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• W2941155406 abstract "Increased hepatic ischemia-reperfusion (IR) injury in steatotic livers is a major reason for rejecting the use of fatty livers for liver transplantation. Necroptosis is implicated in the pathogenesis of fatty liver diseases. Necroptosis is regulated by three key proteins: receptor-interacting serine/threonine-protein kinase (RIPK)-1, RIPK3, and mixed-lineage kinase domain–like protein (MLKL). Here, we found that marked steatosis of the liver was induced when a Western diet was given in mice; steatosis was associated with the inhibition of hepatic proteasome activities and with increased levels of key necroptosis-related proteins. Mice fed a Western diet had more severe liver injury, as demonstrated by increases in serum alanine aminotransferase and necrotic areas of liver, after IR than did mice fed a control diet. Although hepatic steatosis was not different between Mlkl knockout mice and wild-type mice, Mlkl knockout mice had decreased hepatic neutrophil infiltration and inflammation and were protected from hepatic IR injury, irrespective of diet. Intriguingly, Ripk3 knockout or Ripk3 kinase-dead knock-in mice were protected against IR injury at the late phase but not the early phase, irrespective of diet. Overall, our findings indicate that liver steatosis exacerbates hepatic IR injury via increased MLKL-mediated necroptosis. Targeting MLKL-mediated necroptosis may help to improve outcomes in steatotic liver transplantation. Increased hepatic ischemia-reperfusion (IR) injury in steatotic livers is a major reason for rejecting the use of fatty livers for liver transplantation. Necroptosis is implicated in the pathogenesis of fatty liver diseases. Necroptosis is regulated by three key proteins: receptor-interacting serine/threonine-protein kinase (RIPK)-1, RIPK3, and mixed-lineage kinase domain–like protein (MLKL). Here, we found that marked steatosis of the liver was induced when a Western diet was given in mice; steatosis was associated with the inhibition of hepatic proteasome activities and with increased levels of key necroptosis-related proteins. Mice fed a Western diet had more severe liver injury, as demonstrated by increases in serum alanine aminotransferase and necrotic areas of liver, after IR than did mice fed a control diet. Although hepatic steatosis was not different between Mlkl knockout mice and wild-type mice, Mlkl knockout mice had decreased hepatic neutrophil infiltration and inflammation and were protected from hepatic IR injury, irrespective of diet. Intriguingly, Ripk3 knockout or Ripk3 kinase-dead knock-in mice were protected against IR injury at the late phase but not the early phase, irrespective of diet. Overall, our findings indicate that liver steatosis exacerbates hepatic IR injury via increased MLKL-mediated necroptosis. Targeting MLKL-mediated necroptosis may help to improve outcomes in steatotic liver transplantation. Fatty liver disease, including alcoholic liver disease and nonalcoholic fatty liver disease (NAFLD), is a major health issue around the world. NAFLD is associated with obesity, diabetes, and metabolic diseases.1Must A. Spadano J. Coakley E.H. Field A.E. Colditz G. Dietz W.H. The disease burden associated with overweight and obesity.JAMA. 1999; 282: 1523-1529Google Scholar The prevalence of NAFLD is estimated to be about 20% worldwide.2Younossi Z.M. Koenig A.B. Abdelatif D. Fazel Y. Henry L. Wymer M. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes.Hepatology. 2016; 64: 73-84Google Scholar NAFLD is characterized by hepatic steatosis, inflammation, and fibrosis, and can progress to cirrhosis and hepatocellular carcinoma.3Brunt E.M. Wong V.W. Nobili V. Day C.P. Sookoian S. Maher J.J. Bugianesi E. Sirlin C.B. Neuschwander-Tetri B.A. Rinella M.E. Nonalcoholic fatty liver disease.Nat Rev Dis Primers. 2015; 1: 15080Google Scholar Hepatic ischemia-reperfusion (IR) injury is an important type of liver injury that occurs in many clinical conditions, including liver transplantation and liver resection surgeries.4van Golen R.F. Reiniers M.J. Olthof P.B. van Gulik T.M. Heger M. Sterile inflammation in hepatic ischemia/reperfusion injury: present concepts and potential therapeutics.J Gastroenterol Hepatol. 2013; 28: 394-400Google Scholar Hepatic IR injury occurs due to the depletion of oxygen and ATP, excessive inflammatory responses, and the generation of reactive oxygen species after reperfusion.5Luedde T. Kaplowitz N. Schwabe R.F. Cell death and cell death responses in liver disease: mechanisms and clinical relevance.Gastroenterology. 2014; 147: 765-783.e4Abstract Full Text Full Text PDF Scopus (400) Google Scholar Liver transplantation remains the most effective treatment option for end-stage liver diseases. However, the practice of liver transplantation is limited by organ shortage.6Kim W.R. Smith J.M. Skeans M.A. Schladt D.P. Schnitzler M.A. Edwards E.B. Harper A.M. Wainright J.L. Snyder J.J. Israni A.K. Kasiske B.L. OPTN/SRTR 2012 annual data report: liver.Am J Transplant. 2014; 14: 69-96Google Scholar This issue has led to a more aggressive acceptance and usage of liver grafts from extended-criteria donors, which includes using steatotic livers.7Gehrau R.C. Mas V.R. Dumur C.I. Suh J.L. Sharma A.K. Cathro H.P. Maluf D.G. Donor hepatic steatosis induce exacerbated ischemia-reperfusion injury through activation of innate immune response molecular pathways.Transplantation. 2015; 99: 2523-2533Google Scholar Moderate to severe liver donor steatosis (30% to 60% graft macrosteatosis) increases the risk for IR injury, as demonstrated by evidence that fatty liver has a reduced tolerance to IR injury, leading to increased mortality after transplantation.7Gehrau R.C. Mas V.R. Dumur C.I. Suh J.L. Sharma A.K. Cathro H.P. Maluf D.G. Donor hepatic steatosis induce exacerbated ischemia-reperfusion injury through activation of innate immune response molecular pathways.Transplantation. 2015; 99: 2523-2533Google Scholar, 8Li S. Takahara T. Li X.K. Fujino M. Sugiyama T. Tsukada K. Liu C. Kakuta Y. Nonomura N. Ito H. Takahashi K. Nakajima M. Tanaka T. Takahara S. 5-Aminolevulinic acid combined with ferrous iron ameliorate ischemia-reperfusion injury in the mouse fatty liver model.Biochem Biophys Res Commun. 2016; 470: 900-906Google Scholar However, the mechanisms by which fatty liver increases IR injury are not fully understood. Without a clear understanding of these mechanisms, the potential to use fatty livers in transplantation will be limited. Furthermore, therapeutic modalities to improve postsurgical care with extensive fatty liver would also be limited. Limited evidence indicates that necroptosis may play an important role in the pathogenesis of inflammatory liver diseases, including alcoholic liver disease, NAFLD, and hepatic IR injury.9Roychowdhury S. McCullough R.L. Sanz-Garcia C. Saikia P. Alkhouri N. Matloob A. Pollard K.A. McMullen M.R. Croniger C.M. Nagy L.E. Receptor interacting protein 3 protects mice from high-fat diet-induced liver injury.Hepatology. 2016; 64: 1518-1533Google Scholar, 10Gautheron J. Vucur M. Reisinger F. Cardenas D.V. Roderburg C. Koppe C. Kreggenwinkel K. Schneider A.T. Bartneck M. Neumann U.P. Canbay A. Reeves H.L. Luedde M. Tacke F. Trautwein C. Heikenwalder M. Luedde T. A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis.EMBO Mol Med. 2014; 6: 1062-1074Google Scholar, 11Wang S. Ni H.M. Dorko K. Kumer S.C. Schmitt T.M. Nawabi A. Komatsu M. Huang H. Ding W.X. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.Oncotarget. 2016; 7: 17681-17698Crossref Scopus (55) Google Scholar, 12Hong J.M. Kim S.J. Lee S.M. Role of necroptosis in autophagy signaling during hepatic ischemia and reperfusion.Toxicol Appl Pharmacol. 2016; 308: 1-10Google Scholar Necroptosis is a form of cell death also known as programed necrosis, and is mediated by receptor-interacting serine/threonine-protein kinase (RIPK)-1, RIPK3, and the downstream molecule mixed-lineage kinase domain–like protein (MLKL).13Ashkenazi A. Salvesen G. Regulated cell death: signaling and mechanisms.Annu Rev Cell Dev Biol. 2014; 30: 337-356Google Scholar, 14Linkermann A. Green D.R. Necroptosis.N Engl J Med. 2014; 370: 455-465Google Scholar Necroptosis is morphologically similar to necrosis, which is activated by a pathway in common with apoptosis, including death-receptor activation.15de Almagro M.C. Vucic D: necroptosis: pathway diversity and characteristics.Semin Cell Dev Biol. 2015; 39: 56-62Google Scholar, 16Humphries F. Yang S. Wang B. Moynagh P.N. RIP kinases: key decision makers in cell death and innate immunity.Cell Death Differ. 2015; 22: 225-236Google Scholar, 17Newton K. RIPK1 and RIPK3: critical regulators of inflammation and cell death.Trends Cell Biol. 2015; 25: 347-353Google Scholar In the absence of functional caspase-8, RIPK1 and RIPK3 form heterodimers, resulting in the assembly of the necrosome, which is a crucial step that leads to the phosphorylation and activation of MLKL. Activated MLKL forms oligomers or polymers that translocate to the plasma membrane, resulting in disruption of cell membrane and subsequent necrotic cell death.17Newton K. RIPK1 and RIPK3: critical regulators of inflammation and cell death.Trends Cell Biol. 2015; 25: 347-353Google Scholar, 18Moriwaki K. Chan F.K. RIP3: a molecular switch for necrosis and inflammation.Genes Dev. 2013; 27: 1640-1649Google Scholar, 19Vanden Berghe T. Hassannia B. Vandenabeele P. An outline of necrosome triggers.Cell Mol Life Sci. 2016; 73: 2137-2152Google Scholar, 20Liu S. Liu H. Johnston A. Hanna-Addams S. Reynoso E. Xiang Y. Wang Z. MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.Proc Natl Acad Sci U S A. 2017; 114: E7450-E7459Google Scholar In addition, necrosome also increases inflammatory factors including IL-1β and IL-6, which play important roles in alcoholic liver disease, NAFLD, and hepatic IR injury.7Gehrau R.C. Mas V.R. Dumur C.I. Suh J.L. Sharma A.K. Cathro H.P. Maluf D.G. Donor hepatic steatosis induce exacerbated ischemia-reperfusion injury through activation of innate immune response molecular pathways.Transplantation. 2015; 99: 2523-2533Google Scholar There has been intensive ongoing research aiming to identify new preconditioning strategies for diminishing the adverse effects of allograft steatosis after liver transplantation. Specifically, new therapies tested in murine models have demonstrated that parenchymal cell damage and inflammation are major contributors to exacerbated hepatic IR injury in steatotic liver grafts.8Li S. Takahara T. Li X.K. Fujino M. Sugiyama T. Tsukada K. Liu C. Kakuta Y. Nonomura N. Ito H. Takahashi K. Nakajima M. Tanaka T. Takahara S. 5-Aminolevulinic acid combined with ferrous iron ameliorate ischemia-reperfusion injury in the mouse fatty liver model.Biochem Biophys Res Commun. 2016; 470: 900-906Google Scholar, 21Esteban-Zubero E. Garcia-Gil F.A. Lopez-Pingarron L. Alatorre-Jimenez M.A. Ramirez J.M. Tan D.X. Garcia J.J. Reiter R.J. Melatonin role preventing steatohepatitis and improving liver transplantation results.Cell Mol Life Sci. 2016; 73: 2911-2927Google Scholar, 22Sutter A.G. Palanisamy A.P. Ellet J.D. Schmidt M.G. Schnellmann R.G. Chavin K.D. Intereukin-10 and Kupffer cells protect steatotic mice livers from ischemia-reperfusion injury.Eur Cytokine Netw. 2014; 25: 69-76Google Scholar Necrostatin-1, a pharmacologic inhibitor of RIPK1, has been reported to protect against IR injury in heart, kidney, intestine, and liver,12Hong J.M. Kim S.J. Lee S.M. Role of necroptosis in autophagy signaling during hepatic ischemia and reperfusion.Toxicol Appl Pharmacol. 2016; 308: 1-10Google Scholar, 23Koudstaal S. Oerlemans M.I. Van der Spoel T.I. Janssen A.W. Hoefer I.E. Doevendans P.A. Sluijter J.P. Chamuleau S.A. Necrostatin-1 alleviates reperfusion injury following acute myocardial infarction in pigs.Eur J Clin Invest. 2015; 45: 150-159Google Scholar, 24Kim C.R. Kim J.H. Park H.L. Park C.K. Ischemia reperfusion injury triggers TNFalpha induced-necroptosis in rat retina.Curr Eye Res. 2017; 42: 771-779Google Scholar, 25Wen S. Ling Y. Yang W. Shen J. Li C. Deng W. Liu W. Liu K. Necroptosis is a key mediator of enterocytes loss in intestinal ischaemia/reperfusion injury.J Cell Mol Med. 2017; 21: 432-443Google Scholar suggesting that targeting RIPK1-RIPK3-MLKL–mediated necroptosis may be beneficial in IR injury. However, necrostatin-1 is not a specific inhibitor of RIPK1, which has also been demonstrated to inhibit indoleamine-2,3-dioxygenase.26Vandenabeele P. Grootjans S. Callewaert N. Takahashi N. Necrostatin-1 blocks both RIPK1 and IDO: consequences for the study of cell death in experimental disease models.Cell Death Differ. 2013; 20: 185-187Google Scholar Therefore, the exact role of RIPK1-RIPK3-MLKL–mediated necroptosis in hepatic IR injury is still elusive. A murine model of diet-induced hepatic steatosis was used to characterize its effects on hepatic IR injury. Hepatic steatosis was found to be associated with increased hepatic Ripk1, Ripk3, and Mlkl proteins and with exacerbated IR injury. Furthermore, three genetically modified mouse models—Ripk3 knockout (KO), Mlkl KO, and kinase-dead Ripk3 knock-in (KDKI)—were used to examine the effects of necroptosis in hepatic IR injury. Ripk3 KO or Ripk3 KDKI mice were protected from IR liver injury at the late phase but not the early phase. Interestingly, Mlkl KO mice were protected from IR liver injury in both chow-fed and diet-induced obese conditions at both the early and the late phases. These findings may have a significant impact on developing potential therapeutic strategies to attenuate IR injury in fatty livers by modulating RIPKs and MLKL. Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX). Horseradish peroxidase—conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Western diet (WD; catalog number TD.88137) and normal chow diet (CD; catalog number 8604) were from Envigo (Madison, WI). Mlkl KO mice with C57BL/6N background were generated using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas)–mediated genome engineering method by Cyagen (Santa Clara, CA). Briefly, Mlkl exon 2 was selected as the target site. Cas9 mRNA and gRNA generated by in vitro transcription were then injected into fertilized eggs for KO mouse production. Ripk3 KO (C57BL/6N) mice were generously provided by Dr. Vishva Dixit (Genentech, South San Francisco, CA) as we described previously.11Wang S. Ni H.M. Dorko K. Kumer S.C. Schmitt T.M. Nawabi A. Komatsu M. Huang H. Ding W.X. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.Oncotarget. 2016; 7: 17681-17698Crossref Scopus (55) Google Scholar Ripk3 KDKI and Ripk1 KDKI (C57BL/6N) mice were generously provided by Dr. Peter Gough (GlaxoSmithKline, Research Triangle Park, NC).27Kaiser W.J. Daley-Bauer L.P. Thapa R.J. Mandal P. Berger S.B. Huang C. Sundararajan A. Guo H. Roback L. Speck S.H. Bertin J. Gough P.J. Balachandran S. Mocarski E.S. RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition.Proc Natl Acad Sci U S A. 2014; 111: 7753-7758Google Scholar, 28Mandal P. Berger S.B. Pillay S. Moriwaki K. Huang C. Guo H. Lich J.D. Finger J. Kasparcova V. Votta B. Ouellette M. King B.W. Wisnoski D. Lakdawala A.S. DeMartino M.P. Casillas L.N. Haile P.A. Sehon C.A. Marquis R.W. Upton J. Daley-Bauer L.P. Roback L. Ramia N. Dovey C.M. Carette J.E. Chan F.K. Bertin J. Gough P.J. Mocarski E.S. Kaiser W.J. RIP3 induces apoptosis independent of pronecrotic kinase activity.Mol Cell. 2014; 56: 481-495Google Scholar Briefly, Ripk3 KDKI mice were generated by homologous recombination using a targeting construct that mutated the catalytic lysine residue to alanine (K51A) to eliminate all kinase activity. Ripk1 KDKI mice were generated by homologous recombination using a targeting construct that mutated the catalytic lysine residue to alanine (K45A) to eliminate all kinase activity. All mice were housed in cages (five mice per cage) and received a 12-hour light/dark cycle. All animals received humane care. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center (Kansas City, KS). Four-week–old male mice were fed a WD or CD for 8 weeks, followed by hepatic IR surgery as described previously.29Yang M. Antoine D.J. Weemhoff J.L. Jenkins R.E. Farhood A. Park B.K. Jaeschke H. Biomarkers distinguish apoptotic and necrotic cell death during hepatic ischemia/reperfusion injury in mice.Liver Transpl. 2014; 20: 1372-1382Google Scholar Briefly, hepatic ischemia was created by occluding the portal vein, hepatic artery, and bile duct just above the right branch, which provided approximately 70% of the total body's blood supply to the liver, for 45 minutes, followed by reperfusion for different periods of time (1, 6, and 24 hours). Sham control mice underwent the same procedure without vessel occlusion. Mice were euthanized and blood samples and liver tissues were collected. Livers were fixed in 10% formalin and paraffin embedded for histopathologic analysis. Liver injury was determined by measuring serum alanine aminotransferase (Alt). Paraffin-embedded liver sections were stained with hematoxylin and eosin (H&E) for pathologic evaluation. Immunostaining for Mpo-positive neutrophils was performed.30He Y. Feng D. Li M. Gao Y. Ramirez T. Cao H. Kim S.J. Yang Y. Cai Y. Ju C. Wang H. Li J. Gao B. Hepatic mitochondrial DNA/Toll-like receptor 9/MicroRNA-223 forms a negative feedback loop to limit neutrophil overactivation and acetaminophen hepatotoxicity in mice.Hepatology. 2017; 66: 220-234Google Scholar The positive Mpo staining was quantified by counting 20 different fields under microscopy using ×400 magnification. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed as described previously.11Wang S. Ni H.M. Dorko K. Kumer S.C. Schmitt T.M. Nawabi A. Komatsu M. Huang H. Ding W.X. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.Oncotarget. 2016; 7: 17681-17698Crossref Scopus (55) Google Scholar Oil red O staining was performed using liver cryo-sections as described previously.31Williams J.A. Ni H.M. Ding Y. Ding W.X. Parkin regulates mitophagy and mitochondrial function to protect against alcohol-induced liver injury and steatosis in mice.Am J Physiol Gastrointest Liver Physiol. 2015; 309: G324-G340Google Scholar Total liver proteins were extracted using radioimmunoprecipitation assay buffer [1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl (lauryl) sulfate in phosphate-buffered saline]. Plasma membrane fractions of liver tissue were extracted by using a Minute plasma membrane and cell fractionation kit following the manufacturer's instructions (catalog number SM-005; Invent Biotechnologies, Plymouth, MN). Protein (30 μg) was separated on an SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Membranes were probed with appropriate primary and secondary antibodies and visualized with SuperSignal chemiluminescent substrate (catalog number 34577; Thermo Fisher Scientific, Waltham, MA). Densitometry analysis was performed with Un-Scan-It software version 6.1 (Silk Scientific, Orem, UT) and normalized to β-actin or Gapdh for total proteins or sodium potassium ATPase for plasma membrane proteins. All densitometry data are presented as means ± SEM. Proteasome activity was analyzed using Suc-LLVY-AMC and Bz-VGR-AMC substrate (catalog numbers BML-P802 and BML-BW9375; Enzo Life Sciences, Farmingdale, NY) as described previously.11Wang S. Ni H.M. Dorko K. Kumer S.C. Schmitt T.M. Nawabi A. Komatsu M. Huang H. Ding W.X. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.Oncotarget. 2016; 7: 17681-17698Crossref Scopus (55) Google Scholar Briefly, 10 μg of total liver lysates was added to a white 96-well flat-bottom plate. AMC substrate (20 nmol/L) was added to each well, along with 100 μL of assay buffer (50 mmol/L Tris pH 7.5, 25 mmol/L KCl, 10 mmol/L NaCl, and 1 mmol/L MgCl2 diluted in dH2O). Proteasome activity was determined by measuring AMC release using an excitation/emission 380/460 test filter on a plate reader (Tecan Life Sciences, Männedorf, Switzerland) after 1 hour. RNA was extracted from mouse liver using Trizol (Invitrogen, Carlsbad, CA) and reverse-transcribed into cDNA by RevertAid reverse transcriptase (Thermo Fisher Scientific).32Li Y. Chao X. Yang L. Lu Q. Li T. Ding W.X. Ni H.M. Impaired fasting-induced adaptive lipid droplet biogenesis in liver-specific Atg5-deficient mouse liver is mediated by persistent nuclear factor-like 2 activation.Am J Pathol. 2018; 188: 1833-1846Google Scholar Real-time PCR was performed on a CFX384 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) using SYBR Green mix (Bimake, Houston, TX). Expression levels of Actb, Cd68, Ly6g, Il1b, Il6, Mip2, and Tnfa were quantified using quantitative real-time PCR analysis. Primer sequences were as follows: Actb: forward, 5′-TGTTACCAACTGGGACGACA-3′ and reverse, 5′-GGGGTGTTGAAGGTCTCAAA-3′; Ly6g: forward, 5′-TGCGTTGCTCTGGAGATAGA-3′ and reverse, 5′-CAGAGTAGTGGGGCAGATGG-3′; Il1b: forward, 5′- GCCCATCCTCTGTGACTCAT-3′ and reverse, 5′-AGGCCACAGGTATTTTGTCG-3′; Il6: forward, 5′-ACAACCACGGCCTTCCCTACTT-3′ and reverse, 5′-CATTTCCACGATTTCCCAGAGA-3′; Mip2: forward, 5′-CTCAGAGGAAGACGATGAAG T-3′ and reverse, 5′-CTCAGAGGAAGACGATGAAG-3′; and Tnfa: forward, 5′-CGTCAGCCGATTTGCTATCT-3′ and reverse, 5′-CGGACTCCGCAAAGTCTAAG-3′. Murine hepatocytes were isolated by a retrograde, nonrecirculating perfusion of livers with 0.05% Collagenase Type IV (Sigma-Aldrich) as described previously.33Ramachandran A. McGill M.R. Xie Y. Ni H.M. Ding W.X. Jaeschke H. Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice.Hepatology. 2013; 58: 2099-2108Google Scholar Cells were cultured in William's medium E supplemented with 10% fetal bovine serum for 2 hours to allow for attachment and then were switched to the same medium without fetal bovine serum. All cells were maintained in a 37°C incubator with 5% CO2. In vitro hypoxia and reoxygenation experiments were performed as described previously.34Wang J. Koh H.W. Zhou L. Bae U.J. Lee H.S. Bang I.H. Ka S.O. Oh S.H. Bae E.J. Park B.H. Sirtuin 2 aggravates postischemic liver injury by deacetylating mitogen-activated protein kinase phosphatase-1.Hepatology. 2017; 65: 225-236Google Scholar Briefly, murine primary hepatocytes were isolated from CD- and WD-fed mice. Hepatocytes were incubated at 37°C in anaerobic jars with oxygen-absorbing packs for 3 hours, followed by reoxygenation for different periods of time. Cell death was evaluated by quantification of lactate dehydrogenase (Ldh) release into the culture medium with an assay kit following the manufacturer's instructions (Pointe Scientific, Canton, MI). Hepatic triglyceride (TG) extraction was performed as described previously.35Ni H.M. Du K. You M. Ding W.X. Critical role of FoxO3a in alcohol-induced autophagy and hepatotoxicity.Am J Pathol. 2013; 183: 1815-1825Google Scholar Frozen liver tissues (20 to 50 mg) were ground into powder using a mortar and pestle followed by chloroform-methanol extraction. TG analysis was performed using GPO-Triglyceride Reagent Set (Pointe Scientific) following the manufacturer's instructions. Caspase-3 and -8 activities were assessed as described previously.36Ni H.M. Woolbright B.L. Williams J. Copple B. Cui W. Luyendyk J.P. Jaeschke H. Ding W.X. Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy.J Hepatol. 2014; 61: 617-625Google Scholar Briefly, 15 μg of total proteins were combined with 2 μmol/L Ac-IETD-AFC or Ac-DEVD-AFC (Enzo Life Sciences) in a caspase assay buffer [100 mmol/L NaCI, 1 mmol/L EDTA, 20 mmol/L PIPES (piperazine-N,N′-bis), 10% (w/v) sucrose, 0.1% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 10 mmol/L dithiothreitol, pH 7.2] in a final volume of 200 μL and were incubated for 2 hours at 37°C. The change of fluorescence was measured by a plate reader (Tecan Life Sciences). All experimental data are expressed as means ± SEM and were subjected to t-test or one-way analysis of variance with Bonferroni post hoc test where appropriate. P < 0.05 was considered significant. To investigate the role of necroptosis in fatty liver, a mouse model of nonalcoholic fatty liver was first established. Mice fed with WD had both increased macrosteatosis (with large lipid droplets) and microsteatosis (with small lipid droplets) in hepatocytes shown by H&E and oil red O staining (Figure 1, A and B). Western blot analysis revealed increased levels of Ripk1, Ripk3, and Mlkl in WD-fed mice compared to CD-fed mice at as early as 5 weeks and extended up to 12 weeks (Figure 1, C and D). These data indicate that diet-induced hepatic steatosis increases the levels of key necroptosis proteins in mouse livers. Inhibition of proteasome activity by ethanol increases levels of hepatic RIPK3.11Wang S. Ni H.M. Dorko K. Kumer S.C. Schmitt T.M. Nawabi A. Komatsu M. Huang H. Ding W.X. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.Oncotarget. 2016; 7: 17681-17698Crossref Scopus (55) Google Scholar The levels of proteasome core protein subunits Psmα2 and Psmβ5 as well as Psmc1 were decreased in the WD-fed mouse livers compared to those in the CD-fed mice (Figure 2, A and B). Moreover, hepatic trypsin-like proteasome activities were significantly decreased in the WD-fed mice compared to those in the CD-fed mice (Figure 2C). Hepatic chymotrypsin-like proteasome activities also trended to decrease in the WD-fed mice compared with the CD-fed mice (Figure 2C). These results indicate that WD decreases core hepatic proteasome subunit proteins and inhibits proteasome activities in mouse liver. Liver tissue H&E staining revealed that IR was associated with hepatocyte necrosis mainly in the central vein areas but barely in the portal vein areas regardless of CD or WD, although the necrotic areas were much larger in the WD-fed mouse livers than in the CD-fed mouse livers (Figure 3A). Caspase-3 and -8 activities showed no changes after IR in CD- or WD-fed mouse livers (Supplemental Figure S1, D and E). TUNEL staining confirmed the nature of necrotic cell death in the mouse livers after IR, as demonstrated by the diffuse TUNEL staining pattern, similar to acetaminophen-induced necrosis37Ni H.M. McGill M.R. Chao X. Woolbright B.L. Jaeschke H. Ding W.X. Caspase inhibition prevents tumor necrosis factor-alpha-induced apoptosis and promotes necrotic cell death in mouse hepatocytes in vivo and in vitro.Am J Pathol. 2016; 186: 2623-2636Google Scholar (Figure 3B). Since the TUNEL-positive areas are similar to necrotic areas on H&E staining, necrotic areas were quantified using TUNEL staining. The TUNEL-positive areas were significantly decreased in Mlkl KO mice after IR regardless of CD or WD (Figure 3C). The levels of serum Alt activities were also significantly lower in Mlkl KO mice than in wild-type (WT) mice after IR in both CD- and WD-fed mice (Figure 3D), which was consistent with the TUNEL staining data. These data indicate that MLKL-mediated necrosis plays a crucial role in the pathogenesis of IR-induced liver injury. The levels of liver triglyceride were not significantly different between WD-fed WT and Mlkl KO mice, suggesting that MLKL does not affect WD-induced steatosis (Supplemental Figure S1A). Since MLKL oligomer or polymer formation and plasma membrane translocation is a crucial step for triggering necroptosis, the level of Mlkl on the plasma membranes was next determined. The levels of Mlkl plasma membrane translocation were increased with IR in CD-fed mice. The levels of Mlkl plasma membrane translocation were already increased with WD alone, and were not further increased with IR, likely due to the nonspecific degradation of plasma membrane Mlkl as a result of the massive necrosis in the WD-fed mice after IR (Figure 3E). Mlkl oligomer formation was also examined in nonreducing conditions. Levels of Mlkl oligomer formation were increased with IR compared with sham livers, regardless of diet. Levels of Mlkl polymer were increased with IR mouse livers regardless of diet, but the levels of Mlkl polymer were much higher in IR WD-fed mice than in the IR CD-fed mice (Figure 3F). Hepatocytes were next isolated from CD- and WD-fed mice, and these hepatocytes were subjected to hypoxia and reoxygenation for various periods of time. Hepatocytes isolated from WD-fed mice had significantly higher" @default.
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• W2941155406 date "2019-07-01" @default.
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• W2941155406 title "Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers" @default.
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