Decrease in fat de novo synthesis and chemokine ligand expression in non‐alcoholic fatty liver disease caused by inhibition of mixed lineage kinase domain‐like pseudokinase

Abstract

Background and AimReceptor‐interacting serine/threonine kinase 3 and mixed lineage kinase domain‐like pseudokinase (MLKL) have gained attention as apoptosis alternate cell death signaling molecules. We aimed to evaluate the role of MLKL in non‐alcoholic fatty liver disease (NAFLD).MethodsHepatic tissue MLKL expression was compared between NAFLD patients and healthy controls. High‐fat diet was fed to wild‐type and MLKL‐knockout (KO) mice for 12 weeks. Brown adipose fat tissue was measured by [18F]‐fluorodeoxyglucose positron emission tomography. Energy expenditure was measured by indirect calorimetry. Anti‐MLKL effects were also evaluated in in vitro setting using U937 and HepG2 cells.ResultsHepatic tissue MLKL expression increased in NAFLD patients compared with healthy controls. MLKL expression increased according to the degree of steatosis, ballooning, and inflammation. High‐fat diet‐fed MLKL‐KO mice displayed decreased alanine aminotransferase, triglycerides, liver weight, NAFLD activity score (6.3 vs 3.5, P < 0.001), steatosis score (3.0 vs 1.8, P < 0.001), inflammation, and ballooning degeneration compared with wild‐type mice. SREBP1c, fatty acid synthase, and SCD‐1 expressions decreased in MLKL‐KO mice. Adipose tissue F4/80‐positive crown‐like structures were also reduced in MLKL‐KO mice. HepG2 cells treated with necrosulfonamide (an MLKL inhibitor) showed reduced Nile red staining and reduced SREBP1c and SCD‐1 expressions. Stimulation of necroptosis using lipopolysaccharide + caspase inhibitor (zVAD) increased CXCL1/2 expressions in U937 monocyte cells. Lipopolysaccharide + zVAD‐induced increased expressions of CXCL1/2 were reduced with necrosulfonamide treatment.ConclusionsMixed lineage kinase domain‐like pseudokinase inhibition has protective effects in non‐alcoholic steatohepatitis by decreasing hepatic de novo fat synthesis and chemokine (C‐X‐C motif) ligand expressions.

The Role of the Key Effector of Necroptotic Cell Death, MLKL, in Mouse Models of Disease

“…After 18 hours of a choline-deficient methioninesupplemented diet [55] or 8 weeks of a Western diet [17], Mlkl −/− mice are indistinguishable from wild-type controls. Following 12 weeks of a high fat diet (HFD) [56], however, Mlkl −/− mice appear resistant to steatohepatitis given that MLKL-deficiency promotes reduced de novo fat synthesis and chemokine ligand expression [56]. A similar effect is seen following 12 weeks of a high fat, fructose, and cholesterol diet, where Mlkl −/− mice are markedly protected against liver injury, hepatic inflammation and apoptosis attributed to inhibition of hepatic autophagy [57].…”

“…MLKL deficiency provides variable protection against the metabolic syndrome, depending on the challenge. MLKL deficiency appears to protect against dyslipidaemia with reduced serum triglyceride and cholesterol levels following a high fat diet [56] and Western diet [60], respectively. Whilst Mlkl −/− mice appear to have significantly improved fasting blood glucose levels given improved insulin sensitivity following 16 weeks of a high fat diet [59], there is conflicting evidence on the effect at steady state [56,59].…”

“…MLKL deficiency appears to protect against dyslipidaemia with reduced serum triglyceride and cholesterol levels following a high fat diet [56] and Western diet [60], respectively. Whilst Mlkl −/− mice appear to have significantly improved fasting blood glucose levels given improved insulin sensitivity following 16 weeks of a high fat diet [59], there is conflicting evidence on the effect at steady state [56,59]. There is also conflicting evidence on the role of MLKL deficiency in adipose tissue deposition and weight gain [56,59].…”

Targeting programmed cell death in metabolic dysfunction-associated fatty liver disease (MAFLD): a promising new therapy

“…Paradoxically, it was found that neither MLKL nor RIPK1 inhibition reduced inflammation [ 68 ]. Saeed et al found that patients with MAFLD exhibited increased MLKL levels compared to the non-MAFLD group and that MLKL −/− mice induced with a high-fat diet showed decreased transferase levels, triglycerides, MAFLD activity scores, steatosis score, inflammation, balloon degeneration, and expression of de novo lipogenesis (DNL) genes in the liver [ 69 ]. However, Suda found that RIPK1 antisense knockdown caused α-galactosylceramide-treated C57BL/6 mice to undergo large-scale apoptosis-type immune liver injury.…”

Circulating Receptor-Interacting Protein Kinase 3 Are Increased in HBV Patients With Acute-on-Chronic Liver Failure and Are Associated With Clinical Outcome

“…Immunohistochemical staining for RIPK3 (Abcam, #ab194699) and MLKL (Abcam, #ab194699) were performed in these 23 liver tissues, according to the experiment protocol as previously described (Lai et al, 2015). Both RIPK3 and MLKL antibodies used for immunohistochemical staining in our study were carefully selected based on applicability, specificity, and also upon validation from other investigators (Mizumura et al, 2014;Wang et al, 2016;Saeed et al, 2019;Xu et al, 2019). A negative control was coupled with the test in which the antibody was substituted by the primary rabbit negative control.…”