MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcohol-related liver disease (NAFL/NASH). Insufficient autophagy can lead to hepatocellular injury and death. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Here we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy. METHODS: Rip3(+/+), Rip3(-/-), Mlkl(+/+) and Mlkl(-/-) mice were fed Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA). RESULTS: FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency, protected mice from FFC diet-induced liver injury. FFC diet also induced accumulation of p62 and LC3-II, as well as markers of ER stress, in Mlkl(+/+), but not Mlkl(-/-) mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using a mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation to autophagosomes prior to plasma membrane independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to plasma membrane in hepatocytes. CONCLUSIONS: Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury through inhibition of autophagy.