had been pretreated with alcohol. Here, we have conducted quantitative trait locus (QTL) mapping for AFT. DNA sequencing of the progenitor ILS and ISS strains and microarray analyses were also conducted to identify candidate genes and functional correlates.LXS mice were given either saline or alcohol (5 g/kg) on day 1 and then tested for loss of righting reflex AFT on day 2. QTLs were mapped using standard procedures. Two microarray analyses from brain were conducted: (i) naïve LXS mice and (ii) an alcohol treatment time course in the ILS and ISS. The full genomes of the ILS and ISS were sequenced to a depth of approximately 30×.A significant QTL for AFT in the alcohol pretreatment group was mapped to distal chromosome 4; numerous suggestive QTLs were also mapped. Preference drinking and DID have previously been mapped to the chromosome 4 locus. The credible interval of the significant chromosome 4 QTL spanned 23 Mb and included 716 annotated genes of which 150 had at least 1 nonsynonymous single nucleotide polymorphism or small indel that differed between the ILS and ISS; expression of 48 of the genes was cis-regulated. Enrichment analysis indicated broad functional categories underlying AFT, including proteolysis, transcription regulation, chromatin modification, protein kinase activity, and apoptosis.The chromosome 4 QTL is a key region containing possibly pleiotropic genes for AFT and drinking behavior. Given that the region contains many viable candidates and a large number of the genes in the interval fall into 1 or more of the enriched functional categories, we postulate that many genes of varying effect size contribute to the observed QTL effect.