Steatosis is the most common consequence of acute alcohol abuse and may predispose to more severe hepatic disease. Increased lipogenesis driven by the sterol response element binding protein (SREBP) transcription factors is essential for steatosis associated with chronic alcohol ingestion, but the mechanisms underlying steatosis following acute alcohol exposure are unknown. Zebrafish larvae represent an attractive vertebrate model for studying alcoholic liver disease (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 ϳ80 mM intracellular EtOH and upregulation of hepatic cyp2e1, sod, and bip, indicating that EtOH is metabolized and provokes oxidant stress. EtOH-treated larvae develop hepatomegaly and steatosis accompanied by changes in the expression of genes required for hepatic lipid metabolism. Based on the importance of SREBPs in chronic ALD, we explored the role of Srebps in this model of acute ALD. Srebp activation was prevented in gonzo larvae, which harbor a mutation in the membrane-bound transcription factor protease 1 (mbtps1) gene, and in embryos injected with a morpholino to knock down Srebp cleavage activating protein (scap). Both gonzo mutants and scap morphants were resistant to steatosis in response to 2% EtOH, and the expression of many Srebp target genes are down-regulated in gonzo mutant livers. Conclusion: Zebrafish larvae develop signs of acute ALD, including steatosis. Srebp activation is required for steatosis in this model. The tractability of zebrafish genetics provides a valuable tool for dissecting the molecular pathogenesis of acute ALD. (HEPATOLOGY 2009;49:443-452.)

Many etiologies of fatty liver disease (FLD) are associated with hyper-activation of one of the three pathways that comprise the unfolded protein response (UPR), a harbinger of endoplasmic reticulum (ER) stress. The UPR is mediated by pathways initiated by PERK, IRE1a/XBP1and ATF6, and each of these pathways have been implicated as either protective or pathological in FLD. We use zebrafish with FLD and hepatic ER stress to explore the relationship between Atf6 and steatosis. Mutation of the foie gras (foigr) gene causes FLD and hepatic ER stress. Prolonged treatment of wild-type larvae with a dose of tunicamycin that causes chronic ER stress phenocopies foigr. In contrast, acute exposure to a high dose of tunicamycin robustly activates the UPR but is less effective at inducing steatosis. The Srebp transcription factors are not required for steatosis in any of these models. Instead, depleting larvae of active Atf6 either through mbtps1 mutation or atf6 morpholino injection protects against steatosis caused by chronic ER stress whereas it exacerbates steatosis caused by acute tunicamycin treatment. Conclusion ER stress causes FLD. Loss of Atf6 prevents steatosis caused by chronic ER stress but can also potentiate steatosis caused by acute ER stress. This demonstrates that Atf6 can play both protective and pathological roles in FLD.

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.

The trappc11 mutation in zebrafish causes a stressed UPR, leading to fatty liver disease. This phenotype is not due to a trafficking defect but instead results from N-glycosylation defects. Hypoglycosylation and lipid accumulation are also found in patient cells with knockdown or mutated TRAPPC11 genes, suggesting a common etiology with zebrafish.

Fibroblast growth factors (FGFs) are important regulators of retinal development and survival. We examined the expression and distribution of FGF9 and its preferred receptors FGFR2IIIc and FGFR3IIIc in this tissue. FGF9 transcripts in whole rat retina were detected by RT-PCR but were not present in purified cultured Muller glia. Transcripts appeared as 3.2-kb and 4.0-kb bands on Northern blots, and Western blotting of whole retina revealed FGF9-immunoreactive bands at 30 and 55 kDa. FGF9 mRNA demonstrated a biphasic expression profile, elevated at birth and adulthood, but relatively decreased during terminal retinal differentiation (4-14 days postnatal). Antibody labeling broadly reflected these findings: staining in vivo was observed mainly in the inner retina (and outer plexiform layer in adults) whereas FGF9 was not detectable in cultured Muller glia. In adults, FGF9 in situ hybridization also showed a detectable signal in inner retina. FGFR2IIIc and FGFR3IIIc were detected by RT-PCR, and Western blotting showed both FGFRs existed as multiple forms between approximately 100-200 kDa. FGFR2 and FGFR3 antibodies showed prominent labeling in the inner retina, especially in proliferating cultured Muller glia. Exogenous FGF9 elicited a dose-dependent increase in Muller glial proliferation in vitro. These data suggest a role for FGF9 in retinal differentiation and maturation, possibly representing a neuronally derived factor acting upon glial (and other) cells.