Activating transcription factor 6 is necessary and sufficient for alcoholic fatty liver disease in zebrafish.

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. atf6 transcription is significantly upregulated in the liver of zebrafish with alcoholic FLD and morpholino-mediated atf6 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 (fasn), 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 scap morpholino injection neither prevented FLD in nAtf6 transgenics nor synergized with atf6 knockdown to reduce alcohol-induced FLD. In contrast, fasn morpholino injection reduced FLD in nAtf6 transgenic larvae and synergistically interacted with atf6 to reduce alcoholic FLD. Thus, our data demonstrate that Atf6 is required for alcoholic FLD and epistatically interacts with fasn to cause this disease, suggesting triglyceride biogenesis as the mechanism of UPR induced FLD.

Alcohol disrupts endoplasmic reticulum function and protein secretion in hepatocytes.

BACKGROUND: Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo. METHODS: HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP). RESULTS: VL-17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced. CONCLUSIONS: Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD.

Drinks like a fish: using zebrafish to understand alcoholic liver disease.

Steatosis is the most common consequence of acute alcohol abuse, such as occurs during a drinking binge. Acute alcohol induced steatosis may predispose to more severe hepatic disease. We have developed a model of alcoholic liver disease (ALD) in zebrafish larvae to provide a system in which the genes and pathways that contribute to steatosis can be rapidly identified. Zebrafish larvae represent an attractive vertebrate model for studying acute ALD because they possess the pathways to metabolize alcohol, the liver is mature by 4 days post-fertilization (dpf), and alcohol can be simply added to their water. Exposing 4 dpf zebrafish larvae to 2% ethanol (EtOH) for 32 hours achieves ∼80 mM intracellular EtOH and upregulation of hepatic cyp2e1, sod, and bip, indicating that EtOH is metabolized and provokes oxidative stress. EtOH-treated larvae develop ALD as demonstrated by hepatomegaly and steatosis. Increased lipogenesis driven by the sterol response element binding protein (SREBP) transcription factors is essential for steatosis associated with chronic alcohol ingestion but it has not been determined if the same pathway is essential for steatosis following a drinking binge. We report that several Srebp target genes are induced in the liver of zebrafish exposed to EtOH. We used fish which harbor a mutation in the gene encoding the membrane bound transcription factor protease 1 (mbtps1; also called site-1 protease) and embryos in which the Srebp cleavage activating protein (scap) is knocked down to determine the requirement of this pathway in acute ALD. We find that both means of blocking Srebp activation prevents steatosis in response to 2% EtOH. Moreover, this is accompanied by the failure to activate several Srebp target genes in response to alcohol. We conclude that Srebps are required for steatosis in response to acute alcohol exposure. Moreover, these data highlight the utility of zebrafish as a useful new vertebrate model to study ALD.

Exposure to the synthetic FXR agonist GW4064 causes alterations in gene expression and sublethal hepatotoxicity in eleutheroembryo medaka (Oryzias latipes).

The small freshwater teleost, medaka (Oryzias latipes), has a history of usage in studies of chronic toxicity of liver and biliary system. Recent progress with this model has focused on defining the medaka hepatobiliary system. Here we investigate critical liver function and toxicity by examining the in vivo role and function of the farnesoid X receptor alpha (FXRalpha, NR1H4), a member of the nuclear receptor superfamily that plays an essential role in the regulation of bile acid homeostasis. Quantitative mRNA analysis of medaka FXRalpha demonstrates differential expression of two FXRalpha isoforms designated Fxralpha1 and Fxralpha2, in both free swimming medaka embryos with remaining yolk (eleutheroembryos, EEs) and adults. Activation of medaka Fxralpha in vivo with GW4064 (a strong FXRalpha agonist) resulted in modification of gene expression for defined FXRalpha gene targets including the bile salt export protein, small heterodimer partner, and cytochrome P450 7A1. Histological examination of medaka liver subsequent to GW4064 exposure demonstrated significant lipid accumulation, cellular and organelle alterations in both hepatocytes and biliary epithelial cells of the liver. This report of hepatobiliary injury following GW4064 exposure extends previous investigations of the intrahepatic biliary system in medaka, reveals sensitivity to toxicant exposure, and illustrates the need for added resolution in detection and interpretation of toxic responses in this vertebrate.

Paralogous vitamin D receptors in teleosts: transition of nuclear receptor function.

The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we describe the cloning and functional characterization of two novel vitamin D receptor (VDR) paralogs from the freshwater teleost medaka (Oryzias latipes). VDR sequences were identified through mining of the medaka genome database in which gene organization and structure was determined. Two distinct VDR genes were identified in the medaka genome and mapped to defined loci. Each VDR sequence exhibits unique intronic organization and dissimilar 5' untranslated regions, suggesting they are not isoforms of the same gene locus. Phylogenetic comparison with additional teleosts and mammalian VDR sequences illustrate that two distinct clusters are formed separating aquatic and terrestrial species. Nested within the teleost cluster are two separate clades for VDRalpha and VDRbeta. The topology of teleost VDR sequences is consistent with the notion of paralogous genes arising from a whole genome duplication event prior to teleost radiation. Functional characterization was conducted through the development of VDR expression vectors including Gal4 chimeras containing the yeast Gal4 DNA binding domain fused to the medaka VDR ligand binding domain and full-length protein. The common VDR ligand 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D(3)] resulted in significant transactivation activity with both the Gal4 and full-length constructs of medaka (m) VDRbeta. Comparatively, transactivation of mVDRalpha with 1alpha,25(OH)(2)D(3) was highly attenuated, suggesting a functional divergence between these two nuclear receptor paralogs. We additionally demonstrate through coactivator studies that mVDRalpha is still functional; however, it exhibits a different sensitivity to 1alpha,25(OH)(2)D(3), compared with VDRbeta. These results suggest that in mVDRalpha and VDRbeta have undergone a functional divergence through a process of sub- and/or neofunctionalization of VDR nuclear receptor gene pairs.

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.

Defining hepatic dysfunction parameters in two models of fatty liver disease in zebrafish larvae.

Fatty liver disease in humans can progress from steatosis to hepatocellular injury, fibrosis, cirrhosis, and liver failure. We developed a series of straightforward assays to determine whether zebrafish larvae with either tunicamycin- or ethanol-induced steatosis develop hepatic dysfunction. We found altered expression of genes involved in acute phase response and hepatic function, and impaired hepatocyte secretion and disruption of canaliculi in both models, but glycogen deficiency in hepatocytes and dilation of hepatic vasculature occurred only in ethanol-treated larvae. Hepatic stellate cells (HSCs) become activated during liver injury and HSC numbers increased in both models. Whether the excess lipids in hepatocytes are a direct cause of hepatocyte dysfunction in fatty liver disease has not been defined. We prevented ethanol-induced steatosis by blocking activation of the sterol response element binding proteins (Srebps) using gonzo(mbtps1) mutants and scap morphants and found that hepatocyte dysfunction persisted even in the absence of lipid accumulation. This suggests that lipotoxicity is not the primary cause of hepatic injury in these models of fatty liver disease. This study provides a panel of parameters to assess liver disease that can be easily applied to zebrafish mutants, transgenics, and for drug screening in which liver function is an important consideration.

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

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.

Two farnesoid X receptor alpha isoforms in Japanese medaka (Oryzias latipes) are differentially activated in vitro.

The nuclear receptor farnesoid X receptor alpha (FXRalpha, NR1H4) is activated by bile acids in multiple species including mouse, rat, and human and in this study we have identified two isoforms of Fxralpha in Japanese medaka (Oryzias latipes), a small freshwater teleost. Both isoforms share a high amino acid sequence identity to mammalian FXRalpha (approximately 70% in the ligand-binding domain). Fxralpha1 and Fxralpha2 differ within the AF1 domain due to alternative splicing at the fourth intron-exon boundary. This process results in Fxralpha1 having an extended N-terminus compared to Fxralpha2. A Gal4DBD-FxralphaLBD fusion construct was activated by chenodeoxycholic, cholic, deoxycholic and lithocholic acids, and the synthetic agonist GW4064 in transient transactivation assays. Activation of the Gal4DBD-FxralphaLBD fusion construct was enhanced by addition of PGC-1alpha, as demonstrated through titration assays. Surprisingly, when the full-length versions of the two Fxralpha isoforms were compared in transient transfection assays, Fxralpha2 was activated by C(24) bile acids and GW4064, while Fxralpha1 was not significantly activated by any of the compounds tested. Since the only significant difference between the full-length constructs was sequence in the AF1 domain, these experiments highlight a key functional region in the Fxralpha AF1 domain. Furthermore, mammalian two-hybrid studies demonstrated the ability of Fxralpha2, but not Fxralpha1, to interact with PGC-1alpha and SRC-1, and supported our results from the transient transfection reporter gene activation assays. These data demonstrate that both mammalian and teleost FXR (Fxralpha2 isoform) are activated by primary and secondary bile acids.

Protective response of the Ah receptor to ANIT-induced biliary epithelial cell toxicity in see-through medaka.

The adaptive role of the aryl hydrocarbon receptor (Ah receptor or AHR) in protecting against disease-related conditions remains unclear in nonmammalian models, particularly teleosts. Therefore, this study focused on the potential role of AHR in response to biliary epithelial cell toxicity and hepatobiliary alteration in medaka. See-through medaka (STII strain) were exposed for 96 h using the biliary toxicant alpha-naphthylisothiocyanate (ANIT) as a reagent, and fish were evaluated daily using histological and ultrastructural analysis, and by imaging directly through the body wall of living fish. Brightfield and transmission electron microscopy showed that a single ANIT dose (40 mg/kg) specifically induced swelling and apoptosis of bile preductular epithelial cells (BPDECs) as early as 6 h after initial exposure. Following ANIT-induced BPDEC toxicity, in vivo imaging of STII medaka showed significant gallbladder discoloration from 48-72 h. Collectively, these pathologic data suggested that ANIT exposure resulted in acute hepatobiliary changes, lasting < 96 h following initial exposure. We then tested the potential role of AHR in response to ANIT-induced hepatobiliary alteration. Overall, we demonstrated that (1) transient AHR activation and cytochrome P450 1A (CYP1A) induction in livers occurred during ANIT-induced hepatobiliary impairment, (2) pretreatment with an AHR agonist partially protected against acute hepatobiliary alteration, and (3) using a luciferase-based reporter assay, the bile pigment bilirubin weakly activated mouse AHR and binding to medaka-specific CYP1A promoter, resulting in AHR element-driven transcription. Given that bile acids and pigments are present in mammalian and fish liver, these studies collectively suggest that bile-induced AHR activation may be conserved between teleosts and rodents.