Homeostatic generation of reactive oxygen species protects the zebrafish liver from steatosis

Abstract

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.

RETRACTED: Aldehyde dedydrogenase-2 plays a beneficial role in ameliorating chronic alcohol-induced hepatic steatosis and inflammation through regulation of autophagy

“…It would be ideal to stain cytoplasmic lipid droplets using fluorescent Nile Red prior to visualization of their intracellular localization in 3D by confocal microscopy. This method allows a more precise count of the portion of hepatocytes containing one or more lipid droplets [66]. …”

Zebrafish: An Important Tool for Liver Disease Research

“…95,96 ROS has been implicated in nearly every aspect of ALD and oxidation of hepatocyte proteins, membranes, and DNA and is believed to be the major cause of hepatocyte dysfunction in ALD. In contrast to the homeostatic levels of ROS, which were shown to limit fasting-induced steatosis, 64 higher levels of ROS generated by CYP-mediated ethanol metabolism cause mitochondrial dysfunction, which reduces lipid oxidation and contributes to steatosis. 94 In zebrafish, ethanol-induced liver damage is accompanied by oxidative stress (Figure 5).…”

“…The same metabolic perturbations cause FLD in humans and zebrafish 43,61 : a diet high in fat 62 or fructose, 63 fasting, 64–66 toxin exposure, 25,47,66,67 methionine depletion, 68 and genetic factors. 68–71 In rare cases, fatty liver can be caused by inborn errors of metabolism and other genetic disorders, 72 several of which have been modeled in zebrafish and reviewed elsewhere.…”

“…Mutants in the guanine monophosphate synthase ( gmps ) gene increased homeostatic levels of ROS, and this reduced FLD. 64 Combining elegant imaging of fluorescently labeled lipid droplets with chemical inhibitors and genetic tools, researchers showed that mutations in gmps downregulated Rac1 activity, reducing homeostatic levels of ROS, which then reduced a triglyceride hydrolase ( ces3 ), leading to lipid droplet accumulated in hepatocytes 64 (Figure 4). This observation was novel because ROS is considered to be a perpetrator of hepatic injury but was here shown to prevent lipid accumulation, a sign of disease.…”

Three-dimensional structural analysis reveals a Cdk5-mediated kinase cascade regulating hepatic biliary network branching in zebrafish

“…Western blotting and immunohistochemistry were performed as previously described (Lorent et al, 2004;Nussbaum et al, 2013) using the antibodies listed in Table S1.…”