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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Many etiologies of fatty liver disease (FLD) are associated with the hyperactivation of one of the three pathways composing the unfolded protein response (UPR), which is a harbinger of endoplasmic reticulum (ER) stress. The UPR is mediated by pathways initiated by PRKR-like endoplasmic reticulum kinase, inositol-requiring 1A/X box binding protein 1, and activating transcription factor 6 (ATF6), and each of these pathways has been implicated to have a protective or pathological role in FLD. We used zebrafish with FLD and hepatic ER stress to explore the relationship between Atf6 and steatosis. A mutation of the foie gras (foigr) gene caused FLD and hepatic ER stress. The prolonged treatment of wild-type larvae with tunicamycin (TN), which caused chronic ER stress, phenocopied foigr. In contrast, acute exposure to a high dose of TN robustly activated the UPR but was less effective at inducing steatosis. The sterol regulatory element binding protein transcription factors were not required for steatosis in any of these models. Instead, depleting larvae of active Atf6 either through a membrane-bound transcription factor peptidase site 1 mutation or an atf6 morpholino injection protected them against steatosis caused by chronic ER stress, but exacerbated steatosis caused by acute TN treatment. Conclusion: ER stress causes FLD. A 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. (HEPATOLOGY 2011;)

Fatty liver disease (FLD) is emerging as a global epidemic, necessitating a comprehensive understanding of its molecular basis. Interestingly, most etiologies of FLD are associated with the induction of the unfolded protein response (UPR), which is likely attributable to a deficit in the protein folding capacity of the endoplasmic reticulum (ER) in FLD. There is a well-established yet poorly understood link between UPR activation and lipid accumulation in hepatocytes (steatosis).

UPR function is required by all cells to ensure that proteins in the secretory pathway are efficiently processed.1, 2 The three branches of the UPR are connected through the master chaperone binding immunoglobulin protein (Bip). The proximal mediators are as follows:

  • 1
    PRKR-like endoplasmic reticulum kinase (PERK; also called eukaryotic translation initiation factor 2α kinase 3), which phosphorylates eukaryotic translation initiation factor 2 subunit 1α [EIF2S1; also called eukaryotic translation initiation factor 2α (EIF2α)]. This represses protein synthesis and selectively translates activating transcription factor 4 (ATF4) messenger RNA (mRNA).
  • 2
    Inositol-requiring 1A (IRE1A; also called endoplasmic reticulum to nucleus signaling 1), which splices X box binding protein 1 (XBP1) mRNA to encode the spliced X box binding protein (XBP1-s) transcription factor.3
  • 3
    ATF6 transcription factor, which cooperates with ATF4 and XBP1-s to regulate a panel of genes that maintain ER function.1, 2

Accordingly, significant cooperation and crosstalk exist between UPR branches. When the unfolded protein load is mitigated, homeostasis is achieved, and the UPR activity returns to baseline levels. In contrast, when the ER is overwhelmed with unfolded proteins, the UPR is chronically activated in a pathological state termed ER stress. In most cases, UPR activation protects cells by maintaining homeostasis.2 However, prolonged UPR activation with chronic ER stress results in aberrant protein secretion and apoptosis.1, 2

The up-regulation of some or all UPR branches is found in most etiologies of FLD4-8 and contributes to steatosis. Obesity-related steatosis is ameliorated when Eif2s1 phosphorylation is prevented,9 and enhancing protein folding in obese mice results in a reduction of the UPR and improves hepatic insulin resistance.10, 11 In contrast, other studies have indicated that crippling the UPR causes FLD: Xbp1 heterozygosity predisposes mice to developing hepatic insulin resistance,6 and mice lacking Atf6 or DnaJ (Hsp40) homolog subfamily C member 3 (Dnajc3) are unable to resolve steatosis caused by an acute block in protein glycosylation.12, 13 Intriguingly, Bip+/− mice are protected from insulin resistance by compensatory, low-grade UPR activation.14 The theory that UPR activation plays two distinct roles—a protective role in the setting of acute ER stress and a pathological role when the UPR is chronically activated—may unify these seemingly disparate data.

The sterol regulatory element binding protein (SREBP) transcription factors are the master regulators of triglyceride and cholesterol synthesis in hepatocytes, and they are essential for obesity- and alcohol-induced steatosis.15 In some cells, UPR activation causes SREBP activation,16-18 whereas in others, the expression, activation, or function of SREBP is suppressed by ER stress.12, 18-20 Whether UPR activation is coupled to SREBP-driven steatosis remains to be determined.

In this study, we used three methods to induce steatosis in zebrafish larvae and found that each led to ER stress. We demonstrate here that a loss of Atf6 protects against steatosis due to chronic ER stress but increases steatosis if the insult is acute.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Zebrafish.

Wild-type (WT) lines (TAB5 and TAB14) and mutant lines (alleles foigrhi1532b and mbtps1hi1487) were maintained in accordance with the policies of the institutional animal care and use committee of the Mount Sinai School of Medicine. Mutants were genotyped as described.21Tg(fabp10:RFP;ela:GFP) fish were obtained from D. Stainier (University of California at San Francisco).

Morpholinos targeting the anti-thymocyte globulin initiator of atf6 (gene name si:ch211-199m3.9; 5′-ACATTAAATTCGACGACATTGTGCC-3′) or sterol regulatory element binding protein cleavage-activating protein (scap)22 and a nontargeting control (5′-CCT CTTACCTCAGTTACAATTTATA-3′) were ordered from Gene Tools, LLC (Philomath, OR). The morpholinos were diluted in water to a 0.5 mM stock, and approximately 5 pmol was injected into the early embryos. The tunicamycin (TN) treatment protocols are detailed in the Results section.

Oil Red O Staining.

Whole-mount Oil Red O staining was carried out as described.22 Steatosis was scored in larvae with three or more lipid droplets in the liver parenchyma. A Nikon SMZ1500 equipped with a Nikon DS-2M color camera was used to acquire images, which were edited with Photoshop.

The amount of Oil Red O staining per liver cell was quantified with Metamorph software (Molecular Devices) on cryosections stained with Oil Red O and 4′,6-diamidino-2-phenylindole (DAPI). In each bright field image, a region outlining the liver was selected, and Oil Red O–stained particles were selected by color thresholding and were counted. The total area occupied by Oil Red O staining was measured. Each measurement was divided by the number of DAPI-stained nuclei within the region. At least five sections per fish were measured for at least three fish per group.

Histology and Electron Microscopy.

At least four WT and foigr mutant larvae fixed in 4% paraformaldehyde were embedded in plastic as described.23 Four-micrometer sections were incubated in 0.5% periodic acid, washed, stained with Schiff's reagent (5 g/L basic fuchsin, 0.1 N hydrochloric acid, and 0.045 potassium metabisulfite), washed with running tap water, and counterstained with hematoxylin. Images were taken with an Olympus BX41 microscope and a Nikon DS-Ri1 color camera.

Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed with a Roche in situ cell death detection kit as described.24 Hepatocytes were stained with cyanine 3/streptavidin (1:200; Sigma), and nuclei were labeled with DAPI. The percentage of apoptotic hepatocytes was calculated for at least 15 sections (which represented at least three fish per group) through the division of the number of TUNEL-positive hepatocytes by the total number of nuclei in each section.

Samples from larvae at 5 days post-fertilization (dpf) were fixed and processed for transmission electron microscopy as described.25

In Situ Hybridization.

Probes were generated by polymerase chain reaction (PCR) amplification from complementary DNA (cDNA) generated from 5-dpf RNA with the primers listed in Supporting Table 1. The bip probe was generated by the creation of cDNA with the zbip-3a primer. Nucleotides 1235 to 2260 of BC063946.1 were amplified with primers bip-5b and bip-3b. The DNA damage-inducible transcript 3 (chop) probe was amplified with primers zchop-5c and zchop-3, which spanned nucleotides 248 to 976 of NM_001082825.1. The dnajc3 probe was amplified with primers zdnajc3-5p and zdnajc3-3p, which spanned nucleotides 318 to 819 of NM_199610. Each fragment was cloned into PCR II (Invitrogen) and was sequenced. The probes were created with a digoxigenin RNA labeling mix (Roche). Whole-mount in situ hybridizations were performed as described.24

Blotting.

Larvae at 5 dpf were homogenized in a lysis buffer [20 mM trishydroxymethylaminomethane (pH 7.5), 150 mM sodium chloride, 1% Nonidet P40, 2 mM ethylene diamine tetraacetic acid, 10% glycerol, and protease inhibitors]; to a final concentration of 2% sodium dodecyl sulfate and 5% 2-mercaptoethanol. Two embryos were loaded onto a 10% polyacrylamide gel, blotted onto nitrocellulose, and incubated with antibodies recognizing α-tubulin (1:2000; Sigma), Bip (1:3000; Sigma) or phosphorylated eukaryotic translation initiation factor 2 subunit 1α (p-Eif2s1; 1:1000; 9721, Cell Signaling) followed by anti-mouse horseradish peroxidase–conjugated secondary antibody (1:1500; Jackson ImmunoResearch). Blots were visualized by chemiluminescence with a Fujifilm LAS-3000. The band intensities were quantified with Quantity One software (Bio-Rad).

PCR.

RNA was isolated from 5-dpf whole larvae, dissected livers, and liverless carcasses with the Qiagen RNeasy kit. cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen). PCR reactions were performed as described.25 Real-time quantitative polymerase chain reaction (qPCR) was performed in triplicate with Roche SYBR Green on the Roche LightCycler 480 system. The change in the cycle threshold (ΔCt) was calculated for each target gene using the formula (2math image) with ribosomal protein P0 (rpp0) as the reference. The primer specificity (Supporting Table 1) was determined with a melting curve assessment; some amplicons were sequenced. All genes are referred to according to the nomenclature rules for the species under discussion. When no species is specified, zebrafish nomenclature rules are followed.

Statistics.

All experiments were repeated for at least three clutches. For data presented as percentages of control values, we calculated either the average or the median and the standard deviation. The statistical tests included unpaired and paired two-tailed Student t tests, one-sample t tests, analyses of variance (ANOVAs), Fisher's exact test, and chi-square analyses as appropriate.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

foigr Mutants Develop Steatosis and Hepatic Dysfunction.

Nearly all zebrafish larvae with a homozygous mutation in the foigr gene developed hepatomegaly (see Fig. 1A,B and Sadler et al.25) and steatosis by 5 dpf (Fig. 1B,C). The foigr mutants had other defects such as underdeveloped guts, small heads, and eyes, yolk underconsumption, and death by 7 dpf. These phenotypes are common to zebrafish mutants lacking a gene involved in basic cellular processes. However, the phenotype of steatosis in the foigr mutants was unusual.

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Figure 1. foigr mutants develop steatosis and liver damage by 5 dpf. (A) Live foigr larvae expressing Discosoma red fluorescent protein in hepatocytes [Tg(fabp10:dsRed)] had large, round livers in comparison with their WT siblings. (B) Steatosis in foigr livers was detected with whole-mount Oil Red O staining. The liver is circled, and the incompletely consumed yolk in foigr mutants is labeled. The scale bar represents 100 μm (left). (B) Livers dissected from Oil Red O–stained 5-dpf WT and foigr larvae (right). (C) Nearly all foigr mutants developed steatosis by 5 dpf. The percentage of fish that scored positive for steatosis by whole-mount Oil Red O staining was averaged from five clutches (65 mutants and 70 WT fish). *P < 0.001 by a Student t test. (D) qPCR analysis of RNA samples isolated from 5-dpf whole larvae. The target gene expression was normalized to rpp0 to determine ΔCt. The ΔCt for the mutants was divided by ΔCt for their WT siblings to obtain the percentage change in expression in each clutch, and the averages for at least three clutches are shown. All tested genes were significantly down-regulated in mutants (P < 0.01 for all samples by a one-sample t test). (E) Glycogen was detected in sections of WT and foigr livers with the periodic acid–Schiff stain. The scarce glycogen deposits in foigr hepatocytes are marked by white asterisks. The scale bar represents 20 μm. (F) Genes induced by hepatic damage were assessed with qPCR. The fold change for each clutch was calculated by the division of ΔCt for the mutants by ΔCt for their WT siblings, and the results of more than five clutches were averaged (P < 0.05 by a one-tailed t test). (G) The percentages of TUNEL-positive hepatocytes on at least 15 WT (n = 1026) and foigr sections (n = 1005) were averaged. *P = 0.0037 by a Student t test. All error bars represent the standard deviations.

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Impaired hepatic function, liver damage, and hepatocyte death occur in FLD patients. By 5 dpf, the expression of genes involved in key hepatocyte processes (see Supporting Table 1 for the gene names) was decreased in foigr mutants; these processes included carbohydrate metabolism [pyruvate carboxylase (pc) and fructose-1,6-bisphosphatase (fbp)], iron transport [hemopexin (hpx)], and xenobiotic metabolism [cytochrome P450 3A4 (cyp3a4) and carboxylesterase 2 (ces2); Fig. 1D]. Glycogen depletion in foigr mutant hepatocytes (Figs. 1E and 2A) also suggested impaired hepatocyte function. Both serum amyloid A2 (saa2) and thioredoxin (trx) were significantly up-regulated (Fig. 1F), and the 4-fold increase in TUNEL-positive cells (Fig. 1G) in the foigr mutant livers suggested hepatic damage. Together, these data indicate that the foigr mutants developed steatosis, which was accompanied by decreased liver function, liver damage, and hepatocyte apoptosis; this is similar to the situation for patients with FLD. The function of the Foigr protein is unknown, although recent studies have suggested a role in the secretory pathway.26-28 Regardless, the interesting phenotype of the foigr mutants compelled us to investigate the mechanism of steatosis in this new FLD model.

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Figure 2. foigr larvae experience hepatic ER stress. (A) Electron micrographs of 5-dpf WT (left) and foigr livers (right). The boxed regions in the top panels are magnified in the bottom panels. The asterisks indicate characteristic dilated ER. The cytoplasm of WT hepatocytes was full of gray glycogen, whereas only sparse glycogen patches were visible in mutant hepatocytes (glycogen, lipids, mitochondria, and nuclei are indicated by g, L, m, and n, respectively). (B) qPCR analysis of UPR gene expression in 5-dpf whole foigr larvae normalized to the expression in their WT siblings (i.e., set to 1, which is indicated by the gray line). The fold changes in the expression of each gene in the mutants versus their WT siblings were averaged for at least five clutches and were found to be significant (P < 0.01 by a one-sample t test) unless noted otherwise (i.e., NS). Genes are grouped by the pathway or the general function. The inset presents representative western blots of Bip for 5-dpf whole foigr mutants and their WT siblings. (C) qPCR analysis of a subset of UPR target genes in livers dissected from 5-dpf larvae. The average fold changes for five clutches of foigr mutant livers normalized to WT livers are listed in each column. All genes were significantly increased in mutants (P < 0.05 by a one-sample t test). (D) In situ hybridization for bip, chop, and dnajc3 on 5-dpf WT and foigr larvae. The images are representative of at least 20 embryos from two clutches. Staining can be observed in the liver (arrows), jaw, and exocrine pancreas. The scale bar represents 200 μm. (E) PCR analysis of xbp1 splicing with primers for the detection of both unspliced (xbp1-u) and spliced forms (xbp1-s). xbp1 revealed robust splicing in 5-dpf foigr livers and moderate splicing in the liverless carcass. The data are representative of three experiments. (F) p-Eif2s1 was detected with western blotting. The blot was repeated with six batches of 5-dpf WT and foigr samples, and the relative band intensity was normalized to α-tubulin, averaged, and displayed with the standard deviation (P = 0.000008 by a t test). A representative blot is shown. Error bars in all graphs represent the standard deviations. Abbreviations: NS, not significant; QC, protein-folding quality control.

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Foigr Mutation Causes Hepatic ER Stress.

ER stress is marked by UPR induction, compromised ER function, and abnormal ER structures. However, moderate or partial UPR activation may suggest an adaptive response that maintains ER function. To differentiate between these possibilities, we assessed the ER structure and the activation status of each UPR branch in the foigr mutants.

Electron microscopy revealed that the WT hepatocytes had granular cytoplasm full of glycogen, few lipid droplets, and rough perinuclear ER (Fig. 2A). In contrast, the foigr mutant hepatocytes were enlarged with abundant lipid droplets and scarce glycogen patches (Fig. 2A). The most striking feature of the mutant hepatocytes was the grossly dilated ER, which resembled the ER in hepatocytes with ER stress due to a hepatitis C infection4 or TN injection.12

We next assessed the degree to which each branch of the UPR was activated in the foigr mutants. Bip protein (Fig. 2B, inset) and the mRNA of the major players in each UPR branch as well as UPR target genes were up-regulated in mutants. These included chaperones (bip and dnajc3), ER quality control [uridine diphosphate glucose ceramide glucosyltransferase-like 1 (ugcgl1), calnexin (canx), and glucosidase alpha neutral AB (ganab)], ER-associated degradation [Der1-like domain family member 1 (derl1) and endoplasmic reticulum degradation enhancer mannosidase alpha-like 1 (edem1)], and apoptosis [chop and growth arrest and DNA damage-inducible 45 alpha (gadd45a); Fig. 2B]. Many of these genes were expressed at even higher levels in foigr mutant livers (Fig. 2C). In situ hybridization confirmed the enrichment of the UPR target genes bip, chop, and dnajc3 in 5-dpf foigr livers (Fig. 2D, arrow), although moderate induction in other tissues was also found. We found robust xbp1 splicing in 5-dpf foigr livers and, to a lesser extent, in the liverless carcasses of foigr mutants (Fig. 2E) . Although Eif2s1 can be phosphorylated by kinases other than Perk, the marked increase in p-Eif2s1 in 5-dpf foigr mutants (Fig. 2F) suggests Perk activation. The massive up-regulation of each UPR branch and the disruption of the ER structure unequivocally demonstrate that the foigr mutation causes hepatic ER stress.

TN Causes UPR Activation and Steatosis.

Studies in mice suggest that UPR activation can cause steatosis,6, 9, 10, 29 and acute exposure to TN, which blocks protein glycosylation and induces the UPR, causes steatosis in mice.12, 13 We used TN to determine whether ER stress could cause steatosis in zebrafish. Doses exceeding 2.5 μg/mL were acutely toxic to 3- and 4-dpf larvae, and 2 μg/mL was toxic when larvae were treated for more than 12 hours. Treatment with 1 μg/mL TN from 3 to 5 dpf caused no mortality and only moderate phenotypic abnormalities, including hepatomegaly and steatosis (Fig. 3A,B). The expression of genes required for some hepatic functions was reduced (Fig. 3C), and the expression of genes signifying hepatic damage (Fig. 3D) was increased in TN-treated larvae. As expected, prolonged TN treatment induced xbp1 splicing (Fig. 3E) and UPR target genes, including bip and chop (Fig. 3F). These data demonstrate that TN causes ER stress and FLD.

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Figure 3. TN causes steatosis and liver damage. (A) Tg(fabp10:dsRed) larvae that were exposed to DMSO (controls) or 1 μg/mL TN from 3 to 5 dpf were imaged while they were alive (left) or after whole-mount staining with Oil Red O (right) on the fifth day after fertilization. The liver is circled. The scale bar represents 200 μm. (B) TN treatment significantly increased the average percentage of larvae with steatosis (six clutches, n > 100 for each sample) *P < 0.0001 by a Student t test. Error bars represent the standard deviations. The expression of genes implicated in (C) liver function and (D) damage was detected with qPCR in TN-treated embryos and was normalized to DMSO-treated controls. The values represent the average fold changes in three experiments; error bars indicate standard deviations. *P < 0.05 by a one-sample t test. (E) xbp1-u and xbp1-s mRNA was detected in 5-dpf larvae treated for 48 hours with DMSO (−) or TN (+). The image is representative of five experiments. (F) In situ hybridization of 5-dpf larvae for the detection of bip and chop. bip-stained larvae were visualized with ventral (top) and left lateral views (bottom) to show the jaw, pancreas, and liver (arrows). The scale bar represents 200 μm. The images are representative of 20 larvae per sample.

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Srebp Activation Is Not Required for Steatosis Due to TN Treatment or an foigr Mutation.

Srebps and Atf6 are activated by similar mechanisms involving site 1 and 2 proteases (encoded by mbtps1 and mbtps2, respectively; see Ye et al.30 and Fig. 4A). Some studies have demonstrated that the UPR and SREBPs are activated together,16-18 whereas others have reported that UPR activation is accompanied by decreased SREBP activation.12, 13, 20, 31 We found that Atf6 depletion induced Srebp2 target genes (Supporting Fig. 2), and this is consistent with the model proposed by Zeng et al.,20 who found that Atf6 suppresses Srebp2 function. Our finding that Srebp2 target genes [3-hydroxy-3-methylglutaryl coenzyme A reductase A (hmgcra) and farnesyl diphosphate farnesyl transferase 1 (fdft1)] were expressed at lower levels in the foigr mutants (Fig. 4B), in which Atf6 was likely activated, supports this hypothesis.

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Figure 4. Steatosis in response to chronic ER stress does not require Srebp activation. (A) The activation pathways for the nSrebp and nAtf6 transcription factors use common components. Asterisks indicate components targeted in this study with either a morpholino (mo) or a mutant (mt). (B) Srebp1 and Srebp2 target genes were assessed in at least three clutches of RNA isolated from whole larvae (left) and dissected livers (right). The expression in foigr mutants was normalized to their WT siblings (gray bars), and the expression in TN-treated larvae was normalized to their DMSO-treated siblings (black bars). The level of expression in the respective controls was set to 1 (horizontal bars). *P < 0.01 by a one-sample t test. (C) The effects of scap morpholino injections on steatosis in three models (fasting, chronic TN treatment, and foigr mutation) were assessed. The scap morphants and their uninjected siblings were fasted until the sixth day after fertilization (left) or were treated with TN or DMSO from 3 to 5 dpf (center). Moreover, the scap morpholino was injected into foigr mutants and WT siblings (right). All larvae were collected on the fifth day after fertilization, were whole-mount-stained with Oil Red O, and were scored for steatosis. The numbers of larvae that scored positive (black) or negative (white) are plotted. The percentage of fish with steatosis is indicated below each bar. **P < 0.001 by Fisher's exact test. Abbreviations: NS, not significant.

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Although the genes encoding Srebps or their target genes were mostly unchanged in TN-treated whole larvae, whole foigr mutants, and foigr mutant livers (Fig. 4B), acetyl coenzyme A carboxylase α (acc1) and fatty acid synthase (fasn) were up-regulated in foigr livers. We thus explored the possibility that Srebp activation contributes to steatosis due to an foigr mutation or TN treatment with two genetic tools used to block Srebp activation (indicated by asterisks in Fig. 4A). Scap is specific to Srebp processing,32 whereas Mbtps1 and Mbtps2 also cleave other substrates.30, 33 Both are highly effective at blocking steatosis due to other causes, and mbtps1 mutants have significant reductions of Srebp target gene expression.22 A morpholino blocking scap translation was injected either into WT fish treated with TN from 3 to 5 dpf or into foigr mutants and their phenotypically WT siblings. Larvae were collected at 5 dpf, stained with Oil Red O, and scored for steatosis. Uninjected siblings and those injected with a nontargeting control morpholino were used interchangeably as controls because we found no differences in viability, gross appearance, liver size, steatosis, or the expression of the UPR and the Srebp target gene (Supporting Fig. 1A-C) between these two samples. The efficacy of the scap morpholino was demonstrated by resistance to steatosis caused by fasting (Fig. 4C) and alcohol.22 However, scap morphants were not protected from steatosis caused by TN or an foigr mutation (Fig. 4C). Thus, steatosis due to ER stress is independent of Srebp activation.

The mbtps1hi1487 allele had defects in jaw, brain, and liver development and did not develop steatosis without a stimulus (see Fig. 5A and Schlombs et al.34). We found no difference in the expression of Srebp target genes in mbtps1hi1487 mutants in response to TN (Fig. 5B). This supports the hypothesis that Srebps are neither induced by ER stress nor required for steatosis. The mechanism by which the Srebp1c target genes acc1 and fasn are induced in foigr mutant livers is unclear.

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Figure 5. mbtps1hi1487 mutants are protected from steatosis caused by chronic TN treatment. (A) The mbtps1hi1487 mutant larvae had a round liver, but they did not develop more steatosis than their WT siblings. Tg(fabp10:dsRed) WT larvae (top) and mbtps1hi1487 larvae (bottom) were imaged on the fifth day after fertilization and were scored for steatosis with whole-mount Oil Red O staining. (B) The expression of Srebp target genes in 5-dpf WT and mbtps1hi1487 larvae that were treated with TN was analyzed with qPCR. The expression in TN-treated larvae was normalized to the DMSO-treated fish of the same genotype to obtain the fold change for each gene. The average fold changes in WT larvae and mutants from three clutches are plotted; error bars show standard deviations. (C) The mbtps1 mutation induced UPR target genes. cDNA from 5-dpf WT or mbtps1hi1487 whole larvae or dissected livers was analyzed with qPCR. The ΔCt values obtained for each gene are plotted as single points for each individual clutch that was analyzed; the averages are indicated by horizontal bars. *P < 0.05 and **P < 0.005 by a Student t test. (D) DMSO- or TN-treated WT and mbtps1hi1487 larvae were analyzed with qPCR for Atf6 target genes. The ΔCt values for the TN-treated WT and mutant larvae were normalized to DMSO-treated WT larvae to obtain the fold changes. The average fold changes from five clutches are plotted; error bars indicate standard deviations. *P < 0.05 by a one-sample t test. (E) The mbtps1 mutation protected 5-dpf larvae from steatosis caused by chronic TN treatment. WT and mbtps1hi1487 larvae treated from 3 to 5 dpf with 1 μg/mL TN or DMSO were stained with Oil Red O and were scored for steatosis. Steatosis was scored in at least eight clutches, and the median percentages of steatosis are plotted; bars indicate standard deviations. **P < 0.001 by a Student t test. In addition to whole-mount Oil Red O staining, a subset of these samples was sectioned, stained with Oil Red O, and quantified for (F) the number of Oil Red O droplets and (G) the area of Oil Red O per liver cell. The median value for each fish is plotted as a dot; horizontal lines indicate the means. *P < 0.05, **P < 0.005, and ***P < 0.0005 by ANOVA. Abbreviations: NS, not significant.

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We predicted that Atf6 target genes would be expressed at lower levels in mbtps1hi1487 mutants versus WT fish. Surprisingly, the expression of chop, unspliced X box binding protein (xbp1-u), and xbp1-s was increased in mbtps1hi1487 mutants (Fig. 5C). This suggests that Xbp1-s was induced to compensate for Atf6 loss. A similar response occurred in atf6 morphants (Fig. 6A,B). Despite the increase in Xbp1-s, however, some Atf6 target genes in mbtps1hi1487 mutants were not fully activated when they were challenged with TN (Fig. 5D).

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Figure 6. Atf6 knockdown affects UPR activation. (A) An atf6 MO that was injected into Tg(fabp10:dsRed) embryos did not affect their development or steatosis according to whole-mount Oil Red O staining. (B) qPCR was used to analyze cDNA from whole or dissected livers of 5-dpf larvae that were injected with the standard control or the atf6 MO. Each gene was normalized to rpp0; the ΔCt value for each cDNA sample is plotted as a dot, and bars indicate means. P values are indicated (*P < 0.05). (C) Uninjected embryos and atf6 morphants were treated from 24 to 48 hpf with 1 μg/mL TN or DMSO and were collected at 48 hpf; the expression of the Atf6 target gene was examined with qPCR. The average ΔCt values of four clutches are plotted; error bars indicate standard deviations. **P < 0.001 by ANOVA. The fold change for each gene is listed below each column, and numbers with an asterisk represent significant differences in expression between TN-treated fish and DMSO-treated fish of the same genotype. (D) An atf6 MO was injected into embryos generated by the in-crossing of foigr heterozygotes. At 5 dpf, foigr mutants and phenotypically WT siblings with or without the atf6 MO were collected for qPCR analysis. The ΔCt values, which were normalized to rpp0, were calculated from three clutches, averaged, and plotted; error bars indicate standard deviations. *P < 0.01 by ANOVA. The fold change for each gene is listed below each column, and numbers with an asterisk represent significant differences (P < 0.05 by ANOVA) in expression between mutants and WT fish. Abbreviations: hpf, hours post-fertilization; MO, morpholino; XBP1-t, total X box binding protein 1.

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Unexpectedly, both the number of fish and the degree of steatosis caused by TN were significantly reduced in mbtps1hi1487 mutants (only 40% of the mutants developed steatosis after TN treatment; (Fig. 5E). Moreover, WT larvae treated with TN had 3 times more lipid droplets per liver cell (white dots in Fig. 5F) and a 7 times greater area occupied by Oil Red O staining in the liver compared to controls (white dots in Fig. 5G). Both measures of steatosis were significantly reduced in mbtps1hi1487 mutants challenged with TN (black dots in Fig. 5F,G). Because Atf6 target genes (Fig. 5D) but not Srebp targets (Fig. 5B) were decreased in mbtps1hi1487 mutants after TN treatment, we conclude that a loss of Atf6 activity (not Srebps) accounts for the protection of these mutants from steatosis caused by ER stress.

Atf6 Depletion Prevents Steatosis in foigr Mutants.

To determine whether the loss of Atf6 would protect fish from steatosis due to prolonged UPR activation, we injected foigr mutants with a morpholino to block atf6 translation and assessed the effects on UPR target genes and steatosis. As in mice,12, 13 the loss of atf6 did not affect embryo viability, development or the size, shape, or lipid accumulation in the liver (Fig. 6A). Similar to mbtps1hi1487 mutants, the Ire1a/Xbp1 branch was induced in atf6 morphants (Fig. 6B), yet they were impaired in their ability to fully induce the expression of Atf6 target genes in response to TN (Fig. 6C) or foigr mutation (Fig. 6D).

An atf6 morpholino injection into foigr mutants reduced the percentage of mutants with steatosis to 47%; this contrasts with 82% of uninjected mutants and 69% of mutants injected with the control morpholino (Fig. 7A). This finding was confirmed with a splice-blocking atf6 morpholino: less than 30% of the mutants injected with the atf6 splice blocking morpholino developed steatosis, whereas 70% of their uninjected mutant siblings did (not shown).

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Figure 7. atf6 depletion alleviates steatosis in foigr mutants. (A) An atf6 MO decreased the number of 5-dpf foigr larvae that developed steatosis. Embryos generated by the crossing of foigr heterozygotes were not injected (15 clutches) or were injected with the standard control (9 clutches) or the atf6 MO (15 clutches). The larvae were separated according to the phenotype at 5 dpf, stained with Oil Red O, and scored for steatosis. The total number of embryos in each category is plotted, and the percentage of fish with steatosis is listed below each column. *P = 0.01 and ***P < 0.0001 by a χ2 test. (B) An atf6 MO injection reduced the amount of Oil Red O staining in foigr hepatocytes. Cryosections of 5-dpf WT and foigr mutant embryos that either were not injected or were injected with the atf6 MO were stained with Oil Red O and hematoxylin. (C) The number of Oil Red O droplets per liver cell and (D) the area of each liver cell that was stained with Oil Red O were quantified. The medians of at least three sections for each fish are plotted as individual dots; the horizontal bars indicate the means for all fish in each sample. The foigr mutants injected with the atf6 MO were not significantly different from the WT controls. *P < 0.05 and ***P < 0.005 by ANOVA. Abbreviations: MO, morpholino; NS, not significant.

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Steatosis was less severe in foigr mutants that were injected with the atf6 morpholino (Fig. 7B). For the control, uninjected, and atf6 morpholino–injected WT larvae, the median number of lipid droplets per cell ranged from 0.8 to 4, and the overall median number was 2 droplets per cell (Fig. 7C, left); there were more than 12 droplets per cell in foigr mutant livers. Similarly, the area of each cell stained with Oil Red O was more than 5 times greater in foigr mutants versus WT livers (Fig. 7D). Both these measures of hepatic lipid accumulation were significantly reduced in foigr mutants by the injection of the atf6 morpholino (Fig. 7D). Collectively, these data demonstrate that a loss of Atf6 protects against steatosis caused by ER stress due to an foigr mutation or prolonged TN treatment.

Atf6 Depletion Enhances Steatosis Due to Acute TN Treatment.

Acute ER stress induced by an intraperitoneal injection of TN causes steatosis that resolves within 3 days in WT mice but does not resolve in mice lacking Atf6α.12, 13 This contrasts with our finding that a loss of Atf6 provides protection against steatosis due to prolonged ER stress. We hypothesize that the difference is attributable to the acute ER stress experienced by mice injected with TN versus the chronic ER stress occurring in foigr mutants and in larvae bathed in TN for 48 hours.

To test this, we developed a protocol for inducing acute ER stress in zebrafish larvae. Larvae were exposed to 2 μg/mL TN for 12-hour intervals on the fourth and fifth days after fertilization, as outlined in Fig. 8A. In protocols B and C, larvae were collected immediately after exposure. In protocol D, TN was washed out after exposure from 4 to 4.5 dpf, and larvae were collected at 5 dpf. We compared acute and prolonged (i.e., chronic) treatments with TN (Fig. 8A, protocol A) and treatments with dimethyl sulfoxide (DMSO) on UPR activation and steatosis.

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Figure 8. Acute TN treatment causes UPR activation and steatosis, and this is augmented by atf6 depletion. (A) Diagram of the TN exposure protocols. Larvae were treated with DMSO or TN for 48 (chronic treatment; 1 μg/mL) or 12 hours (acute treatment; 2 μg/mL). Samples were collected at the times indicated by each letter for qPCR or Oil Red O staining. Protocol D was the same as protocol C, except that TN was washed out, and the larvae were incubated for another 12 hours. (B) UPR target genes were induced by all four protocols. The fold changes for each gene were calculated with respect to DMSO (set to 1) for at least three clutches and were averaged. Error bars indicate standard deviations. *P < 0.05 and **P < 0.005 by ANOVA. (C) TN was administered to zebrafish according to the chronic and acute protocols outlined in panel A. The number of fish with steatosis was scored with whole-mount Oil Red O–stained larvae. The percentage of steatosis in each sample (three to five clutches) is listed below each column. Control fish were treated with DMSO from 3 to 5 dpf. The difference between the TN-treated fish and the DMSO-treated fish was significant for protocols A and D. ***P < 0.0001 by Fisher's exact test. (D) mbtps1hi1487 mutants were predisposed to steatosis caused by acute TN exposure. The number of fish with steatosis was counted in six clutches of mbtps1hi1487 mutants and their WT siblings. The fish were treated with DMSO from 3 to 5 dpf or with the chronic (protocol A) or acute administration of TN (protocol D). **P < 0.002 and ***P < 0.0001 by Fisher's exact test. (E) atf6 morphants developed more steatosis in response to the acute TN treatment. atf6 morphants, standard control morphants, and uninjected embryos were treated with TN or DMSO according to protocol D. The total number of 5-dpf fish in three clutches that were scored for steatosis according to whole-mount Oil Red O staining is plotted. The percentage of steatosis for each sample is listed below each column. *P < 0.02 by Fisher's exact test.

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The acute and chronic exposure protocols had equivalent effects with respect to the induction of UPR target gene expression (Fig. 8B). Steatosis occurred in 81% of the fish treated with the chronic protocol, but it did not occur after a short exposure (protocols B and C). However, when the TN was washed out (protocol D), 35% of the fish developed steatosis (Fig. 8C).

We then tested whether depleting Atf6 affected steatosis caused by acute TN treatment (protocol D). The percentage of fish with steatosis was significantly reduced among mbtps11487 mutants (45%) versus WT larvae (65%) chronically challenged with TN, but the percentage increased in response to acute TN treatment (85%) in comparison with their WT siblings (42%; Fig. 8D). Similar results were obtained for atf6 morphants: 76% developed steatosis after acute TN treatment, whereas 46% and 52% of the uninjected and control-injected larvae did (Fig. 8D). Thus, Atf6 depletion potentiates steatosis caused by acute ER stress in both zebrafish and mice.12, 13

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We have used zebrafish as a novel tool for understanding the complex relationship between UPR activation and steatosis. Our data demonstrate that both acute and chronic ER stress can lead to steatosis, and they illustrate the opposing roles that Atf6 plays in these different scenarios. We found that Atf6 depletion protects fish from steatosis due to chronic ER stress induced by either foigr mutation or prolonged exposure to TN, but it can accentuate steatosis caused by acute TN treatment. This is an important distinction because most FLD etiologies are likely associated with chronic UPR activation if not frank ER stress. In these cases, attempts to improve protein folding and reduce UPR signaling are predicted to be therapeutic. Exciting data from mouse models suggest the efficacy of this approach.10, 11, 14, 18

How does chronic UPR activation affect lipid metabolism in the liver? One possibility is that components of the UPR may directly modulate lipid metabolism. Although some studies have implicated lipid synthesis directed by Xbp135 or Srebps17, 18, 36, 37 as a factor in steatosis associated with ER stress, we do not believe that lipid synthesis is a major contributing factor to steatosis in our models. We hypothesize that the foigr mutation and TN treatment induce Atf6, and this in turn may suppress Srebp2 activity; this is consistent with data from mammalian cells.20 Although Atf6 depletion caused a slight up-regulation of Srebp2 target genes, this was insufficient to cause steatosis (see Figs. 7A and 8A,C,D). On the contrary, atf6 morphants were protected from steatosis induced by the foigr mutation. Together, our data suggest that triglyceride and cholesterol synthesis is unlikely to significantly contribute to steatosis caused by chronic ER stress.

It is likely that disruption of the secretory pathway prevents lipoprotein secretion. This is supported by the finding of decreased apolipoprotein B levels in the hepatocytes of mice injected with TN.13 In foigr mutant hepatocytes, we observed some lipid droplets within what appeared to be dilated ER; this perhaps reflected lipoprotein retention. Because apolipoprotein B secretion is impaired by treatments causing prolonged ER stress,38 it is feasible that lipoprotein retention in hepatocytes can contribute to steatosis. It is not known whether Atf6 affects lipoprotein secretion or other lipid metabolic pathways in hepatocytes, such as β-oxidation.

A complex mechanism likely accounts for our finding that Atf6 depletion both prevents and accentuates steatosis. We have found that an Atf6 loss results in the up-regulation of other UPR branches. This may be due to direct cross-talk between branches or a response to a transient increase in the unfolded protein load due to the depletion of Atf6. Regardless of the mechanism, the result is that the cells adapt so that they are better equipped to handle the gradual increase in unfolded proteins that likely occurs in foigr larvae or larvae chronically treated with TN. Paradoxically, Atf6 depletion effectively reduces the amount of ER stress caused by these two insults; this is similar to what has been reported for Bip+/− mice.14 We speculate that the reduction of ER stress in turn reduces the amount of steatosis. In contrast, an acute onslaught of unfolded proteins in the ER caused by a short exposure to a high dose of TN requires a robust UPR, which cannot be achieved when Atf6 is depleted. In this acute scenario, the absence of Atf6 exacerbates ER stress and disrupts lipid metabolism via a mechanism that remains elusive.

Foigr is highly conserved, yet its function remains elusive. Recent data suggest that Foigr functions in the secretory pathway,26-28 and this is consistent with our finding of ER dysfunction in foigr mutants. If the foigr mutation causes a defect in the Golgi apparatus,28 a backup of secretory pathway cargo may cause ER stress. If this is the case, treating zebrafish with brefeldin A to disrupt the Golgi apparatus should cause ER stress and phenocopy foigr. Our preliminary studies for testing this are not compelling (not shown). In contrast, the similarities between chronic TN treatment and foigr mutants lead us to speculate that a loss of foigr induces a defect in protein glycosylation. It will be important to define the mechanism by which the foigr mutation leads to UPR activation and to understand the function of Foigr.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors are indebted to Deanna Howarth, Mike Passeri, and Chris Monson for technical assistance. Dr. Friedman, Dr. Krauss, and Dr. Burdine provided helpful comments on the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24396_sm_suppinfofig1.tif2827KSupporting Figure 1. There are no measureable differences between control morpholino injection and uninjected larva. A. Embryo and larval morphology and survival (not shown) are the same in uninjected (top) and control morpholino injected (bottom). Live images are shown. Scale bar = 1 mm. B. Liver size, shape and steatosis is the same in 5 dpf uninjected Tg(fabp10:dsRed) larvae and those injected with the control morpholino. Steatosis was scored based on oil red O staining in 3 clutches. C. UPR and Srebp target gene expression is not affected by control morpholino injection. qPCR analysis of 5 dpf control morphants compared to uninjected controls is plotted as the average fold change from 3 experiments with bars showing the standard deviation.
HEP_24396_sm_suppinfofig2.tif5568KSupporting Figure 2. Srebp2 target genes are upregulated in atf6 morphants. atf6 morphants were collected as whole larvae or the liver was dissected on 5 dpf and analyzed using qPCR. The ΔCt (normalized to rpp0) calculated for each clutch was plotted as an individual dot, with the mean indicated by the horizontal bar. * indicates p<0.05; ** indicates p< 0.005 by students t-test.
HEP_24396_sm_suppinfotab1.doc96KSupporting Information Table 1.

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