Long-Chain ω-3 Levels Are Associated With Increased Alcohol Sensitivity in a Population-Based Sample of Adolescents.

BackgroundThe levels of the ω-3 long-chain polyunsaturated fatty acids (ω-3 LC-PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been associated with alcohol sensitivity in vertebrate and invertebrate model systems, but prior studies have not examined this association in human samples despite evidence of associations between ω-3 LC-PUFA levels and alcohol-related phenotypes. Both alcohol sensitivity and ω-3 LC-PUFA levels are impacted by genetic factors, and these influences may contribute to observed associations between phenotypes. Given the potential for using EPA and DHA supplementation in adjuvant care for alcohol misuse and other outcomes, it is important to clarify how ω-3 LC-PUFA levels relate to alcohol sensitivity.MethodsAnalyses were conducted using data from the Avon Longitudinal Study of Parents and Children. Plasma ω-3 LC-PUFA levels were measured at ages 15.5 and 17.5. Participants reported on their initial alcohol sensitivity using the early drinking Self-Rating of the Effects of Alcohol (SRE-5) scale, for which more drinks needed for effects indicates lower levels of response per drink, at ages 15.5, 16.5, and 17.5. Polygenic liability for alcohol consumption, alcohol problems, EPA levels, and DHA levels was derived using summary statistics from large, publicly available datasets. Linear regressions were used to examine the cross-sectional and longitudinal associations between ω-3 LC-PUFA levels and SRE scores.ResultsAge 15.5 ω-3 LC-PUFA levels were negatively associated with contemporaneous SRE scores and with age 17.5 SRE scores. One modest association (p = 0.02) between polygenic liability and SRE scores was observed, between alcohol problems-based polygenic risk scores (PRS) and age 16.5 SRE scores. Tests of moderation by genetic liability were not warranted.ConclusionsPlasma ω-3 LC-PUFA levels may be related to initial sensitivity to alcohol during adolescence. These data indicate that diet-related factors have the potential to impact humans' earliest responses to alcohol exposure

Contrasting influences of Drosophila white/mini-white on ethanol sensitivity in two different behavioral assays

Background The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white, a derivative of the endogenous gene white. Whether the mini-white transgenic marker or the endogenous white gene influence behavioral responses to acute ethanol exposure in flies has not been systematically investigated. Methods We manipulated mini-white and white expression via (i) transposons marked with mini-white, (ii) RNAi against mini-white and white and (iii) a null allele of white. We assessed ethanol sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of ethanol) and an assay based on ethanol-induced sedation. Results In eRING assays, ethanol-induced impairment of climbing correlated inversely with expression of the mini-white marker from a series of transposon insertions. Additionally, flies harboring a null allele of white or flies with RNAi-mediated knockdown of mini-white were significantly more sensitive to ethanol in eRING assays than controls expressing endogenous white or the mini-white marker. In contrast, ethanol sensitivity and rapid tolerance measured in the ethanol sedation assay were not affected by decreased expression of mini-white or endogenous white in flies. Conclusions Ethanol sensitivity measured in the eRING assay is noticeably influenced by white and mini-white, making eRING problematic for studies on ethanol-related behavior in Drosophila using transgenes marked with mini-white. In contrast, the ethanol sedation assay described here is a suitable behavioral paradigm for studies on ethanol sedation and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-white

Transcriptional analysis of the response of \u3ci\u3eC. elegans\u3c/i\u3e to ethanol exposure

Ethanol-induced transcriptional changes underlie important physiological responses to ethanol that are likely to contribute to the addictive properties of the drug. We examined the transcriptional responses of Caenorhabditis elegans across a timecourse of ethanol exposure, between 30 min and 8 h, to determine what genes and genetic pathways are regulated in response to ethanol in this model. We found that short exposures to ethanol (up to 2 h) induced expression of metabolic enzymes involved in metabolizing ethanol and retinol, while longer exposure (8 h) had much more profound effects on the transcriptome. Several genes that are known to be involved in the physiological response to ethanol, including direct ethanol targets, were regulated at 8 h of exposure. This longer exposure to ethanol also resulted in the regulation of genes involved in cilia function, which is consistent with an important role for the effects of ethanol on cilia in the deleterious effects of chronic ethanol consumption in humans. Finally, we found that food deprivation for an 8-h period induced gene expression changes that were somewhat ameliorated by the presence of ethanol, supporting previous observations that worms can use ethanol as a calorie source

A Small Conductance Calcium-Activated K⁺ Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying

In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. © 2013 Chotoo et al

Genes regulating levels of ω-3 long-chain polyunsaturated fatty acids are associated with alcohol use disorder and consumption, and broader externalizing behavior in humans.

BACKGROUND: Individual variation in the physiological response to alcohol is predictive of an individual's likelihood to develop alcohol use disorder (AUD). Evidence from diverse model organisms indicates that the levels of long-chain polyunsaturated omega-3 fatty acids (ω-3 LC-PUFAs) can modulate the behavioral response to ethanol and therefore may impact the propensity to develop AUD. While most ω-3 LC-PUFAs come from diet, humans can produce these fatty acids from shorter chain precursors through a series of enzymatic steps. Natural variation in the genes encoding these enzymes has been shown to affect ω-3 LC-PUFA levels. We hypothesized that variation in these genes could contribute to the susceptibility to develop AUD. METHODS: We identified nine genes (FADS1, FADS2, FADS3, ELOVL2, GCKR, ELOVL1, ACOX1, APOE, and PPARA) that are required to generate ω-3 LC-PUFAs and/or have been shown or predicted to affect ω-3 LC-PUFA levels. Using both set-based and gene-based analyses we examined their association with AUD and two AUD-related phenotypes, alcohol consumption, and an externalizing phenotype. RESULTS: We found that the set of nine genes is associated with all three phenotypes. When examined individually, GCKR, FADS2, and ACOX1 showed significant association signals with alcohol consumption. GCKR was significantly associated with AUD. ELOVL1 and APOE were associated with externalizing. CONCLUSIONS: Taken together with observations that dietary ω-3 LC-PUFAs can affect ethanol-related phenotypes, this work suggests that these fatty acids provide a link between the environmental and genetic influences on the risk of developing AUD

Long-chain polyunsaturated fatty acids are required for the development of acute functional tolerance to ethanol.

<p>(A) The metabolic pathway for LC-PUFAs in <i>C. elegans</i>. Genes encoding enzymes responsible for each step of the generation of each LC-PUFA are shown over the arrows. Mutations in genes encoding the enzymes FAT<i>-</i>3, FAT<i>-</i>4 and FAT<i>-</i>1 eliminate downstream LC-PUFAs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105999#pone.0105999-Watts1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105999#pone.0105999-KahnKirby1" target="_blank">[26]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105999#pone.0105999-Spychalla1" target="_blank">[28]</a>. (B–C) N2 animals develop acute functional tolerance (AFT) to ethanol, whereas <i>fat-3(wa22), fat-4(wa14),</i> and <i>fat-1(wa9)</i> fail to develop AFT. Animals were treated with 0 or 400 mM ethanol, and locomotion was recorded at 10 and 30 minutes of exposure. Here and in subsequent figures, left graphs show relative speeds (treated/untreated speed). A difference at 10 minutes between N2 and the mutant indicates that the mutant has a change in initial sensitivity; a significant increase in speed from 10 to 30 minutes within a strain is defined as the development of AFT. Right graphs show percent of speed recovered between 10 and 30 minutes within a strain; this is the degree of AFT. <i>fat-3(wa22)</i> is more sensitive than wild type to the intoxicating effects of ethanol at 10 minutes in this set of experiments, although in subsequent experiments, this difference did not reach statistical significance. Error bars represent SEM. *<i>p</i><0.05; **<i>p</i><0.01. <i>n</i> = 6.</p

Nineteen hour treatment of <i>fat</i><i>-</i><i>1(wa9)</i> with EPA rescues AFT but not basal speed defects in <i>fat</i><i>-</i><i>1</i>.

<p>N2 and <i>fat-1(wa9)</i> mutant animals were grown to the L4 stage on NGM plates, then they were moved to NGM plates containing 0.1% NP<i>-</i>40 and supplemented with 0 or 160 µM EPA and allowed to develop into first day adults. After 19 hours of EPA supplementation, adult animals were tested in locomotion assays on 0 or 400 mM ethanol. (A) After 19 hours of EPA supplementation, <i>fat-1(wa9)</i> animals are able to develop AFT, whereas age-matched <i>fat-1(wa9)</i> animals not supplemented with EPA do not develop AFT. (B) 19 hours of EPA supplementation is not sufficient to rescue the slow basal speed of <i>fat-1(wa9)</i> mutant animals. Basal speed (0 mM ethanol) was measured at 10 and 30 minutes after the beginning of the locomotion assay. Error bars represent SEM. **<i>p</i><0.01; ***<i>p</i><0.001 within treatment; ##<i>p</i><0.01 for comparison of supplemented <i>fat-1</i> to supplemented N2. <i>n</i> = 6.</p

Dietary supplementation of AA or EPA can enhance the development of AFT in wild-type animals.

<p>All trials in which N2 was supplemented with AA or EPA are shown, <i>n</i> = 21 for AA, <i>n</i> = 18 for EPA. (A) N2 animals supplemented with AA demonstrate increased initial sensitivity to ethanol and enhanced development of AFT. (B) N2 animals supplemented with EPA demonstrate enhanced AFT. Error bars represent SEM. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p