Figure 4.

Reactive Oxygen Species (ROS) homeostasis is necessary for the prevention of hepatic steatosis. (A and 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%; s.d. 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 N-acetyl cysteine (NAC) (E) visualized for GFP expression and Nile Red (Red) and To-pro-3 (Blue) staining at 7dpf. (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 7dpf was reduced compared to that at 5 dpf (H). Production of ROS in control and hydrogen peroxide-treated larvae was measured at 7 dpf (I). (J and K) Hepatic steatosis in GMP synthetase^s850 mutant larvae was ameliorated by hydrogen peroxide 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 1mM hydrogen peroxide were visualized for 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 hydrogen peroxide-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, hydrogen peroxide-treated wild-type, GMP synthetase^s850 mutant and hydrogen peroxide-treated GMP synthetase^s850 mutant larvae at 7 dpf. Hydrogen peroxide 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 hydrogen peroxide-treated larvae at 7dpf. Hydrogen peroxide treatment suppressed Rac1 inhibitor induced hepatic steatosis. n.s., not significant; *P<0.05, **P<0.01, ***P<0.001; error bars indicate standard deviation.