Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease

SUMMARY Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 μmol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease

Activating Transcription Factor 6 Is Necessary and Sufficient for Alcoholic Fatty Liver Disease in Zebrafish

<div><p>Fatty liver disease (FLD) is characterized by lipid accumulation in hepatocytes and is accompanied by secretory pathway dysfunction, resulting in induction of the unfolded protein response (UPR). Activating transcription factor 6 (ATF6), one of three main UPR sensors, functions to both promote FLD during acute stress and reduce FLD during chronic stress. There is little mechanistic understanding of how ATF6, or any other UPR factor, regulates hepatic lipid metabolism to cause disease. We addressed this using zebrafish genetics and biochemical analyses and demonstrate that Atf6 is necessary and sufficient for FLD. <i>atf6</i> transcription is significantly upregulated in the liver of zebrafish with alcoholic FLD and morpholino-mediated <i>atf6</i> depletion significantly reduced steatosis incidence caused by alcohol. Moreover, overexpression of active, nuclear Atf6 (nAtf6) in hepatocytes caused FLD in the absence of stress. mRNA-Seq and qPCR analyses of livers from five day old nAtf6 transgenic larvae revealed upregulation of genes promoting glyceroneogenesis and fatty acid elongation, including fatty acid synthase (<i>fasn</i>), and nAtf6 overexpression in both zebrafish larvae and human hepatoma cells increased the incorporation of ¹⁴C-acetate into lipids. Srebp transcription factors are key regulators of lipogenic enzymes, but reducing Srebp activation by <i>scap</i> morpholino injection neither prevented FLD in nAtf6 transgenics nor synergized with <i>atf6</i> knockdown to reduce alcohol-induced FLD. In contrast, <i>fasn</i> morpholino injection reduced FLD in nAtf6 transgenic larvae and synergistically interacted with <i>atf6</i> to reduce alcoholic FLD. Thus, our data demonstrate that Atf6 is required for alcoholic FLD and epistatically interacts with <i>fasn</i> to cause this disease, suggesting triglyceride biogenesis as the mechanism of UPR induced FLD.</p></div

nAtf6 overexpression is sufficient to drive steatosis.

<p><b>A:</b> Heatmap of upregulated UPR effector genes in nAtf6 transgenic larvae. The log values based on mRNA-Seq analysis and the median fold change in 3–6 liver samples assessed by qPCR at 5 dpf and 14 dpf. <b>B:</b> Most Atf6 targets are upregulated in the liver in response to ethanol. Genes identified as part of the UPR are significantly induced in the liver of nAtf6 TG and ethanol treated larvae (log value ≥ 0.2; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004335#pgen.1004335.s012" target="_blank">Table S2</a>). <b>C:</b> Oil red O stained WT and nAtf6 transgenic larvae. Livers are circled in the enlarged boxes. <b>D:</b> Steatosis incidence based on scoring of whole mount oil red O stained larvae at 4, 5, and 5.5 dpf. Statistics: chi-square with Fisher's Exact Test. *, p<0.05. “Total n” and “clutch n” corresponds to the number of larvae and number of clutches scored. <b>E:</b> Hepatic triglyceride levels in extracts from pooled livers from WT and nAtf6 transgenic larvae at 5 dpf and 14 dpf, and from single adult livers (∼9 months). Statistics: unpaired t-test. *, p<0.05. Median fold changes are noted.</p

Glyceroneogenesis and fatty acid elongation pathways are dysregulated by nAtf6 overexpression.

<p>Schematic of the glycolysis, gluconeogenesis and glyceroneogenesis pathway (<b>A</b>, adapted from WikiPathways) and heatmap of associated genes (<b>B</b>). Upregulated genes are colored in shades of orange, downregulated genes are colored in shades of green, genes that are unchanged are white and those genes that did not appear in mRNA-Seq analyses are colored gray. Log values and median fold changes from mRNA-Seq analysis and qPCR, respectively, are noted.</p

<i>De novo</i> lipogenesis is enhanced <i>in vitro</i> and <i>in vivo</i> by nAtf6 overexpression.

<p><b>A: </b>¹⁴C-acetate is preferentially incorporated into lipids in 5 dpf nAtf6 TG larvae compared to WT. The percent of ¹⁴C in lipid fraction was divided by ¹⁴C measured in the unextracted lysate from whole fish. The median fold change is noted; n =  number of samples of pooled larvae. <b>B:</b> Oil red O staining of HepG2 cells transfected with GFP or nATF6. Bar  = 10 µm. <b>C:</b> Quantification of lipid droplet number and area (square pixels). Statistics: unpaired t-test. *, p<0.05; n indicates the number of cells quantified over 20 fields from 2 independent experiments. <b>D:</b> Incorporation of ¹⁴C-acetate into lipids by HepG2 cells transfected with GFP or nATF6 normalized to protein. The median fold change is noted; n =  number of separate batches of cells analyzed in 2 independent experiments.</p

Working model by which Atf6 functions as a positive regulator of alcoholic steatosis.

<p>Genetic tools used in this study are illustrated in red.</p

Fasn functions downstream of Atf6 to cause steatosis independent of Srebp activation.

<p><b>A:</b> qPCR analysis of <i>fasn</i> expression in <i>atf6</i> morphants and uninjected larvae treated with EtOH for 32 hours. <b>B:</b> qPCR analysis of <i>fasn</i> expression in nAtf6 TG larvae and HepG2 cells transfected with nATF6. Statistics: unpaired t-test. *, p<0.05. <b>C:</b> Quantification of whole mount oil red O staining of nAtf6 TG larvae injected with <i>scap</i> MO. “Total n” and “clutch n” corresponds to the number of larvae and number of clutches scored, respectively. Statistics: chi-square with Fisher's Exact Test. *, p<0.05. <b>D:</b> Quantification of oil red O staining in nAtf6 TG larvae injected with <i>fasn</i> MO. <b>E:</b> Quantification of oil red O staining in <i>atf6/fasn</i> double morphants treated with 350 mM EtOH for 24 hours. Statistics: chi-square with Fisher's Exact Test. *, p<0.05.</p

Atf6 is required for alcoholic steatosis.

<p><b>A:</b> qPCR analysis of UPR sensors <i>atf6, perk, ire1a,</i> and <i>atf4</i> in the livers of control (EtOH-) and 350 mM ethanol-treated (EtOH+) larvae. dCt values were calculated by normalization to <i>rpp0.</i> Fold changes were calculated based on median dCt values. Statistics: paired t-test. *, p<0.05. <b>B:</b> Expression of <i>atf6</i> in the livers of 350 mM ethanol treated larvae from 0–32 hours. Fold changes were determined by normalizing to control (0 mM EtOH) dCt values. Statistics: one-sample t-test. *, p<0.05. <b>C:</b> Images of oil red O stained whole larvae injected with two different morpholinos targeting <i>atf6</i> (ATG-targeting, <i>atf6</i> ATG, and splice-blocking, <i>atf6</i> SPL) and exposed to either 0 or 350 mM ethanol. Livers are circled in the enlarged boxes. <b>D:</b> Steatosis incidence based on scoring oil red O stained larvae at 5.5 dpf. Statistics: chi-square with Fisher's Exact Test. *, p<0.05. “Total n” and “clutch n” corresponds to the number of larvae and number of clutches scored.</p