Highlights
- •MLKL-mediated signaling contributes to FFC diet-induced liver injury.
- •FFC diet or palmitic acid treatment induces MLKL expression in hepatocytes.
- •Palmitic acid drives MLKL translocation to autophagosomes independently of Rip3.
- •Mlkl, but not Rip3, regulates autophagic flux in a murine model of NAFL/NASH.
- •Pharmacologic inhibition of autophagy induces MLKL expression.
Background & Aims
Autophagy maintains cellular homeostasis and plays a critical role in the development
of non-alcoholic fatty liver and steatohepatitis. 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. Herein, 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 a 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
The 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. The FFC diet also induced
accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress,
in Mlkl+/+ but not Mlkl−/− mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor,
leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA
blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression
and translocation, first to autophagosomes and then to the 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 the plasma membrane in hepatocytes.
Conclusions
Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling
contributes to FFC diet-induced liver injury by inhibiting autophagy.
Lay summary
Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy
contributes to cell injury and death, thus playing a critical role in the pathogenesis
of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis.
Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory
responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism
between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver
disease.
Graphical abstract
Graphical Abstract
Keywords
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Article info
Publication history
Published online: March 24, 2020
Accepted:
March 10,
2020
Received in revised form:
March 6,
2020
Received:
September 19,
2019
Footnotes
Author names in bold designate shared co-first authorship
Identification
Copyright
© 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
