Fig. 4

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.