- •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.
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).
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.
Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy.
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.
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- Unconventional ways to live and die: cell death and survival in development, homeostasis, and disease.Annu Rev Cell Dev Biol. 2018; 34: 311-332
- Decoding cell death signals in liver inflammation.J Hepatol. 2013; 59: 583-594
- TNF-alpha induces two distinct caspase-8 activation pathways.Cell. 2008; 133: 693-703
- Regulation of tumour necrosis factor signalling: live or let die.Nat Rev Immunol. 2015; 15: 362-374
- Hepatocyte caspase-8 is an essential modulator of steatohepatitis in rodents.Hepatology (Baltimore, Md). 2013; 57: 2189-2201
- Impact of pan-caspase inhibition in animal models of established steatosis and non-alcoholic steatohepatitis.J Hepatol. 2010; 53: 542-550
- Necroptosis.N Engl J Med. 2014; 370: 455-465
- Necroptosis in development, inflammation and disease.Nat Rev Mol Cell Biol. 2017; 18: 127-136
- The receptor interacting protein kinases in the liver.Semin Liver Dis. 2018; 38: 73-86
- RIP kinases in liver cell death, inflammation and cancer.Trends Mol Med. 2019; 25: 47-63
- Targeting signal transduction pathways which regulate necrosis in acetaminophen hepatotoxicity.J Hepatol. 2015; 63: 5-7
- Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice.Hepatology. 2013; 58: 2099-2108
- Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis.Clin Sci (Lond). 2015; 129: 721-739
- Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury.Hepatology. 2013; 57: 1773-1783
- Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.Oncotarget. 2016; 7: 17681-17698
- Divergent effects of RIP1 or RIP3 blockade in murine models of acute liver injury.Cell Death Dis. 2015; 6: e1759
- Receptor interacting protein 3 protects mice from high-fat diet-induced liver injury.Hepatology (Baltimore, Md). 2016; 64: 1518-1533
- A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis.EMBO Mol Med. 2014; 6: 1062-1074
- RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL.Nat Commun. 2015; 6: 6282
- MLKL, the protein that mediates necroptosis, also regulates endosomal trafficking and extracellular vesicle generation.Immunity. 2017; 47: 51-65.e57
- The pseudokinase MLKL regulates hepatic insulin sensitivity independently of inflammation.Mol Metab. 2019; 23: 14-23
- Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner.Proc Natl Acad Sci U S A. 2017; 114: E961-E969
- Activated MLKL attenuates autophagy following its translocation to intracellular membranes.J Cell Sci. 2019; 132: jcs220996
- Necroptosis: (Last) message in a bubble.Immunity. 2017; 47: 1-3
- Regulation and functions of autophagic lipolysis.Trends Endocrinol Metab. 2016; 27: 696-705
- Function of autophagy in nonalcoholic fatty liver disease.Dig Dis Sci. 2016; 61: 1304-1313
- Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis.Science. 2014; 343: 1357-1360
- Animal models of nonalcoholic steatohepatitis: eat, delete, and inflame.Dig Dis Sci. 2016; 61: 1325-1336
- The necroptosis-inducing kinase RIPK3 dampens adipose tissue inflammation and glucose intolerance.Nat Commun. 2016; 7: 11869
- Apoptosis and necroptosis in the liver: a matter of life and death.Nat Rev Gastroenterol Hepatol. 2018; 15: 738-752
- The coming decade of cell death research: five riddles.Cell. 2019; 177: 1094-1107
- Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.Proc Natl Acad Sci U S A. 2014; 111: 15072-15077
- Palmitic acid induces production of proinflammatory cytokine interleukin-8 from hepatocytes.Hepatology. 2007; 46: 823-830
- Regulated necrosis: the expanding network of non-apoptotic cell death pathways.Nat Rev Mol Cell Biol. 2014; 15: 135-147
- Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.Cell Death Dis. 2014; 5: e1179
- Live to die another way: modes of programmed cell death and the signals emanating from dying cells.Nat Rev Mol Cell Biol. 2015; 16: 329-344
- Programmed necrosis and autophagy in immune function.Immunol Rev. 2012; 249: 205-217
- Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.Cell. 2009; 137: 1112-1123
- Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.Mol Cell. 2014; 54: 133-146
- Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it.Cell Death Differ. 2016; 23: 253-260
- The role of the LncRNA-FA2H-2-MLKL pathway in atherosclerosis by regulation of autophagy flux and inflammation through mTOR-dependent signaling.Cell Death Differ. 2019; 26: 1670-1687
- TNF-induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members.Cell Death Dis. 2011; 2: e230
- The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis.J Clin Invest. 2016; 126: 4346-4360
- Interferons transcriptionally up-regulate MLKL expression in cancer cells.Neoplasia. 2019; 21: 74-81
- The autophagy machinery controls cell death switching between apoptosis and necroptosis.Dev Cell. 2016; 37: 337-349
- Autophagy regulates inflammatory programmed cell death via turnover of RHIM-domain proteins.eLife. 2019; 8: e44452
- Autophagy regulates lipid metabolism.Nature. 2009; 458: 1131-1135
- Autophagy in nonalcoholic steatohepatitis.Expert Rev Gastroenterol Hepatol. 2011; 5: 159-166
- Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.Cell Metab. 2010; 11: 467-478
- Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice.J Hepatol. 2013; 58: 993-999
- Autophagy regulates adipose mass and differentiation in mice.J Clin Invest. 2009; 119: 3329-3339
- RIPK3 restricts viral pathogenesis via cell death-independent neuroinflammation.Cell. 2017; 169: 301-313.e11
Published online: March 24, 2020
Accepted: March 10, 2020
Received in revised form: March 6, 2020
Received: September 19, 2019
Author names in bold designate shared co-first authorship
© 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.