Potential conflict of interest: Nothing to report.
Supported in part by grants from the NIH (R00 DK078138) and CWRU/Cleveland Clinic CTSA (UL1RR024989) and a startup package from the Cleveland Clinic Foundation to T.F.S. and by grants from the NIH (R01DK60322) and Packard foundation to D.Y.R.S.
Address reprint requests to: Takuya F. Sakaguchi, Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, NE30, Cleveland, OH 44195. E-mail: firstname.lastname@example.org.
Nonalcoholic fatty liver disease is the most common liver disease in both adults and children. The earliest stage of this disease is hepatic steatosis, in which triglycerides are deposited as cytoplasmic lipid droplets in hepatocytes. Through a forward genetic approach in zebrafish, we found that guanosine monophosphate (GMP) synthetase mutant larvae develop hepatic steatosis. We further demonstrate that activity of the small GTPase Rac1 and Rac1-mediated production of reactive oxygen species (ROS) are down-regulated in GMP synthetase mutant larvae. Inhibition of Rac1 activity or ROS production in wild-type larvae by small molecule inhibitors was sufficient to induce hepatic steatosis. More conclusively, treating larvae with hydrogen peroxide, a diffusible ROS that has been implicated as a signaling molecule, alleviated hepatic steatosis in both GMP synthetase mutant and Rac1 inhibitor-treated larvae, indicating that homeostatic production of ROS is required to prevent hepatic steatosis. We further found that ROS positively regulate the expression of the triglyceride hydrolase gene, which is responsible for the mobilization of stored triglycerides in hepatocytes. Consistently, inhibition of triglyceride hydrolase activity in wild-type larvae by a small molecule inhibitor was sufficient to induce hepatic steatosis. Conclusion: De novo GMP synthesis influences the activation of the small GTPase Rac1, which controls hepatic lipid dynamics through ROS-mediated regulation of triglyceride hydrolase expression in hepatocytes. (Hepatology 2013;58:1326–1338)
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The earliest stage of nonalcoholic fatty liver disease (NAFLD), hepatic steatosis, is characterized by excess accumulation of triglycerides (TG) in hepatocytes as lipid droplets. Hepatic steatosis is a risk factor for progression to nonalcoholic steatohepatitis (NASH), which can result in endstage liver disease. There have been no successfully established treatments for NAFLD or NASH, leaving the reduction of known risk factors as the standard of treatment. Thus, understanding the molecular mechanisms that underlie each stage of NAFLD pathogenesis could lead to the development of therapeutic targets to lessen or reverse NAFLD progression. A previous genome-wide association study in humans estimated the heritability of NAFLD to be 26%-27%. However, the number of human genes known to associate with NAFLD is still limited, indicating the importance of finding new genes and pathways responsible for NAFLD pathogenesis.
In NAFLD pathogenesis, hepatic steatosis is induced by a net increase in the rate of TG acquisition and synthesis relative to export and oxidation. The removal of TGs from the liver is achieved by hydrolysis and subsequent β-oxidation of free fatty acids, or by secretion of lipoprotein particles containing TGs. Impairment of pathways regulating lipoprotein particle secretion can thus perturb the balance of TG homeostasis in the liver and lead to hepatic steatosis. Triglyceride hydrolase (TGH also known as Ces3 or Ces1d) is an enzyme involved in the mobilization of stored TGs in hepatocytes to form lipoprotein particles.[4-7]
In the progression of NAFLD from simple steatosis to NASH, it has been proposed that reactive oxygen species (ROS) play an important role. ROS are chemically reactive molecules containing oxygen, and common biological species include hydrogen peroxide (H2O2). ROS have been historically regarded as a toxic byproduct of living cells that induce inflammatory responses and pathological conditions. Accumulating evidence now indicates, however, that ROS, especially the relatively stable H2O2 molecule, can function as intracellular second messengers at normal physiological levels.[9, 10] The physiological role of ROS homeostasis in hepatocytes, however, is largely unknown.
In the cell, ROS can be generated in numerous biological reactions, primarily during mitochondrial metabolism and by ROS-generating enzymes, including the NOX family nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. The NADPH oxidases are multiprotein complexes that generate ROS. The roles of NADPH oxidases have been best characterized in phagocytes; however, this complex is found in many other tissues, including the liver. The regulatory subunit of Nox1 and Nox2 NADPH oxidases is the small GTPase Rac1, a member of the Rho GTPase family that regulates a wide variety of cellular functions. Rac1 works as a molecular switch by cycling between inactive and active states; when guanosine diphosphate (GDP) is bound, Rac1 is inactive, while Rac1 is activated by guanosine triphosphate (GTP) exchange. Therefore, GTP-bound Rac1 is necessary for the activation of Nox1 and Nox2 NAPDH oxidases.
GDP and GTP are generated from guanosine monophosphate (GMP) by transferring phosphate groups from adenosine triphosphate (ATP). In animal cells, GMP is synthesized through two distinct pathways: the de novo synthesis and salvage pathways. Since the salvage pathway is energetically more efficient, it is believed to be the primary supplier of guanine nucleotides. GTP is necessary for NOX2 NAPDH oxidase activation in vitro, but it is unclear how Rac1 and NADPH oxidase-mediated ROS generation is affected when guanosine nucleotides are reduced in vivo.
In this study we implemented a forward genetic approach in zebrafish, which has proved to be a valuable strategy for identifying new genes and pathways that influence hepatic steatosis.[14-18] We identified GMP synthetase mutant larvae as showing a hepatic steatosis phenotype, and subsequently found that they also show down-regulation of Rac1 activation and ROS generation. Accordingly, artificially reducing ROS levels through multiple mechanisms was sufficient to induce hepatic steatosis in wild-type zebrafish larvae, which were then subsequently rescued by artificially increasing ROS levels. These and other data suggest that physiological levels of ROS generation are required to protect the liver from accumulating excess lipid.
Materials and Methods
Zebrafish Husbandry and Transgenic Lines
Zebrafish (Danio rerio) larvae were obtained from crosses of wild-type AB/TL strain or heterozygous mutant fish and raised as described. The following transgenic and mutant lines were used: GMP synthetases850, Tg (fabp10:GFP-CAAX)lri1, and Tg (fabp10:GFP-DNRac1)lri4.
The following molecules were used: Mycophenolic acid (Sigma Aldrich, Product #5255), Rac1 inhibitor (EMD Biosciences, Product #553502), diphenyleneiodonium chloride (DPI, Sigma Aldrich, Product #D2926), dimethyl p-nitrophenylphosphate (E600, Sigma Aldrich, Product #PS613) and N-acetyl-L-cystein (NAC, Sigma Aldrich, Product #A9165). All pharmacological treatments were administered with 1% dimethyl sulfoxide (DMSO) by volume. Concentrations of molecules used in this study are listed in Supporting Table 2.
Oil Red O Staining
Embryos were fixed at 7 days postfertilization (dpf) and treated as described.
Rac1 Activity Assay
The Rac1 Activity Assay Kit (Millipore) was used. Embryos were lysed and incubated with PAK-1 Pak1-binding domain (PBD)-bound beads. After washing, beads were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and blotted with anti-Rac1 (BD Transduction Laboratories, Cat. 610650) and β-tubulin (Abcam, Cat. 75123) antibodies.
Transmission Electron Microscopy
Electron microscopy was performed as described.
Nile Red Staining, Imaging, and Quantification
Embryos were fixed at 7 dpf in 4% paraformaldehyde (PFA) overnight. Livers were removed and soaked in Nile Red (500 ng/mL) along with TO-PRO3 Iodide nuclear stain for 2 hours at room temperature. Livers were then washed three times with phosphate-buffered saline (PBS), mounted in Vectashield mounting media (Vector Laboratories), and imaged with a Leica SP5 confocal microscope (Leica Microsystems). Nile Red and GFP were simultaneously excited using a combination of 488 and 514 nm green argon lasers with emission of 505-520 nm and 570-600 nm, respectively. Images were then processed for 3D rendering using Imaris software (Bitplane Scientific).
The portion of hepatocytes containing lipid droplets was determined by blindly selecting a single z-plane from each confocal z-series and counting the number of cells that were positive or negative for the presence of lipid. Hepatocytes were identified by the expression of Tg (fabp10:GFP-CAAX)lri1.
Immunohistochemistry and In Situ Hybridization
Immunohistochemistry and in situ hybridization were performed as described.
Quantitative RT-PCR was performed as described using the primers listed in Supporting Table 1. The ΔΔCt method was used for relative quantification.
Triglycerides were measured in whole-body extracts of larvae. Total lipids were quantified using the Triglyceride (GPO) Liquid Reagent assay kit (Pointe Scientific). Lipid concentration (mg/mL) was normalized to protein concentration (mg/mL) using the BCA Protein Assay Kit (Pierce, Rockford, IL) according to the manufacturer's instructions.
Following pharmacological treatments, live larvae were incubated in 30 μM 5- (and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (H2DCF) for 90 minutes and culture media was then measured for fluorescence at 485/535 nm on a BMG Labtech Fluorostar Optima Fluorescent Plate Reader (Life Technologies). Background fluorescence was subtracted by measuring fluorescence in identical conditions containing no larva.
Hydrolase Activity Assay
In total, 12-15 larvae were collected following pharmacological treatment and lysed by sonication in 500 μL PBS with 0.1% Triton X-100. Lysates were kept on ice and mixed 2:1 with the following solution: 180 μg/mL SDS, 1% Triton, 350 μg/mL p-nitrophenyl laurate (PNL) in PBS. PNL required incubation at 65°C for 20 minutes to solubilize, and was cooled to room temperature before use. Absorbance (405 nm) was measured as Time30min − Time0min.
All statistical tests were performed by unpaired, two-tailed t test.
Mutation in the GMP Synthetase Gene Causes Hepatic Steatosis in Zebrafish
The s850 mutant was originally identified in a large-scale mutagenesis screen focusing on liver development as a mutant showing reduced liver size (Supporting Fig. 1). A positional cloning approach identified the s850 mutation as a T to A transition in an exon of the GMP synthetase gene that changes the conserved histidine (H189) to glutamine (Q) (Supporting Fig. 2). Subsequently, we found that in GMP synthetases850 mutant larvae hepatocytes start accumulating neutral lipid as indicated by whole-mount Oil Red O (ORO) staining at 7 dpf (Fig. 1A,B). Approximately 30% of GMP synthetases850 mutant larvae showed clear ORO staining in the liver at 7 dpf (Fig. 1E). In GMP synthetases850 mutant larvae, ORO staining in the liver was also observed at 8 and 9 dpf (Fig. 1E). In order to investigate hepatic steatosis in GMP synthetases850 mutant larvae with subcellular-resolution, we developed a method in which we stained cytoplasmic lipid droplets by fluorescent Nile Red and observed their intracellular localization in 3D by confocal microscopy (Fig. 1C,D). This method allowed us to precisely count the portion of hepatocytes containing one or more lipid droplets. In GMP synthetases850 mutant larvae, on average 36.5% of hepatocytes contained Nile Red-positive lipid droplets (SD 13.8; n = 10; P < 0.01), while in their wild-type siblings the percentage of hepatocytes containing Nile Red signal was significantly lower (average 5.5%; SD 6.7; n = 9) (Fig. 1F). Consistently, when we treated GMP synthetases850 mutant larvae with 150 mM GMP, the percentage of hepatocytes containing lipid droplets was reduced (average 13.5%; SD 12.7; n = 9), suggesting that insufficient GMP production might induce hepatic steatosis in GMP synthetases850 mutant larvae. Electron micrographs confirmed the existence of lipid droplets in GMP synthetases850 mutant hepatocytes (Supporting Fig. 3). Consistent with hepatic steatosis, the total triglyceride level is increased in GMP synthetases850 mutant larvae (Fig. 1G).
De Novo GMP Synthesis Is Required to Prevent Hepatic Steatosis
Previous studies indicated that the proliferation of leukocytes in mammals and axon guidance in the Drosophila visual system require de novo GMP synthesis. However, the precise mechanisms by which de novo GMP synthesis regulates these biological processes are not clear. The final two steps of the de novo GMP synthesis pathway are linear and rate-limiting steps in which inosine monophosphate (IMP) dehydrogenase catalyzes the oxidation of IMP to xanthosine monophosphate (XMP) and GMP synthetase catalyzes the amination of XMP to GMP (Fig. 1H). In order to distinguish whether de novo GMP synthesis or unknown signaling or regulatory effects of GMP synthetase are responsible for hepatic steatosis, we treated wild-type zebrafish larvae with mycophenolic acid (MPA), a small molecule inhibitor of IMP dehydrogenase, to downregulate de novo GMP synthesis activity. We found that treating wild-type larvae with 15 μg/mL MPA from 3 to 7 dpf induced hepatic steatosis at 7 dpf (Fig. 1I,J), suggesting inhibition of de novo GMP synthesis is sufficient to induce hepatic steatosis. Consistently, we counted the number of Nile Red-positive hepatocytes in MPA-treated larvae (Average 26.2%; SD 10.5; n = 12) and found significantly more hepatocytes containing lipid droplets (Fig. 1K-M) than in control DMSO-treated larva (average 2.1%; SD 1.7; n = 12). Altogether, these data uncover a new role for de novo GMP synthesis in TG metabolism and hepatic steatosis in zebrafish.
De Novo GMP Synthesis Is Required for Activation of the Small GTPase Rac1
In Drosophila, de novo GMP synthesis is required to activate the small GTPase Rac1. We hypothesized that in zebrafish GMP synthetases850 mutant larvae, the supply of GMP, and hence GTP, is compromised, leading to a decrease in the activity of small GTPases including Rac1. To test this hypothesis, we measured GTP-bound (activated) Rac1 levels using a PBD pull-down assay (Fig. 2A). We found that GTP-bound Rac1 levels are decreased in GMP synthetases850 mutant and MPA-treated larvae (Fig. 2), suggesting that de novo GMP synthesis is required for the full activation of Rac1.
Inhibition of Rac1 Activity in Hepatocytes Induces Hepatic Steatosis
Interestingly, we found that inhibiting Rac1 activity is sufficient to induce hepatic steatosis (Fig. 3A,B). When treated with 50 μg/mL Rac1 inhibitor-containing media for 48 hours from 5 dpf, the activity of Rac1 was down-regulated in larvae (Fig. 2) as expected, and we found that a majority of treated larvae developed hepatic steatosis as indicated by increased Oil Red O staining in liver (Fig. 3B,C). To our knowledge, this are the first in vivo data suggesting a link between small GTPases and the regulation of hepatic steatosis. We counted the number of Nile Red-positive hepatocytes in Rac1 inhibitor-treated larvae (average 35.6%; SD 12.5; n = 9) and found significantly more hepatocytes containing lipid droplets than in DMSO-treated control larvae (average 2.1%; SD 1.7; n = 12) (Fig. 3E,F,H).
After observing that Rac1 is expressed strongly in hepatocytes at 7 dpf (Fig. 3D; Supporting Fig. 4), we hypothesized that Rac1 activity in hepatocytes is required for the prevention of hepatic steatosis. To test this hypothesis, we generated a new transgenic line, Tg (fabp10:GFP-DNRac1)lri4, which expresses dominant negative Rac1 (N17) only in hepatocytes (Supporting Fig. 5). In Tg (fabp10:GFP-DNRac1)lri4 larvae, the percentage of hepatocytes containing lipid droplets stained by Nile Red is significantly higher (average 32.7%; SD 11.9; n = 12) (Fig. 3G,H; Supporting Fig. 5), suggesting that Rac1 activity in hepatocytes is important for the regulation of hepatic steatosis.
Homeostatic Generation of ROS Is Necessary to Prevent Hepatic Steatosis
Historically, the role of Rac1 in actin cytoskeletal reorganization has been extensively studied; however, it is also known that Rac1 forms a protein complex with NADPH oxidases (Nox) to regulate their function in generating the superoxide anion that is quickly dismuted to H2O2 and other ROS molecules.[10, 11, 26] Since accumulating evidence indicates that ROS are important components in cell signaling, we hypothesized that Rac1 regulates hepatic steatosis through Nox-mediated ROS production. To test this hypothesis, we inhibited the activity of Nox by the flavoprotein inhibitor, DPI. We found that larvae treated with 10 μM DPI from 5 dpf showed strong Oil Red O signal in the liver at 7 dpf (Fig. 4A,B). We also confirmed that the percentage of hepatocytes containing lipid droplets stained by Nile Red is significantly higher in DPI-treated larva (average 30.8%; SD 12.5; n = 11) (Fig. 4D,F). These data suggest that down-regulating Nox activity is sufficient to induce hepatic steatosis. To test whether Nox-mediated ROS production is important for the prevention of hepatic steatosis, we treated larvae with the ROS-quenching agent NAC. We found that in larvae treated with 50 μM NAC from 5 dpf, the percentage of hepatocytes containing lipid droplets is significantly increased at 7 dpf (average 47.4%; SD 6.9; n = 9) (Fig. 4E,F). These data suggest that a reduction in ROS production might be responsible for the induction of hepatic steatosis.
To test this hypothesis, we next measured whole-body ROS production in DPI-treated, MPA-treated, Rac1 inhibitor-treated, and GMP synthetases850 mutant larvae. As expected, in 10 μM DPI-treated larva the production of ROS throughout the body was significantly reduced (Fig. 4G). Indeed, we found that ROS production was also reduced in MPA-treated and Rac1 inhibitor-treated and GMP synthetases850 mutant larvae (Fig. 4G,H), supporting the hypothesis that a reduction in ROS production might be responsible for the induction of hepatic steatosis. To further test this hypothesis, we treated larvae with 1 mM H2O2, which increased internal ROS levels as indicated by fluorescence of the ROS indicator, H2DCF (Fig. 4I) without any morphological changes at 7 dpf, and asked if increasing ROS levels would rescue hepatic steatosis. When we treated GMP synthetases850 mutant larvae with 1 mM H2O2 from 4 to 7 dpf, lipid droplets in hepatocytes were significantly decreased (average 10.5%; SD 9.7; n = 10; P < 0.05) (Fig. 4J-L), suggesting that artificially increasing ROS ameliorated hepatic steatosis in GMP synthetase mutant larvae. Consistently, 1 mM H2O2 treatment from 5 to 7 dpf eliminated lipid droplets in hepatocytes of Rac1 inhibitor-treated larvae (average 1.8%; SD 2.9; n = 9; P < 0.01) (Fig. 4M), further supporting the notion that ROS homeostasis is important for the prevention of hepatic steatosis.
ROS Production Influences Triglyceride Hydrolase Gene Expression
To understand the molecular mechanisms by which ROS generation influences hepatic steatosis, we used microarray analysis to look for genes that are affected by both the GMP synthetase mutation and Rac1 inhibitor-treatment in a similar manner. Among the candidates satisfying this criterion, we focused on the triglyceride hydrolase (tgh) gene, which codes for an enzyme responsible for the mobilization of stored triglyceride in hepatocytes.[4, 5, 7] We first verified the down-regulation of tgh gene expression in GMP synthetases850 mutant and Rac1 inhibitor-treated larvae by quantitative RT-PCR (qPCR, Fig. 5A). Since down-regulation of TGH activity is sufficient to induce lipid droplet accumulation in hepatocytes, we hypothesized that the Rac1-mediated ROS production regulates tgh gene expression to control lipid droplet formation in hepatocytes. Supporting this hypothesis, it was found that the expression level of the tgh gene was also reduced in DPI-treated larvae (Fig. 5A). Consistent with these gene expression changes, the enzymatic activity of TGH in GMP synthetases850 mutant, Rac1 inhibitor-treated, and DPI-treated larvae was also reduced (Fig. 5B), suggesting that Rac1-mediated ROS production influences TGH activity by regulating its expression. The tgh gene is expressed only in the yolk syncytial layer (YSL) at 3 dpf (data not shown); however, its expression is restricted to the liver by 5 dpf and it remains expressed only in the liver at 7 dpf (Fig. 5C).
Inhibition of hydrolase activity in cultured hepatocytes by a small molecule inhibitor, Diethyl E600, was shown to attenuate mobilization of TG. Thus, we hypothesized that decreased hydrolase activity causes hepatic steatosis by attenuating the mobilization of transiently stored TG in hepatocytes. To test this hypothesis, we treated zebrafish larvae with 10 μM E600 from 5 to 7 dpf and found that the hydrolase enzymatic activity was reduced in E600-treated larvae (Fig. 5D). We further found that E600-treated larvae developed strong hepatic steatosis (Fig. 5F,H), with more than 20% of hepatocytes containing lipid droplets stained by Nile Red (Fig. 5G-I) in all E600-treated larvae examined (average 42.4%; SD 14.1; n = 10), suggesting that the inhibition of hydrolase activity is sufficient to induce hepatic steatosis in zebrafish larvae.
Since decreased ROS production down-regulates tgh gene expression, we hypothesized that ROS production levels are correlated with tgh gene expression levels. To test this hypothesis, we treated larvae with 1 mM H2O2 from 5 to 7 dpf and examined the expression level of tgh mRNA. We found that tgh mRNA expression was increased in the 1 mM H2O2-treated larvae (Fig. 5J), supporting the hypothesis that ROS levels positively regulate tgh gene expression. Finally, we treated GMP synthetases850 mutant larvae, in which ROS production and tgh gene expression are reduced, with 1 mM H2O2 from 5 to 7 dpf, and examined the expression level of the tgh mRNA. We found that tgh mRNA expression was restored to wild-type levels in H2O2-treated GMP synthetases850 mutant larva (Fig. 5J), suggesting that increasing ROS is sufficient to rescue down-regulation of tgh transcription in GMP synthetases850 mutant larvae.
In this study, we show that de novo GMP synthesis is necessary to prevent hepatic steatosis in zebrafish (Fig. 1). De novo GMP synthesis influences the activation of the small GTPase Rac1, and Rac1 promotes the production of reactive oxygen species (ROS) (Fig. 6), which is important in regulating hepatic lipid dynamics by controlling triglyceride hydrolase gene expression (Fig. 6). Given that down-regulating Rac1 activity, by overexpressing dominant negative Rac1 specifically in hepatocytes, was sufficient to induce hepatic steatosis (Fig. 3G,H), this signaling cascade appears to take place in hepatocytes.
Mycophenolic Acid Influences the Production of ROS
H2O2, a relatively stable ROS that functions as a signaling molecule, mediates communication between wounded tissues and leukocytes. MPA is a Food and Drug Administration (FDA)-approved drug that has been used for immunosuppression; however, the precise molecular mechanisms by which MPA suppresses immune reaction are not clear. In this study we show that in MPA-treated larvae the production of ROS was reduced (Fig. 4F). This reduction in ROS production is most likely due to reduced activation of Rac1 (Fig. 2). Based on these results, we suggest that MPA suppresses the immune response, at least in part, by affecting Rac1 mediated ROS production.
Hepatic Steatosis in GMP Synthetase Mutant Larvae
We observed hepatic steatosis in GMP synthetases850 mutant larvae (Fig. 1). Consistently, we also observed increased total TG levels in these animals (Fig. 1G); however, since we measured the TG level in whole-body, this could be due to increased TG levels in extrahepatic tissues.
Although both intrahepatic biliary and vascular networks exist in GMP synthetases850 mutant larvae at 7 dpf (Supporting Fig. 6), their livers are smaller, likely due to reduced cell proliferation (Supporting Fig. 1). Since liver size is not rescued in H2O2-treated GMP synthetases850 mutant larvae (data not shown), and Rac1 inhibitor-treated, DPI-treated, E600-treated (data not shown) and Tg (fabp10:GFP-DNRac1)lri4 larvae have normal liver size (Supporting Fig. 5), we conclude that the liver cell proliferation phenotype in GMP synthetases850 mutant larvae appears to be independent of the ROS-mediated pathway.
Consistent with a previous study, GMP synthetases850 mutant larvae also display smaller eyes, the absence of xanthopore pigmentation, and dysmorphic branchial arches. However, these phenotypes were not rescued by H2O2 treatment (data not shown), suggesting that these phenotypes are also independent of the ROS-mediated pathway.
Hepatic steatosis is a risk factor for progression to NASH, which is associated with inflammation. In GMP synthetases850 mutant larvae, inflammation is not evident at 7 dpf, as evidenced by a lack of neutrophil infiltration to the liver at this stage (Supporting Fig. 7). However, these data do not exclude the possibility of the presence of other types of immune cells in the livers of GMP synthetases850 mutant larvae.
At 7 dpf, the percentage of GMP synthetases850 mutant larvae showing ORO staining in the liver is relatively low (Fig. 1E). Since MPA treatment to GMP synthetases850 mutant larvae further increased the percentage of ORO staining at 7 dpf (Supporting Fig. 8), maternally deposited GMP synthetase mRNA or protein might be influencing the results, or the s850 allele might not be a null.
We did not observe any hepatic steatosis at 5 or 6 dpf in GMP synthetases850 mutant larvae (data not shown). Similarly, Rac1 inhibitor or DPI treatment from 3 to 5 dpf did not induce hepatic steatosis in wild-type larvae (Supporting Fig. 9). The observation that the tgh gene is expressed in the liver only after 5 dpf (Fig. 5C) may explain why down-regulating ROS production does not induce hepatic steatosis before 5 dpf. Consistent with this hypothesis, Rac1 inhibitor or DPI treatment induces significant hepatic steatosis after 6 dpf both in starved and fed wild-type larvae (Supporting Figs. 9, 10).
tgh Gene Regulation by ROS Levels
We showed that expression of tgh is correlated with ROS levels. In GMP synthetases850 mutant, Rac1 inhibitor-treated, and DPI-treated larvae, in which ROS production is reduced (Fig. 4G, H), relative tgh expression is down-regulated (Fig. 5A). Conversely, in H2O2-treated larvae the expression of tgh was increased (Fig. 5J). Down-regulating TGH activity level by E600 (Fig. 5D) to the level in GMP synthetases850 mutant larvae (Fig. 5B) was sufficient to induce hepatic steatosis (Fig. 5E-I), supporting the hypothesis that reduced tgh expression is responsible for hepatic steatosis in GMP synthetases850 mutant larvae.
A previous study indicated that hepatic TG levels in Tgh-null mice were not statistically different from those in wild-type mice. In mice, Tgh also acts in white adipose tissues and it is likely that the absence of hepatic steatosis in tgh-null mice is due to decreased fatty acids delivery to the liver from adipose tissue, since isolated Tgh-null hepatocytes in culture accumulate more exogenous lipids than wild-type hepatocytes. In contrast, zebrafish white adipose tissues only develop after 12 dpf, potentially explaining why suppressing Tgh activity was sufficient to induced hepatic steatosis at 7 dpf.
In GMP synthetases850 mutant larvae, expression of genes involved in de novo lipogenesis (srebp1, acc1, agapt, and fads2), β-oxidation (aco, cpt1, cyp4a10, and echs1) or lipid uptake (cd36) are not significantly changed at 6 dpf (Supporting Fig. 11). These data also support the hypothesis that reduced tgh expression is responsible for hepatic steatosis in GMP synthetases850 mutant larvae.
Under physiological conditions, ROS produced by β-oxidation of triglyceride-derived free fatty acids may provide feedback to influence Tgh activity, adjusting lipid dynamics in hepatocytes.
ROS and Hepatic Steatosis
ROS are recognized to play important roles in host defense, especially in the innate immune response of leukocytes to pathogens, although the excessive production of ROS frequently results in inflammatory responses in many tissues, including the liver. In the liver, the two-hit model has been proposed for the transition of hepatic steatosis to more severe NASH, in which the first hit is hepatic steatosis and the second hit is ROS-mediated inflammation. Our data provide genetic evidence that physiological ROS levels are also necessary for the prevention of hepatic steatosis in zebrafish larvae (Fig. 6). The ability of H2O2 to rescue hepatic steatosis in GMP synthetases850 mutant, Rac1 inhibitor-treated, and Tg (fabp10:GFP-DNRac1)lri4 larvae (Figs. 4, 6) further supports this idea. Our data do not, however, conflict with the current two-hit model or the notion that excess ROS production is pathological; rather, we propose that a reduction in physiological levels of ROS can be equally pathogenic to increased levels of ROS. These data suggest that proposed antioxidant supplementation for the treatment of NAFLD would require careful dosage control to ensure that ROS levels are not reduced below their physiologically normal levels.
We thank Laura Nagy, Sanjoy Roychowdhury, Jasmine Lau, Takao Sakai, and Thomas McIntyre for critical reading of the article.