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Fig. 4

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ZDB-IMAGE-150612-14
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Figures for Nussbaum et al., 2013
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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 synthetases850 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 synthetases850 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 synthetases850 mutant larvae was ameliorated by H2O2 treatment. Projected confocal images of lipid droplets in the liver stained by Nile Red. GMP synthetases850 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 synthetases850 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 synthetases850 mutant, and H2O2-treated GMP synthetases850 mutant larvae at 7 dpf. H2O2 treatment ameliorated hepatic steatosis in GMP synthetases850 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.

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