Lack of de novo GMP synthesis leads to hepatic steatosis. (A,B) Lateral views of wild-type sibling and GMP synthetase^s850 mutant larvae stained with Oil Red O (ORO) at 7 dpf. GMP synthetase^s850 mutant larvae develop liver steatosis at 7 dpf. Broken lines outline the liver. (C,D) Projected confocal images of the wild-type sibling (C) and GMP synthetase^s850 mutant (D) liver visualized for Nile Red (red) and To-pro-3 (blue) staining and Tg (fabp10:GFP-CAAX)^lri1 (green) expression at 7 dpf. In GMP synthetase^s850 mutant larvae, lipid droplets stained by Nile Red are evident in hepatocytes. (E) The percentage of wild-type and GMP synthetase^s850 mutant larvae showing liver steatosis scored by whole-mount ORO staining at 7, 8, and 9 dpf. ORO staining experiments with wild-type larvae at 7, 8, and 9 dpf were repeated five times (total n = 138 larvae examined and total n = 4 showed ORO signal in the liver), five times (total n = 74 larvae examined and total n = 9 showed ORO signal in the liver), and six times (total n = 67 larvae examined and total n = 13 showed ORO signal in the liver), respectively. ORO staining experiments with GMP synthetase^s850 mutant larvae at 7, 8, and 9 dpf were repeated five times (total n = 124 larvae examined and total n = 36 showed ORO signal in the liver), four times (total n = 34 larvae examined and total n = 15 showed ORO signal in the liver), and three times (total n = 26 larvae examined and total n = 12 showed ORO signal in the liver), respectively. (F) Quantification of liver steatosis measured by the percentage of hepatocytes containing Nile Red positive lipid droplets in wild-type sibling (n = 9), GMP synthetase^s850 mutant (n = 9), and 150 µM GMP-treated GMP synthetase^s850 mutant larvae (n = 9). The percentage of hepatocytes containing lipid droplets is significantly increased in GMP synthetase^s850 mutant larvae. (G) Triglyceride levels measured in whole-body extracts of wild-type and GMP synthetase^s850 mutant larvae at 7 dpf. (H) A schematic of the de novo GMP synthesis pathway. In the linear de novo GMP synthesis pathway, IMP is converted to XMP by IMP dehydrogenase, and subsequently, XMP is converted to GMP by GMP synthetase. MPA inhibits the function of IMP dehydrogenase. The s850 mutation disrupts the function of GMP synthetase. (I) Wild-type larvae exposed to MPA from 3 to 7 dpf were fixed and stained with ORO at 7 dpf. MPA-treated larvae developed liver steatosis. (J) The percentage of control DMSO or MPA-treated larvae showing liver steatosis scored by whole-mount ORO staining at 7 dpf. MPA treatment increased the percentage of larvae showing liver steatosis. ORO staining experiments with MPA-treated larvae were repeated three times with an average n = 35.4 larvae per experiment (total n = 106 larvae examined and total n = 70 larvae showed ORO signal in the liver). (K,L) Projected confocal images of wild-type larvae treated with DMSO (K) or MPA (L) from 3 to 7 dpf visualized for Nile Red (red) and To-pro-3 (blue) staining and Tg (fabp10:GFP-CAAX)^lri1 expression (green). Nile Red stains lipid droplets, and Topro stains nuclei. (M) Quantification of liver steatosis measured by the percentage of hepatocytes containing lipid droplets in DMSO-treated control and MPA-treated larvae at 7 dpf. MPA-treatment significantly increased the percentage of hepatocytes containing lipid droplets. sb, swim bladder. *P < 0.05, **P < 0.01; error bars indicate SD.

The small GTPase Rac1 regulates hepatic steatosis. (A,B) Lateral views of wild-type larvae treated with DMSO (A) or Rac1 inhibitor (B) from 5 to 7 dpf and stained for ORO at 7 dpf. Rac1 inhibitor-treated larvae developed hepatic steatosis. (C) The percentage of DMSO or Rac1 inhibitor-treated larvae showing liver steatosis scored by whole-mount ORO staining. ORO staining experiments with Rac1 inhibitor-treated larvae were repeated 12 times with an average n = 12.1 larvae per experiment (total n = 146 larvae examined and total n = 89 larvae showed ORO signal in the liver). (D) Z-plane confocal image of the dissected liver of wild-type larvae visualized for Rac1 expression at 7 dpf. Rac1 is localized to the entire cell membrane of most cells in the liver. The outlined area is magnified and shown in the bottom right corner. (E,F) Projected confocal images of lipid droplets in the liver stained by Nile Red. Tg (fabp10:GFP-CAAX)^lri1 larvae treated with DMSO (E) or Rac1 inhibitor (F) visualized for GFP expression and Nile Red (red) and To-pro-3 (blue) staining at 7 dpf. (G) Projected confocal image of Tg (fabp10:GFP-DNRac1)^lri4 liver visualized for GFP expression and Nile Red (red) and To-pro-3 (blue) staining at 7 dpf. (H) Quantification of liver steatosis measured by the percentage of hepatocytes containing Nile Red positive lipid droplets in DMSO-treated control, Rac1 inhibitor-treated, and Tg (fabp10:GFP-DNRac1)^lri4 larvae at 7 dpf. *P < 0.05, **P < 0.01, ***P < 0.001; error bars indicate SD.

ROS homeostasis is necessary for the prevention of hepatic steatosis. (A,B) Lateral views of wild-type larvae treated with DMSO (A) or diphenyleneiodonium chloride (DPI) (B) from 5 to 7 dpf and stained for ORO at 7 dpf. Flavoprotein inhibitor, DPI, treated larvae (average 67.2%; SD 12.6; P < 0.001) developed hepatic steatosis. ORO staining experiments with DPI-treated larvae were repeated five times with an average n = 12.2 larvae per experiment (total n = 61 larvae examined and total n = 41 larvae showed ORO signal in the liver). (C-E) Projected confocal images of lipid droplets in the liver stained by Nile Red. Tg (fabp10:GFP-CAAX)^lri1 larvae treated with DMSO (C), DPI (D), or NAC (E) visualized for GFP expression and Nile Red (red) and To-pro-3 (blue) staining at 7 dpf. (F) Quantification of liver steatosis measured by the percentage of Nile Red-positive lipid droplets containing hepatocytes in DMSO, DPI, or NAC-treated larvae at 7 dpf. (G-I) Measurement of whole-body ROS production by H₂DCF fluorescence. Arbitrary units of H₂DCF fluorescence from three different experiments (n > 50) were normalized to control and averaged. Production of ROS in control, MPA-treated, Rac1 inhibitor-treated, and DPI-treated larvae was measured at 7 dpf (G). All tested conditions showed significant reduction of ROS production in (G). Production of ROS in wild-type and GMP synthetase^s850 mutant larvae was measured at 5, 6, and 7 dpf (H). In wild-type larvae, the ROS production levels between 5 and 7 dpf were not significantly changed, while in GMP synthetase^s850 mutant larvae the ROS production level at 7 dpf was reduced compared to that at 5 dpf (H). Production of ROS in control and H₂O₂-treated larvae was measured at 7 dpf (I). (J,K) Hepatic steatosis in GMP synthetase^s850 mutant larvae was ameliorated by H₂O₂ treatment. Projected confocal images of lipid droplets in the liver stained by Nile Red. GMP synthetase^s850 mutant larvae cultured in the absence (J) or presence (K) of 1 mM H₂O₂ were visualized for Nile Red (red) and To-pro-3 (blue) staining and Tg (fabp10:GFP-CAAX)^lri1 expression (green) at 7 dpf. Nile Red positive lipid droplets in the liver are reduced in H₂O₂-treated GMP synthetase^s850 mutant larvae. (L) Quantification of liver steatosis measured by the percentage of hepatocytes containing Nile Red-positive lipid droplet in wild-type, H₂O₂-treated wild-type, GMP synthetase^s850 mutant, and H₂O₂-treated GMP synthetase^s850 mutant larvae at 7 dpf. H₂O₂ treatment ameliorated hepatic steatosis in GMP synthetase^s850 mutant larvae. (M) Quantification of liver steatosis measured by the percentage of hepatocytes containing Nile Red-positive lipid droplets in Rac1 inhibitor-treated and Rac1 inhibitor plus H₂O₂-treated larvae at 7 dpf. H₂O₂ treatment suppressed Rac1 inhibitor induced hepatic steatosis. n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001; error bars indicate SD.

Expression of the tgh gene is regulated by Rac1-mediated ROS. (A) qPCR analysis of tgh mRNA expression levels in wild-type control, GMP synthetase^s850 mutant, Rac1 inhibitor-treated, and DPI-treated larvae at 7 dpf. The averages of at least three independent experiments are shown. tgh mRNA expression level is significantly down-regulated in all tested conditions. (B) Hydrolysis activity toward p-nitrophenyl laurate was measured using lysates of control, GMP synthetase^s850 mutant, Rac1 inhibitor-treated, and DPI-treated larvae at 7 dpf. Measured activity was normalized to control and the average of three different experiments is shown. Hydrolysis activities are significantly decreased in all tested cases. (C) tgh expression in 5 dpf wild-type larvae. Lateral views, anterior to the left. The region around the liver is magnified and shown separately in C′. The expression of tgh is restricted to the liver at this stage. Arrowheads point to the liver. (D) The hydrolysis activity of the homogenate of 7 dpf larvae is measured in the absence or presence of E600. E600 significantly down-regulated hydrolysis activity. (E,F) Lateral views of DMSO- (C) or E600- (D) treated larvae stained for ORO at 7 dpf. Hydrolase inhibitor, E600, treated larvae (average 62.2%; SD 10.6; P < 0.001) developed hepatic steatosis. The black broken lines outline the liver. ORO staining experiments with E600-treated larvae were repeated six times with an average n = 14 larvae per experiment (total n = 84 larvae examined and total n = 52 larvae showed ORO signal in the liver). (G,H) Projected confocal images of lipid droplets in the liver stained by Nile Red. Tg (fabp10:GFP-CAAX)^lri1 larvae treated with DMSO (G) or E600 (H) visualized for GFP expression and Nile Red (red) and To-pro-3 (blue) staining at 7 dpf. (I) Quantification of liver steatosis measured by the percentage of hepatocytes containing Nile Red-positive lipid droplets in DMSO- or E600-treated larvae at 7 dpf. E600-treated larvae developed hepatic steatosis. (J) qPCR analysis of tgh mRNA expression levels in wild-type, H₂O₂-treated wild-type, GMP synthetase^s850 mutant, and H₂O₂-treated GMP synthetase^s850 mutant larvae. tgh mRNA expression levels were restored to the level of wild-type control in H₂O₂-treated GMP synthetase^s850 mutant larvae. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant; error bars indicate SD.

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